WO2025117593A1 - Bioprinted 3d culture scaffolds - Google Patents

Abstract

A three-dimensional scaffold for use in engineered tissue includes a repeated 3D geometry; and at least one of a hydrogel, a biocompatible polymer, and a bioprintable polymer. The repeated 3D geometry may include an open geometry including a plurality of interconnected nodes configured to be seeded with living cells. The scaffold may be formed via bioprinting. The three-dimensional scaffold may also be used for (1) 3D tissue modelling (for liver, kidney, gut, pancreas, heart, lung, spleen, etc.), (2) disease modeling of different tumor types such as breast, colorectal, lung, liver, brain, ovarian and others, (3) drug testing / therapeutic screening of small molecule drugs, peptides, proteins, antibodies, ADCs, gene therapies, RNA-based therapeutics, cell therapies, and tissue therapies, and (4) therapeutic/drug delivery (i.e., via delivery device that can be used to control drug release based on different patterns).

Description

BIOPRINTED 3D CULTURE SCAFFOLDS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/602,984 filed November 27, 2023, the disclosure of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE DISCLOSURE

[0002] Drug and therapy development are generally time-consuming and costly endeavors. In vivo testing of new drugs involves a phased, highly regulated approach that helps to ensure the efficacy of the underlying drugs and/or therapies, and helps to ensure the safety of patients upon whom they are being test, but also often entails multi-billion dollar (and multi-year) regulatory approval processes. In silica modeling of drugs and therapies can also be time-consuming and costly, and can lead to results that are inherently less accurate than those derived from in vivo testing. The success of in silica (i.e., computer) modeling and testing is also dependent on the underlying data upon which those computer models are based. When in vivo testing and in silico modeling result in counter- indications and/or inconclusive results, years of development time and millions (or even billions) of dollars of research and development investments are often lost.

[0003] As it pertains to tissue, living mammalian tissue (for example, bone, muscle, organ, skin, etc. tissue) has proven difficult to develop in a laboratory or simulated environment. Attempts have been made to model or replicate tissue using scaffolds to replicate tissue structure. Conventional scaffold fabrication methodologies, including electrospinning, emulsion templating, freeze drying and/or gas foaming, can lead to high variability in scaffold structure, introducing potentially large sources of variation in comparative studies, and possibly leading to inaccurate or inconsistent results.

SUMMARY OF THE DISCLOSURE

[0004] The present disclosure presents scaffolds, systems, methodologies, and apparatuses that enable quick prototyping and modeling of tissues, for example mammalian tissues such as human tissues. In one aspect, the present embodiments are directed to a three-dimensional scaffold for use in synthetic mammalian tissue that includes a repeated 3D geometry; and at least one of a hydrogel, a biocompatible polymer, and a bioprintable polymer. The repeated 3D geometry may include an open geometry includes a plurality of interconnected nodes configured to be seeded with living cells. The scaffold may be formed via bioprinting.

[0005] In another aspect, the present embodiments are directed to a method of producing a synthetic tissue comprising: bioprinting a scaffold according to the present disclosure; coating the scaffold; seeding the scaffold with at least one living cell; and initiating conditions to simulate an in vivo environment within the seeded scaffold.

[0006] In another aspect, the present embodiments are directed to scaffold designs and/or test rigs (or platforms), which may be used to study inter compartment migration of cells, including a scaffold as described herein disposed within at least one compartment of the multiple compartments.

[0007] In some embodiments, the test rig (or platform) includes at least one hollow base disposed beneath the multiple compartments; the multiple compartments include at least three compartments; the hollow base connects a first compartment of the multiple compartments to a third compartment of the multiple compartments; the multiple compartments include a second compartment disposed between the first compartment and the third compartment; and the second compartment is not fluidly connected to the hollow base.

[0008] In some embodiments, the scaffold includes a porosity in a range from about 40% to about 90%.

[0009] In some embodiments, the scaffold includes a porosity in a range from about 60% to about 80%.

[0010] In some embodiments, the repeated 3D geometry comprises a lattice of interconnected spherical pores.

[0011] In some embodiments, the repeated 3D geometry comprises a fibrous interstitial pattern.

[0012] In some embodiments, the repeated 3D geometry comprises a criss-cross pattern.

[0013] In some embodiments, the repeated 3D geometry is bioprinted.

[0014] In some embodiments, the scaffold comprises a hydrogel comprising an inert polymer.

[0015] In some embodiments, the repeated 3D geometry comprises a plurality of vertically stackable membranes, each membrane comprising a thickness in a range from about 100 pm to about 300 pm.

[0016] In some embodiments, the repeated 3D geometry comprises a vertically stacked membrane with a thickness in a range from about 180 pm to about 220 pm.

[0017] In some embodiments, the membranes comprise a plurality of oppositely-oriented pores. [0018] In some embodiments, the membranes comprise a plurality of pores, the plurality of pores comprising a mean diameter in a range from about 40 pm to about 80 m.

[0019] In some embodiments, the plurality of pores are disposed within the membranes at a density of about 3000-4500 pores per cm².

[0020] In some embodiments, each spherical pore of the lattice of interconnected spherical pores is connected to at least two adjacent spherical pores within the same horizontal plane.

[0021] In some embodiments, each spherical pore of the lattice of interconnected spherical pores is connected to at least one adjacent spherical pore in a next vertical layer higher of spherical pores, and wherein each spherical pore of the lattice of interconnected spherical pores is connected to at least one adjacent spherical pore in a next vertical layer lower of spherical pores.

[0022] In another aspect, the present embodiments are directed to a system (or synthetic tissue) that includes the scaffold as described herein and at least one active cell seeded therein.

[0023] In some embodiments, the system includes hydrogel, wherein the hydrogel is at least one of covalently bonded and ionically bonded to the at least one active cell.

[0024] In some embodiments, the at least one active cell comprises at least one of a mammalian cell.

[0025] In another aspect, the present embodiments are directed to a platform including: multiple compartments; and the scaffold as described herein disposed within at least one compartment of the multiple compartments.

[0026] In some embodiments, the platform includes at least one hollow base disposed beneath the multiple compartments, wherein the multiple compartments include at least three compartments; wherein the hollow base connects a first compartment of the multiple compartments to a third compartment of the multiple compartments; wherein the multiple compartments include a second compartment disposed between the first compartment and the third compartment; and wherein the second compartment is not fluidly connected to the hollow base.

[0027] In another aspect, the present embodiments are directed to a method of producing a synthetic tissue including: bioprinting the scaffold as described herein; coating the scaffold; seeding the scaffold with at least one living cell; and initiating conditions to simulate an in vivo environment within the seeded scaffold.

[0028] In some embodiments, coating the scaffold includes at least one of covalently bonding and ionically bonding at least one extracellular matrix (ECM) protein to the scaffold via at least one linker. [0029] In some embodiments, the linker includes at least one of acrylated polyethylene glycol succinimidyl valerate (acrylated PEG SVA) and polyethylene glycol N-hydroxysuccinimide (PEG-NHS).

[0030] In some embodiments, the method includes at least one post-processing step following bioprinting, the post-processing step including at least one of heat treat, rinsing, hydrating, drying, irradiating, surface smoothing, de-burring, and cooling.

[0031] In some embodiments, initiating conditions to simulate an in vivo environment includes: providing temperature in a range from about 20°C to about 40°C; providing an atmospheric pressure in a range from about 0.95 bars to about 1.05 bars; and providing an oxygen concentration in a range from about 19.5% to about 21.5%.

[0032] In some embodiments, seeding the scaffold with at least one living cell comprises seeding the scaffold with a fluid containing the living cell, the method further including: providing the fluid with a glucose concentration in a range from about 1 mM to about 20 mM; and providing the fluid with pH in a range from about 6 to about 8.

[0033] In another aspect, the present embodiments are directed to a platform including: a first compartment; a second compartment disposed a distance away from the first compartment; and at least one conduit, passageway, or chamber connecting the first compartment to the second compartment.

[0034] In some embodiments, each of the first compartment and the second compartment is vertically oriented, and wherein the conduit, passageway, or chamber is oriented substantially horizontally.

[0035] In some embodiments, each of the first compartment, the second compartment, and the conduit, passageway, or chamber are arranged in a substantially horizontal configuration with the conduit, passageway, or chamber being disposed between the first compartment and the second compartment.

[0036] In some embodiments, the method includes a gap disposed between the first compartment and the second compartment, the gap preventing migration of cells therethrough, wherein the conduit, passageway, or chamber comprises a hollow base disposed beneath the first compartment and the second compartment, the first and second compartment being configured to be seeded with live cells, the hollow base being configured to allow migration of cells from the first compartment to the second compartment, and vice versa, and wherein the gap is not fluidly connected to the hollow base, the first compartment or the second compartment. [0037] In another aspect, the present embodiments are directed to a system including: a platform including: a first compartment; a second compartment disposed a distance away from the first compartment; and at least one conduit, passageway, or chamber connecting the first compartment to the second compartment; and a three-dimensional scaffold disposed within the first compartment, the three-dimensional scaffold including: a repeated 3D geometry; and at least one of a hydrogel, a biocompatible polymer, and a bio-printable polymer; wherein the repeated 3D geometry includes an open geometry comprising a plurality of interconnected nodes configured to be seeded with living cells.

[0038] In some embodiments, the system comprises: a first plurality of live cells disposed within the first compartment; and a second plurality of live cells disposed within the second compartment, wherein the first plurality of cells comprises a different type of cells than the second plurality of cells, and wherein the three-dimensional scaffold is composed of a hydrogel material.

[0039] In some embodiments, the first plurality of live cells includes at least one of primary human hepatocytes (PHH), Jurkat cells, T cells, HUVECs, SK-BR-3 cells, control-treated cells (IgG4), liver cells, Kupffer cells, Immune cells, and IgG-treated cells.

[0040] In some embodiments, the second plurality of live cells includes at least one of necrotic cells, colorectal carcinoma cells, cancer cells, HCT116 cells, and tumor cells.

[0041] In some embodiments, the three-dimensional scaffold includes a plurality of vertically stackable membranes, each membrane including a thickness in a range from about 100 pm to about 300 pm.

[0042] In another aspect, the present embodiments are directed to a method of performing an assay including: providing a platform including: a first compartment; a second compartment disposed a distance away from the first compartment; and at least one conduit, passageway, or chamber connecting the first compartment to the second compartment; disposing a scaffold including a repeated 3D geometry in the first compartment; seeding the first compartment with a first type of live cells; seeding the second compartment with a second type of live cells; placing the first type of cells in fluid and/or operational communication with the second type of cells; exposing at least one of the first type of cells and the second type of cells to at least one stimulus; and assessing at least one property of at least one of the first type of cells and/or the second type of cells.

[0043] In some embodiments, the method includes coating the scaffold prior to seeding the first compartment. [0044] In some embodiments, seeding the first compartment includes seeding the scaffold with live cells.

[0045] In some embodiments, the method includes initiating conditions to simulate an in vivo environment following placing the first type of cells in fluid and/or operational communication with the second type of cells.

[0046] In some embodiments, seeding the first compartment with a first type of live cells includes seeding the first compartment with hepatocytes, and wherein assessing at least one property of at least one of the first type of cells and/or the second type of cells includes assessing production of albumin and/or urea of the hepatocytes.

[0047] In some embodiments, seeding the first compartment with a first type of live cells includes seeding the first compartment with hepatocytes, and wherein assessing at least one property of at least one of the first type of cells and/or the second type of cells includes assessing CYP3A4 activity of the hepatocytes.

[0048] In some embodiments, the method includes seeding the first compartment with a first type of live cells includes seeding the first compartment with hepatocytes, and wherein assessing at least one property of at least one of the first type of cells and/or the second type of cells includes assessing ATP production of the hepatocytes.

[0049] In some embodiments, seeding the first compartment with a first type of live cells includes co-seeding the first compartment with hepatocytes and Kupffer cells, wherein the method further includes co-culturing the hepatocytes and Kupffer cells in the first compartment, wherein assessing at least one property of at least one of the first type of cells and/or the second type of cells includes assessing a level of lipopolysaccharide (LPS) activation in the hepatocytes and/or Kupffer cells.

[0050] In some embodiments, seeding the first compartment with a first type of live cells includes co-seeding the first compartment with hepatocytes and Kupffer cells, wherein the method further includes co-culturing the hepatocytes and Kupffer cells in first compartment, wherein assessing at least one property of at least one of the first type of cells and/or the second type of cells includes assessing a level of TNFalpha and/or IL-6.

[0051] In some embodiments, seeding the first compartment with a first type of live cells includes co-seeding the first compartment with hepatocytes and Kupffer cells, wherein the method further includes co-culturing the hepatocytes and Kupffer cells in first compartment, wherein assessing at least one property of at least one of the first type of cells and/or the second type of cells includes determining a drug induced liver injury ranking.

[0052] In some embodiments, the method includes determining a platform specificity and/or sensitivity based on the at least one property of at least one of the first type of cells and/or the second type of cells.

[0053] In some embodiments, seeding the first compartment with a first type of live cells includes suspending hepatocytes in fluid at a density in a range from about 50,000 cells per ml to about 200,000 cells per ml, and seeding the scaffold with the fluid.

[0054] In some embodiments, the hepatocytes include at least one of Hc-04, HepG2, hepatoma, and imHC.

[0055] In some embodiments, the stimulus includes at least one of: Fialuridine, Entecavir, Troglitazone, Pioglitazone, Clozapine, Olanzapine, Acetaminophen, Metacetamol, Tolcapone, Entacapone, Nefazodone, Buspirone, Trovofloxacin, Levofloxacin, Diclofenac, and Amiodarone. [0056] In some embodiments, seeding the first compartment includes seeding the first compartment with T cells, and wherein the stimulus includes a therapeutic including an antibody, thereby triggering transendothelial migration of at least one T cell from the first compartment to the second compartment.

[0057] In some embodiments, the antibody includes Pembrolizumab.

[0058] In some embodiments, the stimulus includes a first stimulus comprising an isotype control and a second stimulus comprising a therapeutic including an antibody, the method further including: assessing a first T cell response resulting from the first stimulus; assessing a second T cell response resulting from the second stimulus; and comparing the first T cell response to the second T cell response.

[0059] In some embodiments, the isotype control includes IgG4.

[0060] In some embodiments, the three-dimensional scaffold is or includes a porous membrane, wherein seeding the first compartment with a first type of live cells includes lining the porous membrane with an endothelial barrier.

[0061] In some embodiments, the method includes increasing concentrations of Pembrolizumab thereby increasing the transendothelial migration of T-cells from the first compartment to the second compartment.

[0062] In some embodiments, the method includes introducing a second stimulus to the first compartment thereby increasing the rate of transendothelial migration of at least one T cell from the first compartment to the second compartment, the second stimulus comprising a chemoattractant.

[0063] In some embodiments, the chemoattractant includes CXCL12.

[0064] In some embodiments, assessing each of the first T cell response and the second T cell response includes measuring T cell activity via at least one of cytokine release and cytotoxic activity.

[0065] In some embodiments, the cytokine release includes IFNgamma release, and wherein cytotoxic activity includes Granzyme B release.

[0066] In some embodiments, the antibody includes a monoclonal antibody (mAB), the method further including mixing the T-cells with the monoclonal antibody.

[0067] In some embodiments, the monoclonal antibody includes an anti-PD-l-mAB.

