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US20190331662A1 - Organ models - Google Patents

Organ models Download PDF

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US20190331662A1
US20190331662A1 US16/477,067 US201816477067A US2019331662A1 US 20190331662 A1 US20190331662 A1 US 20190331662A1 US 201816477067 A US201816477067 A US 201816477067A US 2019331662 A1 US2019331662 A1 US 2019331662A1
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cells
layer
extracellular matrix
epss
model
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Ibrahim Tarik Ozbolat
Monika Hospodiuk
Dino Joseph Ravnic
Bugra Ayan
Srinivas Koduru
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Penn State Research Foundation
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Penn State Research Foundation
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Publication of US20190331662A1 publication Critical patent/US20190331662A1/en
Assigned to THE PENN STATE RESEARCH FOUNDATION reassignment THE PENN STATE RESEARCH FOUNDATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: AYAN, Bugra, HOSPODIUK, Monika, OZBOLAT, Ibrahim Tarik, KODURU, Srinivas, RAVNIC, Dino Joseph
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/507—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5064—Endothelial cells

Definitions

  • functional pancreas models can be designed to include an extracellular matrix (ECM) containing a plurality (e.g., two or more) of pancreatic islets, and a vascular network.
  • ECM extracellular matrix
  • Type-1 diabetes is a devastating disease caused by malfunction or complete loss of insulin production by beta ( ⁇ )-cells in islets of Langerhans in the pancreas (Atkinson et al., Lancet, 383:69-82 (2014)). As a result, insulin is produced minimally or not at all. In most cases, T1D is caused by an autoimmune response, whereby the immune system attacks ⁇ -cells and destroys them. It is a chronic disease that often leads to severe complications including blindness, limb amputations, kidney failure, neuropathy, and cardiovascular diseases (Faglia et al., Eur. J. Vasc. Endovasc. Surg., 32:484-490 (2006); Leksell et al., Diabetes Res.
  • T1D has been managed by subcutaneous insulin injections and cure has been attempted by the transplantation of cadaveric pancreases or islets (Migliorini et al., Mol. Metab., 3:268-74 (2014)).
  • functional pancreas models can be designed to include an ECM containing a plurality (e.g., two or more) of pancreatic islets, and a vascular network.
  • a functional pancreas model provided herein can be used in vitro to study the biology (e.g., multicellular interactions) of the human pancreas and/or to evaluate therapeutic agents.
  • ⁇ -cell clusters in a three-dimensional (3D) hydrogel culture can be engineered into pancreatic islets with vascularization.
  • murine pancreatic beta cell line e.g., ⁇ -TC3 cells
  • MVECs rat heart microvessel endothelial cells
  • EPSs engineered pancreatic spheroids
  • ADSC adipocyte-derived stem cell
  • ⁇ -cells can be generated and used to bioprint (e.g., 3D bioprint) a functional (e.g., vascularized) pancreas tissue that mimics the physiology and architecture of a human endocrine pancreas.
  • Having the ability to generate a functional human pancreas provides a model that recapitulates the physiology and/or architecture of a human pancreas and provides a unique and unrealized opportunity to evaluate the effect of various therapeutic agents (e.g., T1D drugs) on the human pancreas (e.g., without requiring human testing).
  • various therapeutic agents e.g., T1D drugs
  • a device capable of being used as a pancreas model.
  • a device can include a substrate, a lower layer of ECM disposed on top of the substrate, a strand of matrix (e.g., sacrificial matrix) disposed along an interior region of the lower layer of ECM, an array of pancreatic islets disposed on top of the lower layer of ECM, an upper layer of ECM disposed on the array of pancreatic islets, and a vascular network.
  • the lower layer of ECM can include fibrin, thrombin, fibrinogen, fibrin hydrogel, collagen (e.g., collagen type IV), gelatin, a gelatinous protein mixture (e.g., Matrigel®), and/or laminin.