[0068] In some embodiments, mixing the T-cells with the monoclonal antibody includes exposing peripheral blood mononuclear cells (PBMCs) to anti-CD3, anti-CD28, and/or anti-CD2 antibodies for a first period of time (i.e., about 3 days) and IL-2 antibodies for a second period of time (i.e., about 7 days).

[0069] In some embodiments, the method includes loading the T cells with a fluorescent label prior to seeding the T cells in the first compartment.

[0070] In some embodiments, the fluorescent label includes CellTracker Orange.

[0071] In some embodiments, the T cell includes at least one of a CD3 and a CD8 T cell.

[0072] In some embodiments, seeding the first compartment includes seeding the first compartment with at least one antibody drug conjugate (ADC).

[0073] In some embodiments, the antibody drug conjugate includes a T-Dxd ADC.

[0074] In some embodiments, seeding the second compartment includes seeding the second compartment with breast cancer cells.

[0075] In some embodiments, the method includes using an Alex 647 fluorescent market to identify at least one antibody.

[0076] In some embodiments, seeding the first compartment includes seeding the first compartment with HUVEC (human umbilical vein endothelial cells), the method further including assessing the toxicity of the at least one stimulus by measuring the viability of the HUVEC.

BRIEF DESCRIPTION OF THE DRAWINGS [0077] Fig. 1 illustrates views of 3D culture scaffolds, according to aspects of the present embodiments.

[0078] Fig. 2A illustrates a view of 3D culture scaffolds, according to aspects of the present embodiments.

[0079] Fig. 2B illustrates a view of 3D culture scaffolds, according to aspects of the present embodiments.

[0080] Fig. 3 illustrates fluorescent imaging of liver-on-a-chip tissue, according to aspects of the present embodiments.

[0081] Fig. 4 illustrates a seeding efficiency chart corresponding to the fluorescent imaging of liver-on-a-chip tissue illustrated in Fig. 3, according to aspects of the present embodiments.

[0082] Fig. 5 illustrates fluorescent imaging of liver-on-a-chip tissue, according to aspects of the present embodiments.

[0083] Fig. 6 illustrates fluorescent imaging of liver-on-a-chip tissue, according to aspects of the present embodiments.

[0084] Fig. 7 illustrates a bioprinted scaffold, according to aspects of the present embodiments.

[0085] Fig. 8A illustrates bioprinted scaffolds inserted into a well plate, according to aspects of the present embodiments.

[0086] Fig. 8B illustrates bioprinted scaffolds inserted into a well plate, according to aspects of the present embodiments.

[0087] Figs. 9A-9C illustrate top, front, and side views of a bioprinted scaffold, according to aspects of the present embodiments.

[0088] Fig. 10 illustrates a 3D image of a scaffold design, according to aspects of the present embodiments.

[0089] Fig. 11A illustrates membrane details of the 3D image of a scaffold design, according to aspects of the present embodiments.

[0090] Fig. 11B illustrates a top view of the scaffold design, according to aspects of the present embodiments.

[0091] Fig. 11C illustrates a cross-sectional configuration of a sponge membrane scaffold, according to aspects of the present embodiments.

[0092] Fig. 11D illustrates a cross-sectional configuration of a pore membrane scaffold, according to aspects of the present embodiments. [0093] Fig. 12 illustrates fluorescent imaging of 3D bioprinted liver tissue membrane, according to aspects of the present embodiments.

[0094] Fig. 13 illustrates fluorescent imaging of 3D bioprinted liver tissue membrane, according to aspects of the present embodiments.

[0095] Fig. 14 illustrates fluorescent imaging of 3D bioprinted liver tissue membrane, according to aspects of the present embodiments.

[0096] Fig. 15 illustrates a 3D bioprinted liver microstructure, according to aspects of the present embodiments.

[0097] Fig. 16 illustrates fluorescent imaging of 3D bioprinted liver tissue, according to aspects of the present embodiments.

[0098] Fig. 17 illustrates examples of viable cell types for seeding the microstructure, according to aspects of the present embodiments.

[0099] Fig. 18 illustrates scaffold production technologies, according to aspects of the present embodiments.

[00100] Fig. 19 illustrates a scaffold structure, according to aspects of the present embodiments.

[00101] Fig. 20 illustrates a scaffold structure in a 96 well plate, according to aspects of the present embodiments.

[00102] Fig. 21 shows an exemplary hepatocytes architecture grown using the 3D scaffold design, according to aspects of the present embodiments.

[00103] Fig. 22A shows graphs of albumin production rate by hepatocytes cultures from donor 1, according to aspects of the present embodiments.

[00104] Fig. 22B shows graphs of urea production rate by hepatocytes cultures from donor 1, according to aspects of the present embodiments.

[00105] Fig. 22C shows graphs of CYP3A4 activity by hepatocytes cultures from donor 1, according to aspects of the present embodiments.

[00106] Fig. 23A shows graphs of albumin production rate by hepatocytes cultures from donor 2, according to aspects of the present embodiments.

[00107] Fig. 23B shows graphs of urea production rate by hepatocytes cultures from donor 2, according to aspects of the present embodiments.

[00108] Fig. 23C shows graphs of CYP3A4 activity by hepatocytes cultures from donor 2, according to aspects of the present embodiments. [00109] Fig. 24A shows graphs of albumin production rate by hepatocytes cultures from donor 3, according to aspects of the present embodiments.

[00110] Fig. 24B shows graphs of urea production rate by hepatocytes cultures from donor 3, according to aspects of the present embodiments.

[00111] Fig. 24C shows graphs of CYP3A4 activity by hepatocytes cultures from donor 3, according to aspects of the present embodiments.

[00112] Fig. 25A is a graph of Viability (%) as a function of Multiplier of human Cmax (log 10), for Fial uridine vs Entecavir, according to aspects of the present embodiments.

[00113] Fig. 25B is a graph of Viability (%) as a function of Multiplier of human Cmax (log 10), for Troglitazone vs Pioglitazone, according to aspects of the present embodiments.

[00114] Fig. 25C is a graph of Viability (%) as a function of Multiplier of human Cmax (log 10), for Clozapine vs Olanzapine, according to aspects of the present embodiments.

[00115] Fig. 25D is a graph of Viability (%) as a function of APAP (loglO), for Acetaminophen vs Metacetamol, according to aspects of the present embodiments.

[00116] Fig. 25E is a graph of Viability (%) as a function of Multiplier of human Cmax (log 10), for Tolcapone vs Entacapone, according to aspects of the present embodiments.

[00117] Fig. 25F is a graph of Viability (%) as a function of Multiplier of human Cmax (loglO), for Nefazodone vs Buspirone, according to aspects of the present embodiments.

[00118] Fig. 25G is a graph of Viability (%) as a function of Multiplier of human Cmax (loglO), for Trovafloxacin vs Levofloxacin, according to aspects of the present embodiments.

[00119] Fig. 25H is a graph of Viability (%) as a function of Multiplier of human Cmax (loglO), for Diclofenac, according to aspects of the present embodiments.

[00120] Fig. 251 is a graph of Viability (%) as a function of Multiplier of human Cmax (loglO), for Amiodarone, according to aspects of the present embodiments.

[00121] Fig. 26A is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Fialuridine vs Entecavir, according to aspects of the present embodiments.

[00122] Fig. 26B is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Troglitazone vs Pioglitazone, according to aspects of the present embodiments. [00123] Fig. 26C is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Clozapine vs Olanzapine, according to aspects of the present embodiments.

[00124] Fig. 26D is a graph of albumin normalized response (% of control) as a function of APAP (loglO), for Acetaminophen vs Metacetamol, according to aspects of the present embodiments.

[00125] Fig. 26E is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Tolcapone vs Entacapone, according to aspects of the present embodiments.

[00126] Fig. 26F is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Nefazodone vs Buspirone, according to aspects of the present embodiments.

[00127] Fig. 26G is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Trovafloxacin vs Levofloxacin, according to aspects of the present embodiments.

[00128] Fig. 26H is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Diclofenac, according to aspects of the present embodiments. [00129] Fig. 261 is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Amiodarone, according to aspects of the present embodiments.

[00130] Fig. 27 A shows graphs of Viability (%) as a function of Trovafloxacin concentration, for PHH and co-culture of PHH and KC in the presence of -LPS and +LPS 176, according to aspects of the present embodiments.

[00131] Fig. 27B shows graphs of TNFa production (normalized by cells) as a function of Trovafloxacin concentration, for PHH and co-culture of PHH and KC in the presence of -LPS and +LPS, according to aspects of the present embodiments.

[00132] Fig. 27C shows graphs of IL-6 production (normalized by cells) as a function of Trovafloxacin concentration, for PHH and co-culture of PHH and KC in the presence of -LPS and +LPS, according to aspects of the present embodiments.

[00133] Fig. 28 shows the DILIrank, Garside Rank, and margin of safety (ICso/Cmax) predicted by 2D PHH, Spheroids, Emulate, and 3D scaffold design, for various drugs, according to aspects of the present embodiments. [00134] Fig. 29 is a graph of margin of safety (ICso/Cn™.) as a function of DILI severity category, for 2D, Spheroids, Emulate, and 3D scaffold design, according to aspects of the present embodiments.

[00135] Fig. 30A shows graphs of T cell TEM (%) and CRC killing (%) as a function of IgG4 and Pembrolizumab (pg/mL), for control and CXCL12, according to aspects of the present embodiments.

[00136] Fig. 30B shows graphs of IFNg (ng/mL) and Granzyme B (ng/mL) as a function of IgG4 and Pembrolizumab (pg/mL), for control and CXCL12, according to aspects of the present embodiments.

[00137] Fig. 31 illustrates an example of a cancer immunotherapeutic method using 3D scaffold design, according to aspects of the present embodiments.

[00138] Fig. 32A illustrates an example of mixing of T cells with antibodies, according to aspects of the present embodiments.

[00139] Fig. 32B is a graph of a flow cytometry experiment including CD8 and PD-1 markers, according to aspects of the present embodiments.

[00140] Fig. 32C shows graphs of expression of PD-L-1 on tumor cells, for PD-L-1, isotype control and unstained cells, according to aspects of the present embodiments.

[00141] Fig. 33A shows graphs of INFg secretions (normalized against IgG4) for IgG4, Nivolumab values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments.

[00142] Fig. 33B shows: (1) graphs of T-cell migration (%) for IgG4, and (2) Nivolumab values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments.

[00143] Fig. 33C shows: (1) graphs of INFg secretions (normalized against IgG4) for IgG4, and (2) Nivolumab values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for Transwell®.

[00144] Fig. 33D shows: (1) graphs of T-cell migration (%) for IgG4, and (2) Nivolumab values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for Transwell®.

[00145] Fig. 34A shows: (1) graphs of cleaved Caspase 3 (% positive cells) for IgG4, and (2) Nivolumab values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments. [00146] Fig. 34B shows: (1) graphs of cleaved Caspase 3 (% positive cells) for IgG4, and (2) Pembrolizumad values of 1.0 g/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments.

[00147] Fig. 34C shows: (1) graphs of cleaved Caspase 3 (% positive cells) for IgG4, and (2) Nivolumab values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for Transwell®.

[00148] Fig. 34D shows: (1) graphs of cleaved Caspase 3 (% positive cells) for IgG4, and (2) Pembrolizumad values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for Transwell®.

[00149] Fig. 35A shows an exemplary fluorescent microscopy image in the presence of lgG4 1 ug/mL, for 3D scaffold design, according to aspects of the present embodiments.

[00150] Fig. 35B shows an exemplary fluorescent microscopy image in the presence of Pembrolizumab 0.5 pg/mL, for 3D scaffold design, according to aspects of the present embodiments.

[00151] Fig. 35C shows an exemplary fluorescent microscopy image in the presence of Pembrolizumab 1 pg/mL, for 3D scaffold design, according to aspects of the present embodiments. [00152] Fig. 35D shows graphs of T-cell infiltration (count per 3 ROI) for IgG4, Pembrolizumad values of 0.5 pg/mL and 1 pg/mL, according to aspects of the present embodiments.

[00153] Fig. 35E shows: (1) graphs of mean fluorescence intensity (MFI) of cleaved-caspase 3 for IgG4, and (2) Pembrolizumad values of 0.5 pg/mL and 1 pg/mL, according to aspects of the present embodiments.

[00154] Fig. 36A shows: (1) graphs of INFy (ng/mL) for IgG4, and (2) Cemiplimab values of 1 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments.

[00155] Fig. 36B shows (1) graphs of T-cell migration (%) for IgG4, and (2) Cemiplimab values of 1 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments.

[00156] Fig. 36C shows: (1) graphs of Grz B (ng/mL) for IgG4, and (2) Cemiplimab values of 1 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments.

[00157] Fig. 36D shows: (1) graphs of % Cytotoxicity for IgG4, and (2) Cemiplimab values of 1 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments. [00158] Fig. 37 illustrates an example of a method of simultaneous assessment of safety and efficacy using 3D scaffold design, according to aspects of the present embodiments.

[00159] Fig. 38A shows graphs of HER2 expression (% of positive cells) for SK-BR-3, HCT- 116, A549, and CRC-PDOs, according to aspects of the present embodiments.

[00160] Fig. 38B shows plots of HER2 expression for A549, HCT 116, CRC-PDOs, SK-BR-s, isotype, and unstained, according to aspects of the present embodiments.

[00161] Fig. 39A shows graphs of cleaved caspase 3 (% positive cells) for no Tx (i.e., no therapeutic stimulus), T-Dxd 0.1 pg/mL, T-Dxd 1 g/mL, T-Dxd 10 pg/mL, Trastuzumab 10 pg/mL and Dxd 0.5 pM, according to aspects of the present embodiments.

[00162] Fig. 39B shows graphs of tumor-cell viability (%) for no Tx (i.e., no therapeutic stimulus), T-Dxd 0.1 pg/mL, T-Dxd 1 pg/mL, T-Dxd 10 pg/mL, Trastuzumab 10 pg/mL and Dxd 0.5 pM, for 3D scaffold design, according to aspects of the present embodiments.

[00163] Fig. 39C shows graphs of HUVECs viability (%) for no Tx (i.e., no therapeutic stimulus), T-Dxd 0.1 pg/mL, T-Dxd 1 pg/mL, T-Dxd 10 pg/mL, Trastuzumab 10 pg/mL and Dxd 0.5 pM, for 3D scaffold design, according to aspects of the present embodiments.

[00164] Fig. 40 is a flow chart diagram of a method of performing an assay, according to aspects of the present embodiments.

DETAILED DESCRIPTION OF THE DISCLOSURE

Definitions

[00165] In order for the present disclosure to be more readily understood, certain terms are defined below. Unless defined otherwise herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related.

[00166] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. [00167] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a cell" is understood to represent one or more cells. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[00168] Agent-. As used herein, the term “agent”, may refer to a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety.

[00169] Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[00170] Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). [00171] Biologically active: As used herein, the term “biologically active” refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological or physiological effect on that organism, is considered to be biologically active.

[00172] Biological Sample'. As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g. , fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.

[00173] Biomarker: The term “biomarker” is used herein, consistent with its use in the art, to refer to a to an entity, event, or characteristic whose presence, level, degree, type, and/or form, correlates with a particular biological event or state of interest, so that it is considered to be a “marker” of that event or state. To give but a few examples, in some embodiments, a biomarker may be or comprise a marker for a particular disease state, or for likelihood that a particular disease, disorder or condition may develop, occur, or reoccur. In some embodiments, a bio marker may be or comprise a marker for a particular disease or therapeutic outcome, or likelihood thereof. Thus, in some embodiments, a biomarker is predictive, in some embodiments, a biomarker is prognostic, in some embodiments, a biomarker is diagnostic of the relevant biological event or state of interest. A biomarker may be or comprise an entity of any chemical class, and may be or comprise a combination of entities. For example, in some embodiments, a biomarker may be or comprise a nucleic acid, a polypeptide, a lipid, a carbohydrate, a small molecule, an inorganic agent (e.g., a metal or ion), or a combination thereof. In some embodiments, a biomarker is a cell surface marker. In some embodiments, a biomarker is intracellular. In some embodiments, a biomarker is detected outside of cells (e.g., is secreted or is otherwise generated or present outside of cells, e.g., in a body fluid such as blood, urine, tears, saliva, cerebrospinal fluid, etc. In some embodiments, a biomarker may be or comprise a genetic or epigenetic signature. In some embodiments, a bio marker may be or comprise a gene expression signature.

[00174] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of" (or which "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of" (or "consists of") the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.

[00175] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature. For example, in some embodiments described and/or utilized herein, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, a polypeptide may be considered to be “engineered” if encoded by or expressed from an engineered polynucleotide, and/or if produced other than natural expression in a cell. Analogously, a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). In some embodiments, an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell. As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[00176] “Improve,” “increase”, “inhibit” or “reduce”: As used herein, the terms “improve”, “increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[00177] Interstitial space: As used herein, the term “interstitial space” refers to a void wherein a cell in a fluid(s) and/or a polymer (e.g., a hydrogel) are seeded to create a tissue. [00178] Interstitial infill: As used herein, the term “interstitial infill” refers to a polymer anchor and/or structure (e.g., hydrogel) that enables creation of a two-dimensional or three-dimensional tissue through stimulation of cell association.

[00179] In vitro'. The term “in vitro” as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[00180] In vivo: as used herein refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[00181] Linker: as used herein, is used to refer to that portion of a multi-element agent that connects different elements to one another.

[00182] Operably linked: The term “operably linked,” as used herein, indicates that two or more components are arranged such that the components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. Two molecules are “operably linked” whether they are attached directly or indirectly. [00183] For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5^th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[00184] Patient: As used herein, the term “patient” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disorder or condition. In some embodiments, a patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer, or presence of one or more tumors. In some embodiments, the patient is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition. [00185] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[00186] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or poly anhydrides; and other nontoxic compatible substances employed in pharmaceutical formulations. [00187] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p- toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.

[00188] Physiological conditions: as used herein, has its art-understood meaning referencing conditions under which cells or organisms live and/or reproduce. In some embodiments, the term refers to conditions of the external or internal milieu that may occur in nature for an organism or cell system. In some embodiments, physiological conditions are those conditions present within the body of a human or non-human animal, especially those conditions present at and/or within a surgical site. Physiological conditions typically include, e.g., a temperature range of 20 - 40°C, atmospheric pressure of 1, pH of 6-8, glucose concentration of 1-20 mM, oxygen concentration at atmospheric levels, and gravity as it is encountered on earth. In some embodiments, conditions in a laboratory are manipulated and/or maintained at physiologic conditions. In some embodiments, physiological conditions are encountered in an organism.

[00189] Polypeptide: The term “polypeptide” as used herein refers to a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As is known to those skilled in the art, polypeptides may be processed and/or modified.

[00190] Protein: The term “protein” as used herein refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit, the terms “polypeptide” and “protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” refers to the multiple polypeptides that are physically coupled and function together as the discrete unit.

[00191] Prodrug: As used herein, the term “prodrug” refers to a compound that is a drug precursor which, following administration, releases (e.g., is converted into) the drug in vivo via a chemical or physiological process (e.g., via cleavage as a result of exposure to a particular pH or through action of a particular enzyme or enzymes).

[00192] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[00193] Small molecule: As used herein, the term “small molecule” means a low molecular weight organic and/or inorganic compound. In general, a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not and/or does not comprise a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not and/or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not and/or does not comprise a polysaccharide; for example, in some embodiments, a small molecule is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is a modulating agent (e.g., is an inhibiting/inhibitory agent or an activating agent). In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic agent. Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain small molecule compounds described herein may be provided and/or utilized in any of a variety of forms such as, for example, crystal forms, salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e.g., optical and/or structural isomers), isotopic forms, etc. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more stereoisomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers; in some embodiments, such a small molecule may be utilized in accordance with the present disclosure in a racemic mixture form. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in the form of an individual tautomer, or in a form that interconverts between tautomeric forms. Those of skill in the art will appreciate that certain small molecule compounds have structures that permit isotopic substitution (e.g., ²H or ³H for H; ⁿC, ¹³C or ¹⁴C for 12C; ¹³N or ¹⁵N for 14N; ¹⁷O or ¹⁸O for 160; ³⁶C1 for XXC; ¹⁸F for XXF; 1311 for XXXI; etc). In some embodiments, such a small molecule may be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof. In some embodiments, reference to a particular small molecule compound may relate to a specific form of that compound. In some embodiments, a particular small molecule compound may be provided and/or utilized in a salt form (e.g., in an acid-addition or base-addition salt form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form. In some embodiments, where a small molecule compound is one that exists or is found in nature, that compound may be provided and/or utilized in accordance with the present disclosure in a form different from that in which it exists or is found in nature. Those of ordinary skill in the art will appreciate that, in some embodiments, a preparation of a particular small molecule compound that contains an absolute or relative amount of the compound, or of a particular form thereof, that is different from the absolute or relative (with respect to another component of the preparation including, for example, another form of the compound) amount of the compound or form that is present in a reference preparation of interest (e.g., in a primary sample from a source of interest such as a biological or environmental source) is distinct from the compound as it exists in the reference preparation or source. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a small molecule compound may be considered to be a different form from another salt form of the compound; a preparation that contains only a form of the compound that contains one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form of the compound from one that contains the other conformational isomer ((E) or (Z)) of the double bond; a preparation in which one or more atoms is a different isotope than is present in a reference preparation may be considered to be a different form; etc.

[00194] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[00195] Systemic: The phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” as used herein have their art- understood meaning referring to administration of a compound and/or composition such that it enters the ecosystem. [00196] Tumor Microenvironment: As used herein, the term “tumor microenvironment” refers to a complex ecosystem surrounding a tumor, composed of cancer cells, stromal tissue (including blood vessels, immune cells, fibroblasts and signaling molecules), molecules, and/or the extracellular matrix. Mutual interaction between cancer cells and the different components of the tumor microenvironment support its growth and invasion in healthy tissues which correlates with tumor resistance to current treatments and poor prognosis. A tumor can change its microenvironment, and the microenvironment can affect how a tumor grows and spreads.

[00197] Variant and Mutant: The term “variant” is usually defined in the scientific literature and used herein in reference to an organism that differs genetically in some way from an accepted standard, “Variant” can also be used to describe phenotypic differences that are not genetic (King and Stansfield, 2002, A dictionary of genetics, 6th ed., New York, New York, Oxford University Press.

[00198] The term “mutation” is defined by most dictionaries and used herein in reference to the process that introduces a heritable change into the structure of a gene (King & Stansfield, 2002) thereby producing a “mutant.” The term “variant” is increasingly being used in place of the term “mutation” in the scientific and non- scientific literature. The terms are used interchangeably herein.

Scaffold

Polymer Composition

[00199] Among other things, the present disclosure describes a system, structure, and/or methodology for forming refined physical models of living tissues (i.e., synthetic tissue) that may be used to for drug discovery and for assessing the effectiveness of various therapies. The synthetic tissue may include a structural framework or matrix referred herein as a scaffold. The present disclosure provides technologies that utilize a polymer moiety (and/or materials, such as bioprinted entities, generated from them, and/or ecosystems that include them), for example, for use with scaffolds to model living tissue, including in diagnostic applications (e.g., to identify and validate a new disease target). In some aspects, the present disclosure provides methods to generate polymers (e.g., a hydrogel), that includes control of environmental factors used for assaying synthetic tissue, real-time and/or continuous tracking of biomarkers via sensors, and/or an ability to rapidly image tissues and/or cells. In some embodiments, bioprinting methodologies, systems, and technologies described herein may be overlapping with, similar to, substantially similar to, and/or identical to those described in United States Patent Nos. 10,639,880 and/or 10,828,833, both of which are incorporated herein by reference in their entireties.

[00200] The present disclosure teaches that, in some embodiments, a bioprinted entity provided and/or utilized in accordance with the present disclosure includes a polymer moiety, such as a hydrogel moiety (e.g., PEGDA) and a coating moiety (e.g., a polypeptide), covalently linked to one another, optionally via a linker.

Cells

[00201] The present disclosure teaches that, in some embodiments, a bioprinted entity provided and/or utilized in accordance with the present disclosure includes a polymer moiety, such as a hydrogel moiety (e.g., PEGDA, PEGMA, PEGDMA, PVA, PAAm, HAMA, AlgMA, ColMA, GelMA) a coating moiety (e.g., a polypeptide), covalently linked to one another, optionally via a linker (e.g., acrylated PEG SVA, PEG-NHS, maleimide-PEG- acrylate, acrylated PEG-azide (for click chemistry), thiol -reactive PEG- vinyl sulfone, hydrazone-forming aldehyde-functionalized PEG, and PEG-diacrylate), wherein the coating moiety facilitates the association of a cell.

[00202] In some embodiments, a cell associated with the bioprinted entity may be selected from a naturally occurring cell and/or an engineered cell. In some embodiments, a cell associated with the bioprinted entity may be a naturally occurring cell. In some embodiments a cell associated with the bioprinted entity may be an engineered cell. In some embodiments, one or more cells associated with the bioprinted entity may be a combination of a naturally occurring and an engineered cell.

[00203] In some embodiments, a cell associated with the bioprinted entity and/or seeded therein is selected from a group consisting of an endothelial cell, a biliary endothelial cell, a cholangiocyte, a liver parenchymal cell, a hepatocyte (HC), a primary human hepatocyte (PHH), a heptic stellate cell (HSCs), a Kupffer cell (KC), a mucous cell, a parietal cell, a chief cell, an endocrine cell (e.g., a G cell, a D cell, a enterochromaffin cell, a EC-like cell, a X/A cell), a columnar epithelial cell, a cardiac fibroblast (CF), a cardiomyocyte, a smooth muscle cell, an enterocyte, a goblet cell, a Paneth cell, a stem cell, a neuron, a glia, a keratinocyte, a melanocyte, a Merkel cell, a Langerhan cell, a germ cell, a stromal cell, a seminiferous tubule, a Leydig cell, a tubule epithelial cell, a macula densa cell, a glomerular endothelial cell, a podocyte, a mesangial cell, a parietal epithelial cell, an immortalized cell (e.g. a 3T3 cell, a A549 cell, a HeLa cell, a HEK 293 cell, a HEK 293T cell, a Huh7 cell, a Jurkat cell, a OK cell, a Ptk2 cell, a Vero cell), a patient-derived cell (e.g., a tumor cell), a T cell, a peripheral blood mononuclear cell (PBMC), and/or an induced pluripotent stem cell (iPSC).

Scaffold Formation

[00204] In some embodiments, a bioprinted entity comprises a biocompatible resin and a bioprinted vascularized scaffold. In some such embodiments, a bioprinted entity comprises at least one chamber wherein a cell in media may be pipetted into.

[00205] In some embodiments, the vascularized scaffold may go through the chambers. In some embodiments, aspects of the vascularized scaffold may be controlled (e.g., wall thickness, lumen diameter, porosity, architecture) to provide alternate properties. It is also understood that the vascularized scaffold may comprise more than one independent network (e.g., vasculature, a bile duct). In some embodiments, bioprinting methodologies, systems, and technologies described herein may be overlapping with, similar to, substantially similar to, and/or identical to those described in United States Patent Nos. 10,639,880 and/or 10,828,833.

[00206] Fig. 1 illustrates views of interstitial infill 58, according to aspects of the present embodiments. In some configurations, the interstitial infill 58 may include one or more integral handles 66 and/or other features to facilitate handling of the interstitial infill 58. In some embodiments, the handles 66 are disposed on longitudinal ends of the interstitial infill 58, and are sized to allow for handling via tweezers. In some embodiments, the interstitial infill 58 may include one or more bioprinted orientation features 56 for identifying which side of the interstitial infill 58 or scaffold should face up. For example, in some embodiments, the interstitial infill 58 or scaffold 58 may include one or more rounded features 56 disposed on top of one or more flat platforms such that users may use the bioprinted orientation features 56 as visuals for identifying that the rounded features 56 should face upwards while the interstitial infdl 58 or scaffold is being seeded with live cells, etc.

[00207] Figs. 2 A and 2B illustrate a view of interstitial infill 58, according to aspects of the present embodiments. In the embodiments illustrated in Figs. 2A and 2B, the interstitial infill may include a lattice of interconnected spherical pores 68, connected via a plurality of interconnects 72. In some embodiments, the spherical pores 68 are connect to every adjacent spherical pore 68 via the interconnects 72. In some embodiments, the spherical pores 68 are not connected to every adjacent spherical pore 68, but are connected to 1) at least two adjacent spherical pores 68 in the same horizontal plane, 2) at least one adjacent spherical pore 68 in the next vertical layer higher of spherical pores, and 3) at least one adjacent spherical pore 68 in the next vertical layer lower of spherical pores. Accordingly, in some embodiments, each spherical pore 68 is connected to at least 4 adjacent spherical pores 68 via at least 4 corresponding interconnects 72.

[00208] Referring still to Figs. 2A and 2B, in some embodiments, the framework 58 (or interstitial infill 58) illustrated in Figs. 2A and 2B may be well suited for creating models of (e.g., synthetic) tissue such as liver tissue. In some embodiments, the each of the spherical pores 68 may have a diameter of about 300 pm (i.e., 300 microns) or from about 250 pm to about 350 pm, or from about 200 pm to about 400 pm, or from about 150 pm to about 450 pm. In some embodiments, each of the interconnects 72 may have a maximum dimension (i.e., diameter, length, major axis, etc.) of about 60 pm, or from about 55 pm to about 65 pm, or from about 50 pm to about 70 pm, or from about 45 pm to about 75 pm, or from about 40 pm to about 80 pm. The scaffold 58, framework 58, or interstitial infill 58 may be seeded with active cells via pipette. In some embodiments, the scaffold 58, framework 58, or interstitial infill 58 may be seeded with primary human hepatocyte and/or may be co-cultured with stellate cells and/or Kupffer cells. In some embodiments, the scaffold 58, framework 58, or interstitial infill 58 may be seeded with sinusoidal endothelial cells, hi some embodiments, the scaffold 58, framework 58, or interstitial infill 58 may also be seeded with cholangiocytes. In some embodiments, the scaffold 58, framework 58, or interstitial infill 58 is not vascularized (i.e., not disposed within a vasculature 60). In some embodiments, the scaffold 58, framework 58, or interstitial infill 58 may be seeded at a density of from about 200,000 to about 1 million cell per chip. In some embodiments, a bulk porosity of the scaffold 58 may be in a range from about 40% to about 95%, or from about 40% to about 95%, or from about 50% to about 95%, or from about 60% to about 95%, or from about 65% to about 90%, or from about 70% to about 75%, or from about 60% to about 80%, or from about 50% to about 70%, or from about 55% to about 75%, or from about 65% to about 95%, or from about 70% to about 85%.