  • the lower layer of ECM can include endothelial cells, pericytes, fibroblasts, smooth muscle cells, or combinations thereof.
  • the lower layer of ECM can include calcium chloride (CaCl 2 ).
  • the matrix e.g., sacrificial matrix
  • the matrix can be alginate, agarose, gelatin, sugar, and/or poloxamer (e.g., Pluronic® such as Pluronic® F-127).
  • the pancreatic islets can include ⁇ cells and MVECs.
  • the ⁇ cells can include ADSC-derived 13 cells, induced pluripotent stem cell (IPS) derived beta cells, fibroblast derived beta cells, or any combination thereof.
  • IPS induced pluripotent stem cell
  • the ⁇ cells and the MVECs can be present in a ratio of from about 1:1 to about 10:1.
  • the upper layer of ECM can include fibrin, thrombin, fibrinogen, fibrin hydrogel, collagen (e.g., collagen type IV), gelatin, a gelatinous protein mixture (e.g., Matrigel®), and/or laminin.
  • the upper layer of ECM can include endothelial cells, pericytes, fibroblasts, smooth muscle cells, or combinations thereof.
  • the upper layer of ECM can include CaCl 2 .
  • this document features a method for making a pancreas model.
  • the method can include, or consist essentially of, disposing a first layer of ECM onto a substrate assembled into a model platform, disposing a strand of matrix (e.g., sacrificial matrix) onto an interior region of the first layer of ECM, disposing an array of pancreatic islets onto the first layer of ECM, disposing a layer of ECM onto the array of pancreatic islets, de-crosslinking the matrix (e.g., sacrificial matrix) with a solution to create a channel, seeding the channel with ECs, and perfusing the channel with perfusate.
  • matrix e.g., sacrificial matrix
  • Disposing the first layer of ECM can include alternately disposing a first layer including thrombin, ECs, pericytes, and CaCl 2 , and disposing a second layer including fibrinogen on the first layer.
  • the matrix e.g., sacrificial matrix
  • the pancreatic islets can be EPSs.
  • the EPSs can be engineered by co-culturing ⁇ cells and MVECs.
  • the ⁇ cells can include ADSC-derived ⁇ cells, pluripotent stem cell derived beta cells, fibroblast derived beta cells, or any combination thereof.
  • the ⁇ cells and the MVECs can be co-cultured in a ratio of from about 1:1 to about 10:1.
  • the ⁇ cells and the MVECs can be co-cultured in the presence of a growth factor (e.g., vascular endothelial growth factor, epidermal growth factor, and fibroblast growth factor).
  • a growth factor e.g., vascular endothelial growth factor, epidermal growth factor, and fibroblast growth factor.
  • Disposing the second layer of ECM can include alternately disposing a first layer including thrombin, ECs, pericytes, and CaCl 2 , and disposing a second layer including fibrinogen on the first layer until the model platform is full.
  • the solution can be sodium citrate.
  • the channel can be perfused with laminar flow.
  • the channel can be perfused for about 14 days at a rate of about 0.1 dyne/cm 2 for about 6 hours, followed by a rate of about 1 dyne/cm 2 for about 18 hours, followed by a rate of about 10 dyne/cm 2 for about 13 days.
  • the perfusate can be serum-free media.
  • the pancreas model can be a patient-specific pancreas model.
  • FIG. 1 shows a schematic (A) and a photograph (B) of 3D printed agarose molds.
  • FIG. 2 shows EPS fabrication and morphology.
  • FIG. 2A contains photographs of an agarose mold in a Petri dish surrounded by cell culture media (left), an EPS on the top view (middle), and the cut-away view of an agarose mold showing EPS at the bottom of a microwell on day 3 (right).
  • FIG. 2B contains bar graphs showing that EPS diameter changed over time (1, 5, and 10 days) for three different cell seeding ratios: ⁇ TC3-only (left), a 1:1 ratio of ⁇ TC3 cells to RHMVEC cells (middle), and a 2:1 ratio of ⁇ TC3 cells to RHMVEC cells (right).