Interstitial Infill and Interstitial Space [00209] The present disclosure teaches that, in some embodiments, an ecosystem that produces a platform for creating physical, synthetic models of an organ, a tissue, and/or a cell, via bioprinting of a scaffold comprises a bioprinted entity, vasculature, an interstitial infill, and/or an interstitial space.

[00210] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, the interstitial space may be a void wherein a cell in a fluid(s) and/or a polymer (e.g., a hydrogel) are seeded to create a tissue. In some such embodiments, the cell in a fluid(s) may be an endothelial cell optionally coupled to live tissues and/or a cell other than an endothelial cell.

[00211] In some embodiments, the interstitial infill may be a polymer anchor and/or structure (e.g., a hydrogel) that enables creation of a two-dimensional or three-dimensional tissue through stimulation of cell association. In some such embodiments, the cell in a fluid(s) may be an endothelial cell optionally coupled to live tissues and/or a cell other than an endothelial cell.

[00212] In some embodiments, the interstitial infill comprises an interstitial pattern that approximates the geometries of the cell and/or tissue(s) native format. In some embodiments, the interstitial pattern is selected from a fibrous and/or a spherical pattern.

[00213] In some embodiments, the interstitial pattern is a fibrous pattern. In some embodiments, the fibrous pattern may be modified according to different parameters (e.g., density, thickness, orientation).

[00214] The present disclosure teaches, that at least in some embodiments, the interstitial space is perfused with a coating moiety through a chamber. In some such embodiments, the coating moiety is a cell and/or plurality of cells, which may be the same or different. In further embodiments, the cell is an endothelial cell and a cell other than an endothelial cell. In some embodiments, the cell is an endothelial cell. In some embodiments, the cell is other than an endothelial cell.

[00215] In some embodiments, the interstitial pattern is a spherical pattern, which may comprise pores and/or windows. In some embodiments, the spherical pattern may be modified according to different parameters (e.g., size, wall thickness, number of microspheres).

Liver System

[00216] In some embodiments, the model organ is a liver. In some aspects, a model liver comprises a cell chamber (to metabolize a biologically active material e.g., a drug), an access point (to enable delivery or sampling of a fluid(s) comprising a cell and/or a biologically active material), an inlet, and an outlet. In some embodiments, the model liver is vascularized. In some embodiments, the model liver is optionally vascularized. In some embodiments, the model liver is not vascularized.

[00217] Fig. 3 -6 illustrate a scaffold used in connection with a model liver, according to aspects of the present embodiments. The model liver may include a vasculature associated with a cell that comprises a scaffold including an interstitial pattern which may be characterized by fluorescence imaging, optionally wherein the interstitial pattern approximates the geometries of the cell and/or tissue(s) native format. In some embodiments, the interstitial pattern is spherical. In some embodiments, the interstitial pattern is fibrous. In some embodiments, the present disclosure provides a liver model (e.g., liver-on-a-chip) that shows improved cellular distribution relative to a comparable liver model (e.g., when comparing interstitial patterns via fluorescence imaging).

[00218] Fig. 4 illustrates a seeding efficiency chart corresponding to the fluorescent imaging of liver-on-a-chip tissue illustrated in Fig. 3, according to aspects of the present embodiments. As illustrated in Fig. 3, live cells 78 appear in a green color and dead cells 82 appear in a red color. The live cells 78 (green) also generally have a much larger volume and/or cross-sectional area than the dead cells 82. Optimal microstructure was obtained through assessments of primary human hepatocyte seeding efficiency and survival rates. Seeding efficiency not only surpassed industry benchmarks but also contributed to robust cell survival. As shown in Fig. 4, the live cells 78 demonstrated a much higher structure than the dead cells 82, even at similar seeding efficiencies. Both populations (live cells 78 and dead cells 82) were seeded at efficiencies from about 55% to about 70%, as shown in Fig. 4. As shown in Figs. 5 and 6, according to the present embodiments, the live cells may form arrangements (i.e., interstitial patterns) that include repeating patterns of approximate geometric shapes such as circles, ovals, octagons, hexagons, and/or other shapes.

[00219] Fig. 7 illustrates a bioprinted scaffold, according to aspects of the present embodiments. The bioprinted scaffold may be inserted into well plates for 3D culture applications. The bioprinted scaffold 58 may include several different configuration and internal lattice patterns including those shown in Figs. 1, 2A, 2B, 9A, 9B, 9C, and 15.

[00220] Figs. 8 A and 8B illustrate bioprinted scaffolds inserted into a well plate, according to aspects of the present embodiments. In some embodiments, the well plate may include various numbers of wells, (for example, from about 6 wells to about 1536 wells) and may include bioprinted hydrogel scaffolds inserted in the wells, the scaffolds being coated with two reagents. [00221] Figs. 9A-9C illustrate top, front, and side views of a bioprinted scaffold, according to aspects of the present embodiments. For example, as shown in Fig. 9A, the bioprinted scaffold may include spheres of uniform size. As shown in Fig. 9B, the bioprinted scaffold may include three or more different sphere sizes arranged in various patterns, according to the vertical layer in which they are located. As shown in Fig. 9C, the bioprinted scaffold may include two sphere sizes arranged in an alternating pattern.

[00222] Fig. 10 illustrates a 3D image of a scaffold design 90 (or test rig) (or platform), that may be used to study inter compartment migration of cells, according to aspects of the present embodiments. In some embodiments, the scaffold design 90 (or test rig) (or platform) may include a generally cylindrical outer shape with one or more compartment, passages and/or channels disposed longitudinally therethrough. The one or more longitudinal compartments may include: a first compartment 84, which may include a generally circular or oval-shaped cross section; a gap 86 adjacent to, but spaced slightly from the first compartment 84, the gap including a curved cross section that is concave from the perspective of the first compartment 84; and, a second compartment 88 adjacent to, but spaced slightly from the gap 86, the second compartment 88 including a curved cross section that is concave from the perspective of the gap 86. The gap 86 may be disposed between the first compartment 84, and the second compartment 88. While the gap 86 may be concave from the perspective of the first compartment 84, the gap 86 may also be convex from the perspective of the second compartment 88.

[00223] Referring still to Fig. 10, each of the first longitudinal compartment 84, the gap 86, and the second longitudinal compartment 88 may extend longitudinally (for example, vertically in the configuration shown in Fig. 10) from a hollow base 98 that connects each of the first and second longitudinal compartments 84, 88, but not to the gap 86. This scaffold design 90 or configuration allows cells to initially be seeded in the first compartment 84, and then microscopy can be used over time to identify cell migration into the second compartment 88 via the hollow base 98. The gap 86 includes or comprises a void (i.e., it is air-filled) thereby preventing cell migration thereacross, even for cells that have migrated through the cell wall. Accordingly, cells that have migrated from the first compartment 84 to the second compartment 88 have done so via the hollow base 98. (The gap 86 is sealed off from and fluidly disconnected from the hollow base 98, and therefore prevents diffusion of cells between the first compartment 84 and the second compartment 88). The scaffold design 90 may also include a support pedestal 96 disposed within the hollow base 98 to help support the walls between the first longitudinal compartment 84, the gap 86, and the second longitudinal compartments 88. In some embodiments, the walls of the scaffold 90 may include a porosity in a range of about 1% to about 30% (for example, from about 1-5%, 1-10%, 1- 15%, 1-20%, etc.)

[00224] Still referring to Fig. 10, cells in the first and second compartments 84, 88 may be functionally coupled such that interaction between the two (or more) populations of cells is possible. For example, in some embodiments, cells in the first compartment 84 may be fluidly coupled to cells in the second compartment 88. In some embodiments, cells in the first compartment 84 may be operationally coupled (i.e., functionally coupled) to cells in the second compartment 88 via extra cellular matrices, scaffolds, hydrogel structures, etc. In some embodiments, cells in the first compartment 84 may be operationally coupled (i.e., functionally coupled) to cells in the second compartment 88 even when the first compartment 84 is not fluidly coupled to the second compartment 88.

[00225] Fig. 11A illustrates membrane details of the 3D image of a scaffold design of Fig. 10, according to aspects of the present embodiments. The membranes may include a solid non- permeable membrane 102 (i.e., a grayscale membrane), a sponge design 104, and/or a pore design 106, with different patterns, pore densities, and spacings, as shown in Fig. HA. In some embodiments, each of the grayscale membrane 102, the sponge membrane 104, and the pore membrane may include a thickness of about 200 microns, or from about 175pm to about 225pm, or from about 150pm to about 250pm, or from about 100pm to about 300pm. In some embodiments, each of the grayscale membrane 102, the sponge membrane 104, and/or the pore membrane 106 may include a cross-sectional area of about 0.33 cm², or from about 0.30 cm² to about 0.35 cm², or from about 0.25 cm² to about 0.40 cm². In some embodiments, the grayscale membrane may include cross-links with varying lengths. In some embodiments, each of the grayscale, sponge, and pore membranes repeat their respective structural patterns ever layer (for example, about every 200 microns (+/- 20%) of vertical linear distance).

[00226] Fig. 11B illustrates a top view of the scaffold design shown in Fig. 10, according to aspects of the present embodiments. In some embodiments, a maximum width (i.e., maximum radial width) 92 of the second compartment 88 may be about 3 to 10 times (for example, 3 to 8 times, 4 to 8 times, 5 to 10 times, 5 to 8 times, 5 to 7 times, 5 to 6 times, etc.) a maximum width (i.e., maximum radial width) 94 of the gap 86. As shown in Fig. 1 IB, each of the first and second longitudinal compartments 84, 88 may include a cross sectional area that is larger at the top end of the compartment than at the bottom end of the compartment. [00227] Fig. 11C illustrates a cross-sectional configuration of a sponge membrane scaffold 104, according to aspects of the present embodiments. According to aspects of the present embodiments, the sponge membrane scaffold 104 may include various geometries. In some embodiments, the sponge membrane scaffold 104 may include cylindrical pores and/or various microspherical patterns. In some embodiments, the sponge membrane 104 may include a plurality of oppositely disposed (or oriented) pores 108. The pores 108 may be spaced in an alternating configuration with each pore 108 disposed in, or protruding from, an opposite wall (for example an upper internal wall 120 or lower internal wall 118 of the membrane 104) from an adjacent pore 108. Each pore 108 may include a nominally triangular (for example, equilateral triangle) shape with a nominal height of about 100 pm, or from about 80 pm to about 120 pm, or from about 50 pm to about 150 pm. In some embodiments, the sponge membrane 104 may include a lateral spacing 110 (center- to-center of the pores 108) between adjacent pores 108 protruding from the same wall of about 300 pm, or from about 275 pm to about 325 pm, or from about 250 pm to about 350 pm, or from about 200 pm to about 400 pm, or from about 150 pm to about 450 pm. In some embodiments, each pore 108 is connected to the respective lower or upper wall 118, 120 at a tip of the triangularshaped pore 108 with a flat edge of the pore 108 protruding into the interior of the sponge membrane 104, the flat edge that is protruding into the interior of the sponge membrane 104 being oriented such that it is substantially parallel to the lower wall 118 and/or the upper wall 120. In some embodiments, the sponge membrane 104 may include a point-to-point (or tip-to-tip, or vertex-to-vertex) spacing 112 between each pore 108 and the closest adjacent pore 108 (which would be protruding from an opposite wall 118, 120) of about 60 pm, or from about 55 pm to about 65 pm, or from about 50 pm to about 70 pm, or from about 45 pm to about 75 pm, or from about 40 pm to about 80 pm.

[00228] Fig. 11D illustrates a cross-sectional configuration of a pore membrane scaffold 106, according to aspects of the present embodiments. The pore membrane 106 may include a plurality of pores 116, each pore 116 including a diameter of about 60 pm, or from about 55 pm to about 65 pm, or from about 50 pm to about 70 pm, or from about 45 pm to about 75 pm, or from about 40 pm to about 80 pm. The pore membrane 106 may include a spacing 114 between pores of about 210 pm, or from about 195 pm to about 225 pm, or from about 180 pm to about 240 pm, or from about 150 pm to about 270 pm. In some embodiments, the pore membrane 106 may include a pore density of about 3500/cm² to about 4000/cm², or from about 3700/cm² to about 4200/cm², or from about 3300/cm² to about 4500/cm², or from about 3000/cm² to about 4500/cm². [00229] Fig. 12 illustrates fluorescent imaging of 3D bioprinted liver tissue membrane, according to aspects of the present embodiments. The images were captured using a solid, non-permeable membrane with the scaffold design 90 of Fig. 10. As shown in Fig. 12, the cells attached at the top of the first channel 84 or compartment. There was no evidence of cells reaching the gap 86 or the second channel (or compartment) 88. The solid, non-permeable membrane imaged in Fig. 12 was used as a negative control for TEM studies. The cells demonstrated a polygonal morphology with cell-cell interaction, and no evidence of contamination.

[00230] Fig. 13 illustrates fluorescent imaging of 3D bioprinted liver tissue membrane, according to aspects of the present embodiments. The images were captured using a porous, slightly permeable membrane with the scaffold design 90 of Fig. 10. Cells attached mainly on the top of the first channel/compartment 84, with some cells also migrating to the second compartment 88. The cells demonstrated a polygonal morphology with cell-cell interaction. In some images, cells that appear to have reached the gap 86 are actually disposed within the hollow base 98 located beneath the gap 86.

[00231] Fig. 14 illustrates fluorescent imaging of 3D bioprinted liver tissue membrane, according to aspects of the present embodiments. The images were captured using a permeable microsphere membrane with the scaffold design 90 of Fig. 10. Cells migrated through the microsphere windows from the first compartment 84 into each of the hollow base 98 and the second compartment 88. No cell attachment was observed on the tops of the channels. Cells were observed attaching mainly on the bottom of the compartments or channels 84, 88. In addition, due to imaging effects the concentration of cells appearing to be disposed within the void 102 within the gap 86 (but which were actually located directly thereunder in the hollow base 98) was observed as being qualitatively lower (or less visible) than within the first and second compartments 84, 88. In some embodiments, as shown in each of the two bottom panels of Fig. 14, the 3D bioprinted liver tissue membrane may include a repeated geometric structure, for example, a repeated geometric structure that is substantially square shaped.

[00232] Fig. 15 illustrates a 3D bioprinted microsphere scaffold used as a liver microstructure in connection with the liver model (for example, a liver lobule model), according to aspects of the present embodiments. According to aspects of the present embodiments, the microsphere scaffold may include micro-spherical pores that are both uniform and size-controllable, may house PHH spheroids, and may include windows that connect adjacent pores. In some embodiments, the microsphere scaffold may include a hexagonal close-packed arrangement. In some embodiments, the microsphere scaffold may include chemistry that enables toe covalent binding of extracellular matrix (ECM) proteins.

[00233] Fig. 16 illustrates fluorescent imaging of 3D bioprinted liver tissue, according to aspects of the present embodiments. In some embodiments, as shown in Fig. 16, the cell morphology may result in high cell concentration clusters disposed on top of a 3D bioprinted liver tissue membrane a repeated geometric structure (i.e., a repeated hexagonal geometric structure).

[00234] Fig. 17 illustrates examples of viable cell types for seeding the microstructure, according to aspects of the present embodiments. In some embodiments, the microstructure may be compatible with a range of cell types, due to the versatile nature of bioprinted hydrogels, thereby facilitating a number of diverse applications. For example, in some embodiments, the microstructure may be compatible with a human breast cancer cell, a human lung fibroblast, and/or a human colorectal carcinoma cell. In some embodiments, perfusion capability of the microstructure may allow for extended study duration, enable analysis of cellular response(s), cell morphology, and/or assessment of drug safety and/or efficacy. In some embodiments, seeding the microstructure may enable assays to study a cell of interest.

[00235] Fig. 18 illustrates conventional scaffold production technologies, including electrospinning, emulsion templating, freeze drying and/or gas foaming. As shown in Fig. 18, conventional scaffold production technologies can lead to high variability in scaffold structure, introducing potentially large sources of variation in comparative studies that use conventional scaffold production technologies. By contrast, the 3D culture scaffolds of the present embodiments are formed via 3D bioprinting, thereby achieving precise porosity control and/or reproducibility, and enhancing the consistency of the scaffold fabrication process.

[00236] Fig. 19 illustrates a scaffold structure, according to aspects of the present embodiments. According to aspects of the present embodiments, the scaffold structure may be precisely controlled and/or reproducible through a bioprinting process. In some embodiments, the scaffold structure may be bioprinted to fit a cell culture and/or cell culture container. In some embodiments, the scaffold may be bioprinted to meet production scale capacity (e.g., to meet high volume demand).

[00237] Fig. 20 illustrates scaffold structures inserted in a 96 well plate, according to aspects of the present embodiments. The 96 well plate allows high-throughput culturing of the scaffolds, as described herein. For example, as shown in Fig. 20, according to aspects of the present embodiments, each well may contain cells cultures on different scaffold patterns and orientations to enable parallel, simultaneous cell culturing of various scaffold configurations.

[00238] In some embodiments, a scaffold, as described in the present disclosure may be precisely controlled and/or reproducible through a bioprinting process.

[00239] In some embodiments, a scaffold, as described in the present disclosure, may be produced at a high-volume demand/capacity for a cell culture and/or cell culture container. In some such embodiments, a high- volume demand/capacity may be at least 2,000 cell culture and/or cell culture containers per year. In some such embodiments, a high-volume demand/capacity may be at least 4,000 cell culture and/or cell culture containers per year. In some such embodiments, a high-volume demand/capacity may be at least 8,000 cell culture and/or cell culture containers per year. In some such embodiments, a high- volume demand/capacity may be at least 16,000 cell culture and/or cell culture containers per year. In some such embodiments, a high-volume demand/capacity may be at least 32,000 cell culture and/or cell culture containers per year. In some such embodiments, a high-volume demand/capacity may be at least 64,000 cell culture and/or cell culture containers per year.

[00240] In some embodiments, a scaffold, as described in the present disclosure may be free from animal derived materials (i.e., fully synthetic). The 3D printed scaffolds of the present embodiments may be biocompatible (that is, they may be compatible with w variety of cell types include human primary cells, cancer cells, etc.

[00241] In some embodiments, a scaffold, as described in the present disclosure may be compatible with one or more cell types. In some such embodiments, a cell type may include a cell as described in the present disclosure.

EXEMPLIFICATION

[00242] The following examples are intended to illustrate but not limit the disclosed embodiments. The following examples are useful to confirm aspects of the disclosure described above and to exemplify certain embodiments of the disclosure.

[00243] These non- limiting examples demonstrate particular features and advantages of provided technologies - e.g., of provided 3d culture scaffolds.

Example 1: Methods, Seeding Hepatocytes. [00244] According to aspects of the present embodiments, hepatocytes (e.g., Hc-04, HepG2, hepatoma, imHC) may be re-suspended at approximately 100,000 cells per mL (for example, in a range from about 75,000 cells per ml to about 150,000 cells per mL, or from about 50,000 cells per ml to about 200,000 cells per ml) before seeding into a 96 well plate containing cell chambers 120 (e.g., as shown in Figs. 8A-B and 20). Each cell chamber 120 contains a bioprinted scaffold with a specific internal lattice pattern (for example, the microspheres, i.e., spherical pores 68, and/or other patterns, for example as shown in Figs. 1, 2A-B, 7, 9A-C, and 15) which serves as an interstitial network (e.g., design) that can function as vasculature when hepatocytes are added. This vasculature can associate with hepatocytes to create a liver microstructure. Hepatocytes can be seeded in (for example, within the spherical pores 68 of) the scaffold depending on the experimental goal.

[00245] Plasmodium falciparum sporozoites may be obtained from the salivary glands of infected mosquitos. Extracted sporozoites may be filtered through a nylon screen and placed in Hanks Balanced Salt Solution (HBSS) containing 2% Fetal Bovine Serum (FBS). Sporozoites may then be centrifuged at 12 x 000 g before resuspending in HBSS with 2% serum at approximately lOOul for every 100,000 sporozoites collected. Approximately 50,000 sporozoites may be added to the microspheres (i.e., spherical pores 68) within each cell chamber 120. The plate may then be gently rocked for 2 minutes before placing in a growth chamber set at 37° Celsius and 5% CO2. After 1 hour, hepatocytes may be washed 3 times before replacing with hepatocyte culture media. Optionally, fresh media may be perfused through the interstitial space (i.e., microspheres) of each cell chamber 120 every 24 hours after seeding. This method allows the pathogenesis of Malaria causing sporozoites to be studied in a culture system that replicates the 3 -dimensional environment sporozoites establish an infection in.

Example 2: Cell chambers containing a scaffold pattern enable the identifying of neutralizing antibodies.

[00246] This example demonstrates the capacity for cell chambers 120 with scaffold patterns described herein to support hepatocyte cultures (e.g., liver microstructure, liver system) for the purpose of measuring the neutralizing capacity of antibodies.

[00247] According to aspects of the present embodiments, separate buffers each containing an individual antibody in varying concentrations (e.g. , titrations) may be administered to hepatocytes through an access point of each cell chamber 120 (for example, by pipetting the buffer compositions into each well manually from above). 15 minutes after antibody addition, 50,000 sporozoites may be added to the spherical pores 68 of each cell chamber 120. Experimental groups may include cell chambers 120 exposed to either an antibody of interest (e.g., serum/polyclonal, monoclonal) or a control (e.g., does not include an antibody, or includes only an antibody that does not target pathogen).

[00248] Referring still to Example 2, 24 hours post sporozoite infection, hepatocytes may be washed twice in cold PBS and re suspended in TRlzol for RNA extraction following manufacturer’s protocol. In some embodiments, Quantitative PCR (qPCR) may be performed using probes against P. falciparum circumsporozoite protein (CSP). qPCR on known concentrations of sporozoites may be used to correlate CSP transcripts with sporozoite number. A comparison of sporozoite number in each experimental condition may be used to identify antibodies capable of inhibiting infection. Optionally, serum may be collected from non-human primates at least 21 days after a vaccination dose. According to aspect of the present embodiments, serum found to inhibit sporozoite infection may indicate that a vaccine is efficacious.

[00249] To further study the neutralizing capacity of antibodies and the extent to which hepatocyte seeding has occurred in one or more cell chambers 120, the present methodology may be used in connection with the scaffold design (i.e., test rig or platform) shown in Fig. 10. For example, microscopy may be used to confirm retention of hepatocytes only in the first compartment 84 (Fig. 12). After this confirmation, fluorescent sporozoites may be added to a second compartment (e.g., compartment 88 shown in Fig. 12) that contains a buffer comprising an antibody of choice. This method allows the pre-incubation of sporozoites with an antibody within the cell chamber, but in a compartment separate from hepatocytes. 24 hours post sporozoite addition to the second compartment 88, cells within the first compartment may be collected and lysed so that qPCR can be utilized to quantify the approximate number of parasites that have successfully invaded the hepatocytes. Hepatocytes measured to have a reduction in parasite burden, as determined by qPCR and relative to a negative control (e.g., no antibody, or off-target antibody), are exposed to sporozoites that were incubated with neutralizing antibodies.

Example 3: Cell Chambers containing a scaffold pattern enable identification of compounds that disrupt pathogen growth. [00250] This example demonstrates the capacity for cell chambers with scaffold patterns described herein to support hepatocyte cultures (e.g., liver microstructure, liver system) for the purpose of identifying drugs capable of disrupting the life cycle of Malaria causing parasites (e.g., Plasmodium spp. Sporozoites).

[00251] According to aspects of the present embodiments, hepatocyte seeding may be carried out as described in Example 1. Drug(s) may be administered to the access point of at least 3 cell chambers 120 containing hepatocytes at least 24 hours after seeding. At least 15 minutes after drug addition, sporozoites may be added to each cell chamber 120 as described in example 1. Sporozoites may then be allowed to grow in hepatocytes for 5 days, with fresh media perfused through the cell-seeded scaffold every 24 hours. After 5 days, cells may be harvested, and qPCR carried out as described in Example 2. A negative control may include cell chambers 120 that did not receive a drug but were similarly infected with sporozoites. Cell chambers with a decrease in sporozoite burden relative to the negative control are determined to contain drugs with potential to inhibit sporozoite growth. Cell chambers may optionally be barcoded to make identification of cell chambers 120 and their associated drug easier to identify in larger scale settings.

Example 4: Cell chambers containing a scaffold pattern enable pathogen motility to be assessed in the presence and absence of therapeutics.

[00252] This example demonstrates the capacity for cell chambers 120 with scaffold patterns described herein to support hepatocyte cultures (e.g., liver microstructure, liver system) to study pathogen motility in a vascularized, 3 -dimensional environment.

[00253] According to aspects of the present disclosure, hepatocyte seeding may be carried out as described in Example 1. Each plate may then be placed into a growth chamber of a microscope capable of live cell imaging. 3uM Fluorescent beads may be used to image and localize the vasculature of each chamber’s liver microstructure. Fluorescent sporozoites may be incubated with an antibody on ice for about 45 minutes (for example, from about 30 minutes to about 60 minutes) before being added to the same compartment of each cell chamber 120 where hepatocyte seeding occurred. Images may then be continuously acquired through a Z-plane. Fluorescent parasites that have entered the vasculature may be identified through colocalization with the vasculature’s fluorescent labeling. Distance traveled, velocity, and the time it takes for sporozoites traveling in the vasculature to invade a hepatocyte may be recorded and then determined using ImageJ (and/or other suitable image processing programs (for example, Java-based image processing programs)) and/or a velocity/distance measurement tool plugin.

[00254] To study sporozoite motility further, hepatocyte seeding may be initiated in a first compartment (e.g., compartment 84), with microscopy confirming retention of hepatocytes only in this first compartment 84 (Fig. 12). After this confirmation, fluorescent sporozoites may be added to a buffer within the second compartment (e.g., compartment 88). After sporozoite addition, live cell microscopy may be utilized to observe sporozoite motility from the second compartment 88 to the hepatocyte containing first compartment 84. The buffer in the first compartment 84 may optionally contain compounds that enhance or inhibit motility, so that sporozoites are pre-exposed to such a compound before attempting motility and encountering hepatocytes.

[00255] The examples described above can be conducted using non hepatic cell types (e.g., endothelial, fibroblasts, immortalized cells, etc.) and non-Malaria causing pathogens (e.g., parasites, viruses, bacteria) with slight modifications to the protocol.

Example 5: Methods; seeding and differentiation of monocytes in cell chambers with a scaffold pattern.

[00256] According to aspects of the present embodiments, monocytes may be isolated from spleens of non-human primates or through leukapheresis of human donor blood. Monocyte enrichment may occur through continuous counter-flow centrifugation elutriation as described in literature. After isolation, monocytes may be centrifuged at 300 x g for 5 minutes at room temperature. To differentiate monocytes into macrophages, supernatant may be aspirated, and monocytes gently re-suspended in media suitable for monocyte differentiation, (for example, RPMI 1640 medium with 2mM L-glutamine, 50 ng/mL M-CSF, 25 ng/mL IL-10, and 10% complete FBS). Monocyte suspension may then be seeded into one or more compartments of a cell chamber containing a scaffold pattern as described herein and placed in a 37°C cell culture incubator with 5% CO2 and 95% air. 48 hours after seeding, the vasculature and/or interstitial space (i.e., a microstructure) of each cell chamber may be gently perfused with RPMI 1640 medium prewarmed to 37 °C. Following perfusion, fresh complete medium is perfused through the microstructure every 48 hours until monocyte differentiate and proliferate reach confluency. Example 6: Cell chambers with a scaffold pattern enable interrogation of immune cell chemotaxis.

[00257] This example demonstrates that cell chambers containing scaffold patterns described herein may be utilized to observe immune cell migration (e.g., chemotaxis).

[00258] Macrophages differentiated according to Example 5 are seeded into a first compartment (e.g., compartment 84) of the cell chamber scaffold design (e.g., FIG. 10, scaffold design 90). After seeding, fluorescently labeled antigen coated liposomes may be added to a second compartment (e.g., 88). Macrophages may be fluorescently identified through labeling of CD68 for human macrophages or CD64 for mouse derived macrophages. Time-lapse fluorescent microscopy may then be utilized to observe the migration of activated macrophages from a first compartment 84 to the liposome containing second compartment 88. Since the distance between compartments is known, kinetics of macrophage migration may be measured. Compounds believed to enhance or inhibit chemotaxis can optionally be added to the gap 86 that is between the first and second compartments 84, 88 or optionally an access point, or one or more compartment. Migration velocity and/or proportion of cells migrating in the presence of different stimuli (e.g., antigen, chemical stimuli) may then be measured and calculated, as described herein.

Example 7: Cell chambers with a scaffold pattern enable interrogation of immune cell phagocytosis in a 3-dimensional environment.

[00259] This example demonstrates that cell chambers containing scaffold patterns described herein can be utilized to measure immune cell phagocytic potential.

[00260] According to aspects of the present embodiments, monocytes may be seeded into cell chambers, as described in Example 5. Fluorescent liposomes, optionally comprised of antigens on their surface, may be added to the same compartment as monocytes. Live cell microscopy may then be utilized as described in Example 6 to observe, in real time, the phagocytic potential of macrophages. The proportion of macrophages in a field of view that are capable of phagocytosis may be observed and calculated. In some embodiments, at least 10 field of views may be utilized to calculate the proportion of macrophages with phagocytic potential. Macrophage activating or inhibition stimuli (e.g., interferon gamma, IL-4, IL- 10, other cytokines) may additionally be added to the macrophage containing compartment, an intervening compartment, or an access point, to activate macrophages. Measurements of the proportion of macrophages, in response to stimuli, capable of phagocytosis may then be calculated as previously described.

Example 8: 3D scaffold design enables functional hepatocyte cultures

[00261] This example demonstrates that 3D scaffold design described herein enables functional hepatocyte cultures.

[00262] Functional hepatocytes are important in testing for phenotypes, since the functionality of a cell is a requirement to test whether it is affected by a variable. For example, if cells function poorly and/or are dying, it may be challenging to test how variables affect the functioning of the cells. The functionality of hepatocytes may be evaluated by measuring the production of albumin, the production of urea, and/or the activity of CYP3A4 (an enzyme found in hepatocytes). The functionality of hepatocyte cultures grown by 3D scaffold design was compared to the functionality of hepatocytes grown by 2D and 2Dsw systems, for three different donors. 2D and 2Dsw, in this context, refers to systems with scaffolds with two-dimensional and two-dimensional sandwiched architectures, respectively.

[00263] Primary human hepatocytes (PHHs) were introduced to the 3D scaffold design, 2D, and 2Dsw systems, and preserved under the same culture media and conditions. Effluents were collected at different timepoints to monitor albumin and urea production rate. PHHs were subjected to relevant CYP450 substrates to evaluate the metabolic activity.