  • C FIG.
  • 2C contains SEM images of three-day cultured EPSs shown with a magnification of the surface (morphology) for ⁇ TC3-only (left), a 1:1 ratio of ⁇ TC3 cells to RHMVEC cells (middle), and a 2:1 ratio of ⁇ TC3 cells to RHMVEC cells (right).
  • FIG. 3 contains bar graphs showing cell viability of two dimensional (2D) co-cultures.
  • FIG. 4 shows EPS cell viability.
  • FIG. 4A contains bar graphs showing a quantification of cell viability on free-standing EPSs (left) and EPSs embedded in fibrin (middle) over 1, 5, and 10 days. The right panel depicts the proliferation rate conducted on MTT assay over the same time points.
  • FIG. 4B contains representative pictures of live/dead staining at 1 (top row), 5 (middle row), and 10 (bottom row) days of free-standing EPSs (left panel) and EPSs embedded in fibrin (right panel).
  • EPSs were cultures using ⁇ TC3-only (left), a 1:1 ratio of ⁇ TC3 cells to RHMVEC cells (middle), or a 2:1 ratio of ⁇ TC3 cells to RHMVEC cells (right).
  • FIG. 5 shows EPS functionality.
  • FIG. 5A contains microscope images of ultra-morphology of aggregates showing the vesicles containing insulin granules in each ratio: ⁇ TC3-only (left), a 1:1 ratio of ⁇ TC3 cells to RHMVEC cells (middle), and a 2:1 ratio of ⁇ TC3 cells to RHMVEC cells (right). The arrows demonstrate immature (black) and mature (grey) insulin granules.
  • FIG. 5B contains microscope images of immunocytochemistry of EPSs with RHMVECs stained for PECAM, ⁇ TC3 insulin, and nuclei.
  • FIG. 6 shows EPSs seeded within the fibrin hydrogel.
  • FIG. 6A contains time laps images of EPSs containing a 1:1 ratio of ⁇ TC3 cells to RHMVEC cells (top) or a 2:1 ratio of ⁇ TC3 cells to RHMVEC cells (bottom) over a 65-hour period and observed in a live cell imaging chamber.
  • the solid arrows indicate the void space caused by the contraction of EPSs and the dotted arrows demonstrate the endothelial sprouting.
  • FIG. 6B contains microscope images of immunocytochemistry showing endothelial cells sprouting.
  • FIG. 6C contains microscope images of H&E staining of EPSs containing ⁇ TC3-only (left), a 1:1 ratio of ⁇ TC3 cells to RHMVEC cells (middle), or a 2:1 ratio of ⁇ TC3 cells to RHMVEC cells (right), cultured for five days in fibrin, and observed at magnification of 20 and 40.
  • FIG. 6D contains bar graphs showing capillary sprouting length (top) and capillary number (bottom) over a period of five days in culture.
  • FIG. 7 shows that ADSC-derived ⁇ -cells are functional.
  • A A flow cytometry dot-plot of ADCS-derived ⁇ -cells double stained against insulin and NKX 6.1.
  • B A flow cytometry dot-plot of ADCS-derived ⁇ -cells double stained against C-peptide and NKX 6.1.
  • C A bar graph of insulin release in ADCS-derived ⁇ -cells in response to glucose.
  • FIG. 8 contains a schematic showing a step-by-step fabrication of an exemplary pancreas-on-a-chip model.
  • FIG. 9 contains a schematic showing bioprinting of an exemplary perfusable pancreas-on-a-chip model.
  • A The perfusable model platform (B) lined up with a single-layer of bone marrow endothelial cells (BMECs) under the proposed perfusion settings.
  • C Encapsulated islets in fibrin sprouted robust capillaries in 2 weeks.
  • D1 Sprouted capillaries allowed intravasation of MDA-MB-231 metastatic cancer cells when they were seeded originally within the cell aggregates.