[00264] Fig. 21 shows an exemplary hepatocytes architecture 150 grown using the 3D scaffold design, according to aspects of the present embodiments. In some embodiments, architecture 150 includes hepatocyte plates 152, portal triads 154, central veins 156, and liver scaffold 158. In some embodiments, hepatocytes architecture 150 is a schematic representation of the in vivo environment in which primary human hepatocytes are separated by vascular channels, surrounded by ECM for support, and organized in the form of a plate (for example, with arrays of hepatocytes arranged such that they extend radially outward from the central vein (which runs “into the page” in the illustration of Fig. 21) to the liver scaffold, which may include a hexagonal configuration) The portal triads may be disposed within the liver scaffold at the interface between a first hepatocyte plate and an adjacent second hepatocyte plate. Fig. 3, an exemplary image corresponding to the architecture 150, shows the primary human hepatocytes interacting with: (i) the surface of the coated regions of the interstitial space within a single pod, (ii) each other within a single pod (called microsphere), and (iii) other hepatocytes in neighboring pods via interconnections (called windows).

[00265] Figs. 22A-C demonstrate the measurements performed on hepatocyte culture from donor

1, according to aspects of the present embodiments. Fig. 22A shows graphs of albumin production rate (ug/day/l e6 PHH) on day 3, day 5, day 7 and day 9, by hepatocytes which were grown by 3D scaffold design 162, 2D system 164, and 2Dsw system 166. The dotted line 160 indicates the human level of albumin production. Fig. 22B shows graphs of urea production rate (mg/day/le6 PHH) by hepatocytes which were grown by 3D scaffold design, 2D system, 2Dsw system. Fig. 22C shows graphs of CYP3A4 activity (D-Luc nM/le6 PHH) by hepatocytes which were grown by 3D scaffold design, 2D system, 2Dsw system. The 3D architecture results in higher albumin production and CYP4A4 activity than the 2D and 2Dsw systems. The 3D and 2Dsw systems result in higher urea production than the 2D system.

[00266] Figs. 23A-C demonstrate the measurements performed on hepatocyte culture from donor

2, according to aspects of the present embodiments. Fig. 23A shows graphs of albumin production rate ( g/day/l e6 PHH) on day 3, day 5, day 7 and day 9, by hepatocytes which were grown using 3D scaffold design 162, 2D system 164, 2Dsw system 166. The dotted line 160 indicates the human level of albumin production. Fig. 23B shows graphs of urea production rate (mg/day/le6 PHH) by hepatocytes which were grown using 3D scaffold design, 2D system, 2Dsw system. Fig. 23C shows graphs of CYP3A4 activity (D-Luc nM/le6 PHH) by hepatocytes which were grown using 3D scaffold design, 2D system, 2Dsw system. The 3D architecture results in higher albumin production, urea production, and CYP4A4 activity than the 2D and 2Dsw systems. Statistical significance between data series is indicated with the bars and star annotations on the respective data plots.

[00267] Figs. 24A-C demonstrate the measurements performed on hepatocyte culture from donor

3, according to aspects of the present embodiments. Fig. 24A shows graphs of albumin production rate (pg/day/le6 PHH) on day 3, day 5, day 7 and day 9, by hepatocytes which were grown using 3D scaffold design 162, 2D system 164, 2Dsw system 166. The dotted line 160 indicates the human level of albumin production. Fig. 24B shows graphs of urea production rate (mg/day/le6 PHH) by hepatocytes which were grown using 3D scaffold design, 2D system, 2Dsw system. Fig. 24C shows graphs of CYP3A4 activity (D-Luc nM/le6 PHH) by hepatocytes which were grown using 3D scaffold design, 2D system, 2Dsw system. The 3D architecture results in higher albumin production, urea production, and CYP4A4 activity than the 2D and 2Dsw systems. Statistical significance between data series is indicated with the bars and star annotations on the respective data plots.

[00268] Referring still to Figs. 22-24, the hepatocyte culture grown by 3D scaffold design generally exhibits greater albumin production, urea production, and activity of CYP3A4, compared to the same culture grown using 2D and 2Dsw systems, across three donors, demonstrating superior functionality. These results may indirectly indicate superior viability of the hepatocyte culture grown by 3D scaffold design, as more viable hepatocytes may also lead to greater albumin production, urea production, and/or CYP3A4 activity. Since the cell seeding, culture and assay are consistent across different culture systems, the higher performance of 3D scaffold design is driven by the architecture and niche factors. The 3D scaffold design offers a more physiologically relevant, three-dimensional architecture which enables cell-surface as well as cell-cell interactions. Recapitulating these features allows the cells to experience a more in vivo-like environment, compared to that of 2D or 2Dsw systems, thereby exhibiting better function.

Example 9; 3D scaffold design enables accurate prediction of liver toxicity

[00269] This example demonstrates the dynamic range of 3D scaffold design in measuring liver toxicity.

[00270] Liver injury by drugs that are (and are not) FDA approved is a pervasive and serious problem, as liver toxicity is not always discovered in the drug discovery and/or approval process and is often discovered after approval once patients report liver issues. In this example, the ability of 3D scaffold design to test the liver toxicity of drugs was evaluated. The hepatocytes grown within the 3D scaffold design were exposed to various drugs that are known to be toxic to hepatocytes, and the viability and functionality of the hepatocytes were measured.

[00271] Figs. 25A-I demonstrate hepatocyte viability as measured by total ATP content, according to aspects of the present embodiments. In some embodiments, a pair of drugs is present, where one drug (indicated with circles) is thought to be a toxicant for the liver and one drug (indicated with squares) is thought to be a safer counterpart. Fig. 25A is a graph of Viability (%) as a function of Multiplier of human Cmax (log 10), for Fialuridine vs Entecavir. Fig. 25B is a graph of Viability (%) as a function of Multiplier of human Cmax (log 10), for Troglitazone vs Pioglitazone. Fig. 25C is a graph of Viability (%) as a function of Multiplier of human C_max (log 10), for Clozapine vs Olanzapine. Fig. 25D is a graph of Viability (%) as a function of APAP (loglO), for Acetaminophen vs Metacetamol. Fig. 25E is a graph of Viability (%) as a function of Multiplier of human Cmax (log 10), for Tolcapone vs Entacapone. Fig. 25F is a graph of Viability (%) as a function of Multiplier of human Cmax (log 10), for Nefazodone vs Buspirone. Fig. 25 G is a graph of Viability (%) as a function of Multiplier of human Cmax (loglO), for Trovafloxacin vs Levofloxacin. Fig. 25H is a graph of Viability (%) as a function of Multiplier of human Cmax (loglO), for Diclofenac. Fig. 251 is a graph of Viability (%) as a function of Multiplier of human Cmax (loglO), for Amiodarone.

[00272] Figs. 26A-1 demonstrate hepatocyte health as a function of albumin production, according to aspects of the present embodiments. In some embodiments, a pair of drugs is present, where one drug (indicated with circles) is thought to be a toxicant for the liver and one drug is thought to be a safer counterpart (indicated with squares). Fig. 26A is a graph of albumin normalized response (% of control) as a function of Multiplier of human C_ma.x (loglO), for Fialuridine vs Entecavir. Fig. 26B is a graph of albumin normalized response (% of control) as a function of Multiplier of human C_max (loglO), for Troglitazone vs Pioglitazone. FIG. 26C is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Clozapine vs Olanzapine. Fig. 26D is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Acetaminophen vs Metacetamol. Fig. 26E is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Tolcapone vs Entacapone. Fig. 26F is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Nefazodone vs Buspirone. Fig. 26G is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Trovafloxacin vs Levofloxacin. Fig. 26H is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Diclofenac. Fig. 261 is a graph of albumin normalized response (% of control) as a function of Multiplier of human Cmax (loglO), for Amiodarone.

[00273] The experimental results illustrated in Figs. 25-26 demonstrate the ability of 3D scaffold design to effectively test the liver toxicity of drugs. The results shown from the safer counterparts of more toxic drugs confirm the dynamic range of 3D scaffold design in measuring liver toxicity. As a result, 3D scaffold design may detect hepatocyte toxicity of safer counterparts, and thus enable testing for safer drugs. For example, a compound may not to be highly toxic for detection of the detrimental effects on hepatocytes. 3D scaffold design demonstrates the ability to measure both minor and significant toxic effects by compounds on hepatocytes. [00274] Drug metabolism primarily relies on certain enzymes, such as, the CYP group of enzymes that participate in reactions, including oxidation, reduction, and hydrolysis. As discussed in Example 8, 3D scaffold design outperforms the controls in exhibiting CYP levels, and therefore allow higher sensitivity, as seen by the ATP and albumin content.

Example 10: 3D scaffold design captures immune-mediated hepatotoxicity

[00275] This example demonstrates 3D scaffold design achieves the ability to study immune- mediated hepatotoxicity (i.e., toxicity of hepatocytes).

[00276] Immune-mediated liver toxicity is associated with activated immune cells. Drug induced liver injury (DILI), may be exacerbated in the presence of activated immune cells, which are anticipated when using immunotherapy. To evaluate the ability of 3D scaffold design to measure immune-mediated hepatotoxicity, Lipopolysaccharide (LPS) and Trovafloxacin were used. LPS is a bacterial endotoxin, which activates immune cells. Trovafloxacin is a DILI drug (i.e., known to cause liver injury). Trovafloxacin and LPS are known to synergistically play a role in immune- mediated hepatotoxicity. Co-culture of primary human hepatocytes (PHHs) and Kupffer cells (KCs, liver immune cells) were used to examine chemical-induced inflammatory reactions resulting in hepatocellular toxicity.

[00277] Fig. 27A shows graphs of Viability (%) as a function of Trovafloxacin concentration, for PHH 170 and co-culture 172 of PHH and KC in the presence of -LPS 174 (i.e., squares), +LPS 176 (i.e., circles), according to aspects of the present embodiments. The co-culture 172 in the presence of LPS 174, 176 which activates KC, leads to death of cells at a lower concentration of Trovafloxacin. These results demonstrate the ability of 3D scaffold design to measure classical immune-mediated toxicity of hepatocytes, and particularly the synergistic effect of Trovafloxacin and activated immune cells.

[00278] Fig. 27B shows graphs of TNFa production (normalized by cells) as a function of Trovafloxacin concentration, for PHH 170 and co-culture 172 of PHH and KC in the presence of -LPS 174, +LPS 176, according to aspects of the present embodiments. TNFa is a cytokine that can be released by both PHH and KC. FIG. 27C shows graphs of IL-6 production (normalized by cells) as a function of Trovafloxacin concentration, for PHH 170 and co-culture 172 of PHH and KC in the presence of -LPS 174, +LPS 176, according to aspects of the present embodiments. IL- 6 is a cytokine that can be released by both PHH and KC. These results demonstrate the ability of 3D scaffold design to study the shifts in TNFa and IL-6 profiles, cytokines known to play a role (along with DILIs) in liver toxicity. These studies are made possible due to 3D scaffold design ability to support the culture of functional Kupffer cells.

Example 11: 3D scaffold design enables consistent stratification of drugs

[00279] This example demonstrates that measurements performed by using 3D scaffold design lead to consistent stratification of drugs.

[00280] When testing for compounds that cause DILI, culture systems that may faithfully predict the compounds that are likely to cause liver injury are highly desirable. DILIrank is the largest reference drug list ranked by the risk for developing drug-induced liver injury in humans. A compound with a higher ranking is more likely to cause liver injury. Garside rank classifies the severity of DILI, i.e., a compound with a lower number has higher likelihood of causing liver injury. A reliable culture system to measure DILI may result in values that correlate with DILIrank and Garside Rank values.

[00281] Fig. 28 shows the DILIrank, Garside Rank, and margin of safety (ICso/Cmax) predicted by 2D PHH, Spheroids, Emulate, and 3D scaffold design, for various drugs, according to aspects of the present embodiments. DILIrank values range from 1 to 8, corresponding to Steatosis, Cholestasis, ALT/AST, HB, Jaundice, Necrosis, ALF, Fatal, respectively, as shown in Fig. 28. Garside rank values range from 5 to 1, corresponding to No, ALT/AST, Low, High, and Severe, respectively, as shown in Fig. 28. Margin of safety is defined as the ratio of IC50 to Cmax, where IC50 is the concentration of a drug that is required to kill 50% of cells. A low IC50 value indicates a low concentration of drug is required to kill hepatocytes, and therefore the drug may be extremely dangerous to hepatocytes. For example, ICso/Cmax values in a range from 0 to about 10 may qualify as severe. ICso/Cmax values in a range from about 10 to about 50 may qualify as high. ICso/Cmax values of about 50 and above may qualify as low risk. A reliable assay to measure DILI results in ICso/Cmax values that correlate with DILI and Garside rankings, i.e., a drug with a higher DILI rank, has a lower ICso/Cmax value relative to a drug with a lower DILI rank. Measurements performed by 3D scaffold design result in a clear and consistent stratification of drugs (based on IC50 value) that are relatively more dangerous. Stated otherwise, the ICso/Cmax values measured using the 3D scaffold design correlate closely with the corresponding DILIrank and Garside Rank drug rankings. [00282] Fig. 29 is a graph of margin of safety (ICso/Cmax) as a function of DILI severity category, for 2D 180, Spheroids 182, Emulate 184, and 3D scaffold design 186, according to aspects of the present embodiments. The graph of Fig. 29 includes four subgraphs: false negative 188, true negative 190, true positive 192, and false positive 194. 3D scaffold design shows a low ICso/Cmax ratio (i.e., true positive 192) for a compound with any DILI concern, except for one value at the low DILI concern category, and show a high ICso/Cmax ratio (i.e., true negative 188) for a compound in ASL/ALT and No Dili categories. This example demonstrates the ability of 3D scaffold design to accurately predict what compounds are likely to have low DILI values, and thus are considered better candidates for therapeutics.

[00283] Table 1 shows the data used in Fig. 29, including the number of true positive, true negative, false negative, and false positive, as well as sensitivity and specificity of 2D, Spheroids, Emulate, and 3D scaffold design. This example reveals the ability of 3D scaffold design to achieve greater sensitivity and specificity compared to other systems.

Table 1

Example 12: 3D scaffold design enables T cell migration

[00284] This example demonstrates that T cell migration and cytotoxicity measurements in response to a therapeutic can be achieved using 3D scaffold design.

[00285] Response to Pembrolizumab, an antibody that induces transendothelial migration (TEM) and tumor killing, by colorectal cancer cells (CRCs) was studied in presence of CXCL 12, a chemoattractant. Fig. 30A shows graphs of T cell TEM (%) and CRC killing (%) as a function of IgG4 and Pembrolizumab ( g/mL), for control 196 and CXCL12 198, according to aspects of the present embodiments. IgG4 is an isotype control (i.e., non-specific antibody that does not induce TEM and tumor killing). TEM (i.e., crossed the endothelial barrier lining the porous membrane) was enhanced in the presence of CXCL2 198 and with increasing concentrations of Pembrolizumab. CRC killing was enhanced in the presence of CXCL2 198 and with increasing concentrations of Pembrolizumab. CRC killing is only accessible if the T cells migrate across the porous membrane, which occurs in the presence of a chemoattractant.

[00286] Fig. 30B shows graphs of IFNg (ng/mL) and Granzyme B (ng/mL) as a function of IgG4 and Pembrolizumab (pg/mL), for control 196 and CXCL12 198, according to aspects of the present embodiments. IFNg release (a cytokine) and Granzyme B release (a cytotoxic activity) are two different measurements of T cell activity. These results demonstrate that Pembrolizumab induces T cell activation regardless of a chemoattractant presence, and this activation further results in tumor killing only if T cells are induced to migrate to the tumor microenvironment comprised of CRC. Accordingly, 3D scaffold design enables testing of a known cancer immunotherapeutic to determine the effects of the therapeutic (i.e., Pembrolizumbab) on T-cell migration and cytotoxicity, and may provide a platform to test other potential therapeutics.

Example 13: 3D scaffold design enables cancer immunotherapeutic method

[00287] This example demonstrates a cancer immunotherapeutic method, mixing T cells with anti-PD-l-mAB, that was achieved using 3D scaffold design.

[00288] Mixing T cells with a mAB (monoclonal antibodies) that bind programmed death- 1 receptor (PD-1), and which is expressed by activated T cells, allows the T cells to stay active (e.g., retain tumor targeting/killing capacity) in a tumor microenvironment. Tumor cells express PDL-1 (programmed death ligand 1) that can bind to PD-1 on activated T cells, promoting the death of T cells through PD-1/PDL1- interaction, and diminishing the ability for T cells to combat cancer. By binding T-cell’s PD-1 with an antibody, the tumor cannot bind PD-1 with its PDL-1, and thus the T-cell will remain active, and exert cytotoxic effect on tumor cells (e.g., tumor killing).

[00289] Fig. 31 illustrates an example of a cancer immunotherapeutic method 200 using 3D scaffold design 90, according to aspects of the present embodiments. In some embodiments, the method 200 includes dosing 202 inside first compartment 84 of 3D scaffold design 90 and sampling 204 inside second compartment 88 of 3D scaffold design 90 (the first and second compartments 84, 88 being separated via space 86). In some embodiments, preparation of 3D scaffold design 90 includes applying a coating, linker, and ECM proteins to the porous membrane 106. Endothelial cells 208 are subsequently seeded on the coated porous membrane 106 (e.g., top surface) and allowed to grow to confluency, creating a selectively permeable biological barrier. In some embodiments, sampling 204 includes seeding tumor cells 210 through the second compartment 88, which results in recreating a tumor microenvironment on the other side of the coated porous membrane 106 (e.g., bottom surface). In this example, tumors cells are colorectal cancer cells (CRCs) 206. In this setup, T cells, when active, are able to cross between endothelial cells 208 that make up the barrier and infiltrate the tumor microenvironment 206. In some embodiments, the dosing 202 includes mixing T cells 210 with anti-PD-l-mAB 212. In this example, T cells 210 are PBMC derived T cells (i.e., a collection of various immune cells).

[00290] Fig. 40 is a flow chart diagram of a method 270 of performing an assay, according to aspects of the present embodiments. At step 272, the method 270 may include applying a coating to a porous membrane disposed in a first compartment of 3D scaffold design. At step 274, the method 270 may include applying a linker to the porous membrane. At step 276, the method 270 may include applying ECM proteins to the porous membrane. At step 278, the method 270 may include seeding endothelial cells on the coated porous membrane. At step 280, the method 270 may include seeding tumor cells in a second compartment of the 3D scaffold design. In some embodiments, the tumors cells are colorectal cancer cells. At step 282, the method 270 may include allowing a tumor microenvironment to form in the first compartment. At step 284, the method 270 may include mixing T cells with anti-PD-l-mAB in the first compartment. In some embodiments, T cells are PBMC derived T cells. In some embodiments, anti-PD-l-mAB is anti-CD3/CD28/CD2 antibodies. In some embodiments, anti-PD-l-mAB is IL-2 antibodies. At step 286, the method 270 may include allowing T cells to cross between endothelial cells and infiltrate the tumor microenvironment.

[00291] Fig. 32A illustrates an example of mixing 220 of T cells 206 with antibodies, according to aspects of the present embodiments. In this example, antibodies include anti-CD3/CD28/CD2 214 and IL-2 216. T cells 206 are exposed to anti-CD3/CD28/CD2 antibodies 214 for 3 days and IL-2 antibodies 216 for 7 days to activate and proliferate T cells within this PBMC population.

[00292] To validate the immunotherapeutic method 200 to activate/proliferate T cells, flow cytometry was used to assess the population of cells that express CD8 (a T cell marker) and PD-1 (marker for activated-cells). Fig. 32B is a graph of a flow cytometry experiment including CD8 and PD-1 markers, according to aspects of the present embodiments. The quadrant 222 shows the population of PD-1 positive CD8 positive T cells, validating the method 200 to create these T cells. [00293] Fig. 32C shows graphs of expression of PD-L-f on tumor cells, for PD-L-1 226, isotype control 226 and unstained cells 228, according to aspects of the present embodiments. In this example, tumor cells are HCT 116, a colorectal carcinoma cell line commonly used to study colon cancer. The PD-L-1 226 peak shows (using an anti-PDLl antibody) the amount of PDL-1 expressed by the tumor cells. The isotype control 226 and unstained cells 228 are negative controls.

Example 14: 3D scaffold design exhibits enhanced TEM of T cells in response to anti-PD-1

[00294] This example demonstrates that T cells in 3D scaffold design achieve enhanced TEM relative to a different system, Transwell®.

[00295] Secretion of IFNg, a measurement of T cell activity, in the presence of Nivolumab, an anti-PDl antibody, was compared for 3D scaffold design and Transwell®. Fig. 33 A shows graphs of INFg secretions (normalized against IgG4) for IgG4, Nivolumab values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments. Fig. 33B shows: (1) graphs of T-cell migration (%) for IgG4, and (2) Nivolumab values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments. Fig. 33C shows: (1) graphs of INFg secretions (normalized against IgG4) for IgG4, and (2) Nivolumab values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for Transwell®. Fig. 33D shows: (1) graphs of T-cell migration (%) for IgG4, and (2) Nivolumab values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for Transwell®.

[00296] In the presence of Nivolumab, T cells in both the 3D scaffold design and Transwell® become active and secrete IFNg (Figs.33A and 33C). T cell migration, however, only occurs (to a statistically significant level) in the 3D scaffold design (Fisg. 33B and 33D). These results further demonstrate that the reversal of T cell dysfunction in response to stimuli (e.g., therapeutics) can be measured using 3D scaffold design.

Example 15: 3D scaffold design enables T cell-mediated tumor killing in response to anti-

PD-1

[00297] This example demonstrates that 3D scaffold design achieves greater sensitivity in measuring tumor killing activity, relative to a different system, Transwell®. [00298] Cleaved caspase 3, a marker of apoptosis (i.e., programmed cell death), in the presence of two different therapeutics, Nivolumab, an anti-PDl antibody, and Pembrolizumbad, which activates migration and tumor killing, was measured for 3D scaffold design and Transwell®.

[00299] Fig. 34A shows: (1) graphs of cleaved Caspase 3 (% positive cells) for IgG4, and (2) Nivolumab values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments. Fig. 34B shows: (1) graphs of cleaved Caspase 3 (% positive cells) for IgG4, and (2) Pembrolizumad values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments. Fig. 34C shows: (1) graphs of cleaved Caspase 3 (% positive cells) for IgG4, and (2) Nivolumab values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for Transwell®. Fig. 34D shows: (1) graphs of cleaved Caspase 3 (% positive cells) for IgG4, and (2) Pembrolizumad values of 1.0 pg/mL, 10 pg/mL and 20 pg/mL, for Transwell®. [00300] Only T cells grown in the 3D scaffold design achieved a dose-dependent increase in percent of cells positive for cleaved caspace 3 (Fig. 34 A), while the percent positive cells associated with cleaved caspace 3 in the Transwell® are quite high without a shift from a lower percentage to a higher percentage (Fig. 34C). This lack of shift indicates there is no inactive states vs active state of T cells, confirming Transwell® model did not measure such activation in response to a drug. This example confirms the ability of 3D scaffold design including the features described herein following the methods described herein to measure active and inactive states of T cells, in response to stimuli.

[00301] Figs. 35A-C show exemplary fluorescent microscopy images in the presence of IgG4 1 ug/mL, Pembrolizumab 0.5 pg/mL, and Pembrolizumab 1 pg/mL, respectively, for 3D scaffold design, according to aspects of the present embodiments. Fluorescent markers for CD3 230 (a marker of T cells), cleaved-caspace 3 232 (a marker for apoptosis that indicates T cell cytotoxicity), and spheroids 234 (a 3d structure of cell aggregates that exist in tumor microenvironments) are shown in Figs. 35A-C. The white circles 236 indicate the locations where T cells have infiltrated the tumor microenvironment. A quantification of the white circles 236, i.e., how many T cells have infiltrated the tumor microenvironment across the three regions of interest (ROI) (as shown in Fig. 35D). Each square image (Fig. 33A, Fig. 33B, Fig. 33C) represents an ROI. Fig. 35D shows graphs of T-cell infiltration (count per 3 ROI) for IgG4, Pembrolizumad values of 0.5 pg/mL and 1 pg/mL, according to aspects of the present embodiments. A dose dependent increase in T cell tumor microenvironment infiltration is observed in the presence of T cell activator antibody Pembrolizumab. This infiltration quantification indicates that migration is required for T cells to infiltrate the tumor microenvironment, further demonstrating 3D scaffold design can achieve an environment that allows T cells to migrate while observing such migration and associated cytotoxicity, in response to a stimulus, and in the presence of a tumor microenvironment.

[00302] Fig. 35E shows: (1) graphs of mean fluorescence intensity (MFI) of cleaved-caspase 3 for IgG4, and (2) Pembrolizumad values of 0.5 pg/mL and 1 pg/mL, according to aspects of the present embodiments. FIG. 35E is the quantification of T cell cytotoxicity from images shown in Figs. 35A-C. High MFI values indicate brighter signals by cleaved-caspace 3 marker 232, corresponding to greater T-cell cytotoxicity against tumors. A dose dependent increase in MFI of cleaved-caspase 3 is observed in the presences of Pembrolizumab. These results demonstrate that 3D scaffold design recapitulates the physiological environment faithfully and achieves the ability to measure T cell migration and cytotoxicity in response to stimuli (e.g., antibodies), and in the presence of a tumor microenvironment.

Example 16: 3D scaffold design enables cancer immunotherapeutic effects of Cemiplimab

[00303] This example demonstrates immunotherapeutic effect of Cemiplimab to induce TEM and cytotoxicity can be achieve by 3D scaffold design.

[00304] Release of IFNy, a cytokine indicating T cell activation, Grz B, a protese indicating T cell cytotoxicity, and T cell migration were measured, in the presence of Cemiplimab, in 3D scaffold design.

[00305] Fig. 36A shows: (1) graphs of INFy (ng/rnL) for lgG4, and (2) Cemiplimab values of 1 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments. Fig. 36B shows (1) graphs of T-cell migration (%) for IgG4, and (2) Cemiplimab values of 1 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments. Fig. 36C shows: (1) graphs of Grz B (ng/mL) for IgG4, and (2) Cemiplimab values of 1 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments. Fig. 36D shows: (1) graphs of % Cytotoxicity for IgG4, and (2) Cemiplimab values of 1 pg/mL, 10 pg/mL and 20 pg/mL, for 3D scaffold design, according to aspects of the present embodiments. These results indicate that increasing T cell activation by Cemiplimab increases the cytotoxic capacity of T cells, but this observation is not dose dependent, as observed in other phenotypes. Example 17: 3D scaffold design enables testing of anti-tumor efficacy and endothelial toxicity of T-Dxd in HER2 overexpressing breast cancer cells

[00306] This example demonstrates the ability of 3D scaffold design to test the anti-tumor efficacy of an antibody drug conjugate (ADC), and the unintended off-target harmful effects of the ADC on cells that should not be targeted.

[00307] An ADC carries a toxic compound, that is inert while conjugated to the antibody. Once an ADC binds and enters a specific cell (e.g., a cancer cell), the changes in the environment cause the toxic compound to be cleaved from the antibody and exert its toxic effect on the cancer cell only. This type of ADC prevents widespread toxic effects of a compound by using an antibody for highly targeted intracellular delivery to a specific cancer cell. Evaluating the efficacy of ADCs against the target, and the off-target toxicity of the compound are of great interest.

[00308] Fig. 37 illustrates an example of a method 240 of simultaneous assessment of safety and efficacy using 3D scaffold design 90, according to aspects of the present embodiments. In some embodiments, the method 240 includes dosing 202 inside first compartment 84 of 3D scaffold design 90 and sampling 204 inside second compartment 88 of 3D scaffold design 90. In some embodiments, porous membrane 106 is prepared to create a selectively permeable biological barrier including endothelial cells 208, as described in Example 13. In some embodiments, sampling 204 includes seeding SK-BR-3 BC cells 242 through the second compartment 88, which results in recreating a tumor microenvironment on the other side of the coated porous membrane 106 (e.g., bottom surface). In some embodiments, the dosing 202 includes adding an ADC to the first compartment 84, where they must traverse the endothelial lining 208 on the porous membrane 106. In this example the ADC is T-Dxd 244 which targets HER2 overexpressing breast cancer cells. Subsequently, endothelial cells, tumor cells and supernatant are collected, and measurements are taken to measure the harmful effects of the ADC on the endothelial cells (i.e., unintended effects), and on the tumor cells (i.e., intended consequence).

[00309] Different cell types that serve as models in breast cancer studies were compared by using two different methods. Fig. 38A shows graphs of HER2 expression (% of positive cells) for SK- BR-3, HCT-116, A549, and CRC-PDOs, according to aspects of the present embodiments. Fig. 38B shows plots of HER2 expression for A549 250, HCT 116252, CRC-PDOs 254, SK-BR-s 256, isotype 258, and unstained 260, according to aspects of the present embodiments. HER2 expression was measured by using an antibody against HER2, followed by Alexa647, a fluorescent marker against the antibody. The perforated line 248 establishes the lowest HER2 expression for a cancerous cell. The cell types below line 248 (i.e., to the left of line 248) are the negative isotype control 258 (non-specific antibody), and unstained 260 (represents background or signal noise). These results demonstrate the appropriateness of the chosen cell line to study the effects of therapeutics that target breast cancer.

[00310] Fig. 39A shows graphs of cleaved caspase 3 (% positive cells) for no Tx (i.e., no therapeutic stimulus), T-Dxd 0.1 pg/mL, T-Dxd 1 pg/mL, T-Dxd 10 ug/mL, Trastuzumab 10 g/mL and Dxd 0.5 pM, according to aspects of the present embodiments. The cleaved caspase 3 measurements indicate the percent of cells that are undergoing apoptosis induced by the ADC.

[00311] Fig. 39B shows graphs of tumor-cell viability (%) for no Tx (i.e., no therapeutic stimulus), T-Dxd 0.1 pg/mL, T-Dxd 1 pg/mL, T-Dxd 10 pg/mL, Trastuzumab 10 pg/mL and Dxd 0.5 pM, for 3D scaffold design, according to aspects of the present embodiments. Tumor-cell viability indicates the percent of tumor cells that are alive (viable) when each anti-cancer drug is added.