  • D2 Capillaries also sprouted from tumor spheroids made of breast cancer cells (MDA-MB-231), human umbilical vascular endothelial cells (HUVECs), and fibroblasts.
  • vascularized organ models e.g., pancreas models
  • functional pancreas models can be designed to include an ECM containing a plurality (e.g., two or more) of pancreatic islets, and a vascular network.
  • a vascularized human pancreas model can be used in vitro to study the biology of the human pancreas and/or to evaluate therapeutic agents.
  • a pancreas model provided herein can include an ECM containing a plurality (e.g., two or more) of vascularized islets connected by one or more anastomoses.
  • a pancreas model provided herein can include a plurality (e.g., two or more) nerves.
  • a pancreas model can include a lower layer of ECM, a plurality of pancreatic islets connected by a vascular network, and an upper layer of ECM.
  • Organ models provided herein can be 3D organ models.
  • an organ model provided herein can be a functional (e.g., vascularized and/or innervated) organ model.
  • An organ model provided herein can be a model of any appropriate organ (e.g., pancreas, skin, heart, liver, kidney, and lung). In some cases, an organ model provided herein is a pancreas model.
  • An organ model provided herein can be a tumor model.
  • a tumor model can be a model of any appropriate cancer type (e.g., pancreatic cancer, breast cancer, lung cancer, prostate cancer, ovarian cancer, and melanoma).
  • a tumor model provided herein is a breast cancer model.
  • An organ model provided herein can be a model of an organ from any appropriate mammal (e.g., a human, mouse, rat, dog, and cat). In some cases, an organ model provided herein is a human organ model.
  • organ models provided herein can include (e.g., be on and/or within) a device (e.g., a model platform). Organ models present on and/or within a model platform can also be referred to an organ-on-a-chip (e.g., a pancreas-on-a-chip) model.
  • a model platform can be made of any appropriate material. Examples of materials that can be used for a model platform include, without limitation, glass, plastic (e.g., polystyrene), and rubber (e.g., silicone).
  • a material used for a model platform can be a sterile material.
  • a material used for a model platform can be a biocompatible material.
  • a platform can include an opening such that a liquid (e.g., a perfusate) can be perfused through the model platform.
  • a model platform can have two-parts (e.g., having a bottom portion and an upper portion).
  • a bottom portion of a model platform can include a bottom face.
  • a bottom face of a bottom portion of a model platform can be a different material from the rest of the device.
  • a bottom face can be optically transparent (e.g., to facilitate imaging).
  • a model platform can include a well (e.g., in a multiwell plate).
  • a model platform can include a substrate (e.g., a chip such as a microfluidics chip).
  • the upper portion can provide outer walls (e.g., within which organ models provided herein (e.g., pancreas models) can be contained) and can be open on its top and bottom surfaces.
  • an upper portion that is open on its top can have the opening sealed (e.g., to aid in maintaining sterile conditions) with, for example, glass (e.g., a glass coverslip).
  • the two-parts can be assembled using a press-fit assembly.
  • Organ models provided herein can be any appropriate size.
  • an organ model can have a width of about 0.5 mm to about 5 mm (e.g., about 0.8 mm to about 5 mm, about 1.0 mm to about 5 mm, about 1.3 mm to about 5 mm, about 1.5 mm to about 5 mm, about 1.8 mm to about 5 mm, about 2.0 mm to about 5 mm, about 2.5 mm to about 5 mm, about 0.5 mm to about 4.5 mm, about 0.5 mm to about 4.2 mm, about 0.5 mm to about 4.0 mm, about 0.5 mm to about 3.8 mm, about 0.5 mm to about 3.5 mm, or about 0.5 mm to about 3.0 mm).