[00312] The unintended, off target effects of each drug on endothelial cells were evaluated, by measuring the viability of HUVEC (human umbilical vein endothelial cells). Fig. 39C shows graphs of HUVECs viability (%) for no Tx (i.e., no therapeutic stimulus), T-Dxd 0.1 pg/mL, T- Dxd 1 pg/mL, T-Dxd 10 pg/mL, Trastuzumab 10 pg/mL and Dxd 0.5 pM, for 3D scaffold design, according to aspects of the present embodiments. These data demonstrate that 3D scaffold design achieves simultaneous measurement of anti-tumor efficacy of various anti-cancer drugs (i.e., on target effects) and the safety of each drug as measured by its off-target unintended effects on endothelial cells.

Characterization of a Scaffold

[00313] Those skilled in the art, reading the present specification, will appreciate that it may be desirable to characterize one or more features of bioprinted entities independent of a cell and/or associated with a cell, and/or of components or combinations thereof, for example when designing (e.g., selecting appropriate components of) or producing a provided system and/or when monitoring or assessing a preparation thereof. Alternatively or additionally, in some embodiments, it may be desirable to assess one or more features of a provided system as administered, for example in order to monitor a subject and/or treatment thereof. [00314] In some embodiments, cellular distribution is a characteristic property of a system and/or bioprinted entity associated with a cell and/or seeded therein. In some embodiments, the present disclosure provides systems and/or bioprinted entities that show improved cellular distribution relative to a comparable system and/or bioprinted entity.

[00315] Those skilled in the art, reading the present specification, will appreciate that it may be desirable to characterize one or more features of bioprinted entities, and/or of components or combinations thereof, for example when designing (e.g., selecting appropriate components of) or producing a provided system and/or when monitoring or assessing a preparation thereof. Alternatively or additionally, in some embodiments, it may be desirable to assess one or more features of a provided system as administered, for example in order to monitor a subject or treatment thereof.

Claims

CLAIMS WE CLAIM:

1. A method of performing an assay comprising: providing a platform comprising: a first compartment; a second compartment disposed a distance away from the first compartment; and at least one conduit, passageway, or chamber connecting the first compartment to the second compartment; disposing a scaffold comprising a repeated 3D geometry in the first compartment; seeding the first compartment with a first type of live cells; seeding the second compartment with a second type of live cells; placing the first type of cells in fluid and/or operational communication with the second type of cells; exposing at least one of the first type of cells and the second type of cells to at least one stimulus; and assessing at least one property of at least one of the first type of cells and/or the second type of cells.

2. The method of claim 1 , further comprising coating the scaffold prior to seeding the first compartment.

3. The method of claim 1, wherein seeding the first compartment comprises seeding the scaffold with live cells.

4. The method of claim 1 , further comprising initiating conditions to simulate an in vivo environment following placing the first type of cells in fluid and/or operational communication with the second type of cells.

5. The method of claim 1, wherein seeding the first compartment with a first type of live cells comprises seeding the first compartment with hepatocytes, and wherein assessing at least one property of at least one of the first type of cells and/or the second type of cells comprises assessing production of albumin and/or urea of the hepatocytes.

6. The method of claim 1 , wherein seeding the first compartment with a first type of live cells comprises seeding the first compartment with hepatocytes, and wherein assessing at least one property of at least one of the first type of cells and/or the second type of cells comprises assessing CYP3A4 activity of the hepatocytes.

7. The method of claim 1, wherein seeding the first compartment with a first type of live cells comprises seeding the first compartment with hepatocytes, and wherein assessing at least one property of at least one of the first type of cells and/or the second type of cells comprises assessing ATP production of the hepatocytes.

8. The method of claim 1 , wherein seeding the first compartment with a first type of live cells comprises co-seeding the first compartment with hepatocytes and Kupffer cells, wherein the method further comprises co-culturing the hepatocytes and Kupffer cells in the first compartment, wherein assessing at least one property of at least one of the first type of cells and/or the second type of cells comprises assessing a level of lipopolysaccharide (LPS) activation in the hepatocytes and/or Kupffer cells.

9. The method of claim 1, wherein seeding the first compartment with a first type of live cells comprises co-seeding the first compartment with hepatocytes and Kupffer cells, wherein the method further comprises co-culturing the hepatocytes and Kupffer cells in first compartment, wherein assessing at least one property of at least one of the first type of cells and/or the second type of cells comprises assessing a level of TNFalpha and/or IL-6.

10. The method of claim 1, wherein seeding the first compartment with a first type of live cells comprises co-seeding the first compartment with hepatocytes and Kupffer cells, wherein the method further comprises co-culturing the hepatocytes and Kupffer cells in first compartment, wherein assessing at least one property of at least one of the first type of cells and/or the second type of cells comprises determining a drug induced liver injury ranking.

11. The method of claim 1 , further comprising determining a platform specificity and/or sensitivity based on the at least one property of at least one of the first type of cells and/or the second type of cells.

12. The method of claim 1, wherein seeding the first compartment with a first type of live cells comprises suspending hepatocytes in fluid at a density in a range from about 50,000 cells per ml to about 200,000 cells per ml, and seeding the scaffold with the fluid.

13. The method of claim 12, wherein the hepatocytes comprise at least one of Hc-04, HepG2, hepatoma, and imHC.

14. The method of claim 1, wherein the at least one stimulus comprises at least one of: Fialuridine, Entecavir, Troglitazone, Pioglitazone, Clozapine, Olanzapine, Acetaminophen, Metacetamol, Tolcapone, Entacapone, Nefazodone, Buspirone, Trovofloxacin, Levofloxacin, Diclofenac, and Amiodarone.

15. The method of claim 1, wherein seeding the first compartment comprises seeding the first compartment with T cells, and wherein the at least one stimulus comprises a therapeutic comprising an antibody, thereby triggering transendothelial migration of at least one T cell from the first compartment to the second compartment.

16. The method of claim 15, wherein the antibody comprises Pembrolizumab.

17. The method of claim 1, wherein the at least one stimulus comprises a first stimulus comprising an isotype control and a second stimulus comprising a therapeutic comprising an antibody, the method further comprising: assessing a first T cell response resulting from the first stimulus; assessing a second T cell response resulting from the second stimulus; and comparing the first T cell response to the second T cell response.

18. The method of claim 17, wherein the isotype control comprises IgG4.

19. The method of claim 1, wherein the three-dimensional scaffold is or comprises a porous membrane, wherein seeding the first compartment with a first type of live cells comprises lining the porous membrane with an endothelial barrier.

20. The method of claim 16, further comprising increasing concentrations of Pembrolizumab thereby increasing the transendothelial migration of T-cells from the first compartment to the second compartment.

21. The method of claim 15, further comprising introducing a second stimulus to the first compartment thereby increasing the rate of transendothelial migration of at least one T cell from the first compartment to the second compartment, the second stimulus comprising a chemoattractant.

22. The method of claim 21, wherein the chemoattractant comprises CXCL12.

23. The method of claim 17, wherein assessing each of the first T cell response and the second T cell response comprises measuring T cell activity via at least one of cytokine release and cytotoxic activity.

24. The method of claim 23, wherein cytokine release comprises IFNgamma release, and wherein cytotoxic activity comprises Granzyme B release.

25. The method of claim 15, wherein the antibody comprises a monoclonal antibody (mAB), the method further comprising mixing the T-cells with the monoclonal antibody.

26. The method of claim 15, wherein the monoclonal antibody comprises an anti-PD-1- mAB.

27. The method of claim 25, wherein mixing the T-cells with the monoclonal antibody comprises exposing peripheral blood mononuclear cells (PBMCs) to anti-CD3, anti-CD28, and/or anti-CD2 antibodies for a first period of time and IL-2 antibodies for a second period of time.

28. The method of claim 15, further comprising loading the T cells with a fluorescent label prior to seeding the T cells in the first compartment.

29. The method of claim 28, wherein the fluorescent label comprises CellTracker Orange.

30. The method of claim 15, wherein the T cell comprises at least one of a CD3 and a CD8 T cell.

31. The method of claim 1, wherein seeding the first compartment comprises seeding the first compartment with at least one antibody drug conjugate (ADC).

32. The method of claim 31, wherein the at least one antibody drug conjugate comprises a T-Dxd ADC.

33. The method of claim 32, wherein seeding the second compartment comprises seeding the second compartment with breast cancer cells.

34. The method of claim 33, further comprising using an Alex 647 fluorescent market to identify at least one antibody.

35. The method of claim 1, wherein seeding the first compartment comprises seeding the first compartment with HUVEC (human umbilical vein endothelial cells), the method further comprising assessing the toxicity of the at least one stimulus by measuring the viability of the HUVEC.

36. A three-dimensional scaffold for use in synthetic mammalian tissue, the scaffold comprising: a repeated 3D geometry; and at least one of a hydrogel, a biocompatible polymer, and a bioprintable polymer; wherein the repeated 3D geometry comprises an open geometry comprising a plurality of interconnected nodes configured to be seeded with living cells.

37. The scaffold of claim 36, comprising a porosity in a range from about 40% to about 90%.

38. The scaffold of claim 36, comprising a porosity in a range from about 60% to about 80%.

39. The scaffold of claim 36, wherein the repeated 3D geometry comprises a lattice of interconnected spherical pores.

40. The scaffold of claim 36, wherein the repeated 3D geometry comprises a fibrous interstitial pattern.

41. The scaffold of claim 36, wherein the repeated 3D geometry comprises a criss-cross pattern.

42. The scaffold of claim 36, wherein the repeated 3D geometry is bioprinted.

43. The scaffold of claim 36, wherein the scaffold comprises a hydrogel comprising an inert polymer.

44. The scaffold of claim 36, wherein the repeated 3D geometry comprises a plurality of vertically stackable membranes, each membrane comprising a thickness in a range from about 100 m to about 300 pm.

45. The scaffold of claim 36, wherein the repeated 3D geometry comprises a vertically stacked membrane with a thickness in a range from about 180 pm to about 220 pm.

46. The scaffold of claim 44, wherein the membranes comprise a plurality of oppositely- oriented pores.

47. The scaffold of claim 44, wherein the membranes comprise a plurality of pores, the plurality of pores comprising a mean diameter in a range from about 40 pm to about 80 pm.

48. The scaffold of claim 47, wherein the plurality of pores are disposed within the membranes at a density of about 3000-4500 pores per cm².

49. The scaffold of claim 39, wherein each spherical pore of the lattice of interconnected spherical pores is connected to at least two adjacent spherical pores within the same horizontal plane.

50. The scaffold of claim 49, wherein each spherical pore of the lattice of interconnected spherical pores is connected to at least one adjacent spherical pore in a next vertical layer higher of spherical pores, and wherein each spherical pore of the lattice of interconnected spherical pores is connected to at least one adjacent spherical pore in a next vertical layer lower of spherical pores.

51. A system (or synthetic tissue) comprising the scaffold of claim 36 and at least one active cell seeded therein.

52. The system of claim 51, comprising hydrogel, wherein the hydrogel is at least one of covalently bonded and ionically bonded to the at least one active cell.

53. The system of claim 51, wherein the at least one active cell comprises at least one of a mammalian cell.

54. A platform comprising: multiple compartments; and the scaffold of claim 36 disposed within at least one compartment of the multiple compartments.

55. The platform of claim 54, comprising at least one hollow base disposed beneath the multiple compartments, wherein the multiple compartments comprise at least three compartments; wherein the hollow base connects a first compartment of the multiple compartments to a third compartment of the multiple compartments; wherein the multiple compartments comprise a second compartment disposed between the first compartment and the third compartment; and wherein the second compartment is not fluidly connected to the hollow base.

56. A method of producing a synthetic tissue comprising: bioprinting the scaffold of claim36; coating the scaffold; seeding the scaffold with at least one living cell; and initiating conditions to simulate an in vivo environment within the seeded scaffold.

57. The method of claim 56, wherein coating the scaffold comprises at least one of covalently bonding and ionically bonding at least one extracellular matrix (ECM) protein to the scaffold via at least one linker.

58. The method of claim 57, wherein the at least one linker comprises at least one of acrylated polyethylene glycol succinimidyl valerate (acrylated PEG SVA) and polyethylene glycol N-hydroxysuccinimide (PEG-NHS), maleimide-PEG-acrylate, acrylated PEG-azide, thiol-reactive PEG-vinyl sulfone, hydrazone-forming aldehyde-functionalized PEG, and PEG- diacrylate.

59. The method of claim 56, comprising at least one post-processing step following bioprinting, the at least one post-processing step comprising at least one of heat treat, rinsing, hydrating, drying, irradiating, surface smoothing, de-burring, and cooling.

60. The method of claim 56, wherein initiating conditions to simulate an in vivo environment comprises: providing temperature in a range from about 20°C to about 40°C; providing an atmospheric pressure in a range from about 0.95 bars to about 1.05 bars; and providing an oxygen concentration in a range from about 19.5% to about 21.5%.

61. The method of claim 60, wherein seeding the scaffold with at least one living cell comprises seeding the scaffold with a fluid containing the at least one living cell, the method further comprising: providing the fluid with a glucose concentration in a range from about 1 mM to about 20 mM; and providing the fluid with pH in a range from about 6 to about 8.

62. A platform comprising: a first compartment; a second compartment disposed a distance away from the first compartment; and at least one conduit, passageway, or chamber connecting the first compartment to the second compartment.

63. The platform of claim62, wherein each of the first compartment and the second compartment is vertically oriented, and wherein the at least one conduit, passageway, or chamber is oriented substantially horizontally.

64. The platform of claim 62, wherein each of the first compartment, the second compartment, and the at least one conduit, passageway, or chamber are arranged in a substantially horizontal configuration with the at least one conduit, passageway, or chamber being disposed between the first compartment and the second compartment.

65. The platform of claim 63, further comprising a gap disposed between the first compartment and the second compartment, the gap preventing migration of cells therethrough, wherein the at least one conduit, passageway, or chamber comprises a hollow base disposed beneath the first compartment and the second compartment, the first and second compartment being configured to be seeded with live cells, the hollow base being configured to allow migration of cells from the first compartment to the second compartment, and vice versa, and wherein the gap is not fluidly connected to the hollow base, the first compartment or the second compartment.

66. A system comprising: a platform comprising: a first compartment; a second compartment disposed a distance away from the first compartment; and at least one conduit, passageway, or chamber connecting the first compartment to the second compartment; and a three-dimensional scaffold disposed within the first compartment, the three- dimensional scaffold comprising: a repeated 3D geometry; and at least one of a hydrogel, a biocompatible polymer, and a bio-printable polymer; wherein the repeated 3D geometry comprises an open geometry comprising a plurality of interconnected nodes configured to be seeded with living cells.

67. The system of claim 66, further comprising: a first plurality of live cells disposed within the first compartment; and a second plurality of live cells disposed within the second compartment, wherein the first plurality of cells comprises a different type of cells than the second plurality of cells, and wherein the three-dimensional scaffold is composed of a hydrogel material.

68. The system of claim 66, wherein the first plurality of live cells comprises at least one of primary human hepatocytes (PHH), Jurkat cells, T cells, HUVECs, SK-BR-3 cells, control- treated cells (IgG4), liver cells, Kupffer cells, vein endothelial cells, Immune cells, and IgG-treated cells.

69. The system of claim 66, wherein the second plurality of live cells comprises at least one of necrotic cells, colorectal carcinoma cells, cancer cells, HCT116 cells, and tumor cells.

70. The system of claim 66, wherein the three-dimensional scaffold comprises a plurality of vertically stackable membranes, each membrane comprising a thickness in a range from about 100 pm to about 300 pm.