  • an organ model can have a depth of about 0.5 mm to about 10 mm (e.g., about 1 mm to about 10 mm, about 2 mm to about 10 mm, about 3 mm to about 10 mm, about 4 mm to about 10 mm, about 5 mm to about 10 mm, about 6 mm to about 10 mm, about 0.5 mm to about 9 mm, about 0.5 mm to about 8 mm, about 0.5 mm to about 7 mm, about 0.5 mm to about 6 mm, about 0.5 mm to about 5 mm, about 1 mm to about 8 mm, about 3 mm to about 7 mm, or about 4 mm to about 6 mm).
  • 0.5 mm to about 10 mm e.g., about 1 mm to about 10 mm, about 2 mm to about 10 mm, about 3 mm to about 10 mm, about 4 mm to about 10 mm, about 5 mm to about 10 mm, about 6 mm to about 10 mm, about 0.5 mm to
  • an organ model can have a height of about 0.5 mm to about 1 mm (e.g., about 0.5 mm to about 0.9 mm, about 0.5 mm to about 0.8 mm, about 0.5 mm to about 0.7 mm, about 0.5 mm to about 0.6 mm, about 0.6 mm to about 1 mm, about 0.7 mm to about 1 mm, about 0.8 mm to about 1 mm, or about 0.9 mm to about 1 mm).
  • an organ model can be from about 0.5 mm ⁇ 0.5 mm ⁇ 0.5 mm to about 5 ⁇ 10 mm ⁇ 1 mm in size (e.g., width ⁇ depth ⁇ height).
  • the pancreas model can include any appropriate number of pancreatic islets (e.g., EPSs).
  • a pancreas model provided herein can include between about 20 islets per mm 3 and about 50 islets per mm 3 (e.g., between about 25 islets per mm 3 and about 50 islets per mm 3 , between about 30 islets per mm 3 and about 50 islets per mm 3 , between about 35 islets per mm 3 and about 50 islets per mm 3 , between about 40 islets per mm 3 and about 50 islets per mm 3 , between about 20 islets per mm 3 and about 45 islets per mm 3 , between about 20 islets per mm 3 and about 40 islets per mm 3 , between about 20 islets per mm 3 and about 35 islets per mm 3 , or between about 20 islets per mm 3 and about 30 islets per mm 3 ).
  • An islet can be any appropriate size. In some cases,
  • An organ model provided herein can be vascularized.
  • an organ model e.g., a functional, 3D, human pancreas model
  • the islets can include vascularization (e.g., neovascularization) within the islets.
  • vascularization e.g., neovascularization
  • the islets can be connected by one or more anastomoses (e.g., connections between blood vessels; also referred to as a vascular network).
  • a vascular network can be a microvascular network or a macrovascular network.
  • organ models provided herein can be stable (e.g., can maintain organ physiology and architecture).
  • an organ model can be viable and/or perfusable while on a model platform.
  • the pancreas model can be perfusable in the device for at least about 5 (e.g., at least about 7, at least about 8, at least about 10, at least about 12, at least about 15, at least about 18, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, or at least about 26) days.
  • the pancreas model can be perfusable in the device for at least about 1 month (e.g., at least about 2, at least about 3, at least about 4, at least about 5, or at least about 6 months).
  • an organ model provided herein can be a heterocellular organ model.
  • a heterocellular organ model can include multiple (e.g., two or more) different cell types, cell tissues, and/or organoids.
  • the pancreas model can include an ECM, a plurality of islets (e.g., EPSs), and a vascular network.
  • organ models described herein e.g., human pancreas models and human tumor models.
  • organ models provided here are fabricated by accurate disposing of biomaterials (e.g., cells, organoids, ECM components, growth factors, signaling molecules, genes, nanoparticles, cytokines, and/or other functional components) on a model platform to recapitulate the native physiology and/or architecture of an organ.
  • biomaterials e.g., cells, organoids, ECM components, growth factors, signaling molecules, genes, nanoparticles, cytokines, and/or other functional components
  • pancreas models can be fabricated by disposing pancreatic biomaterials on a model platform to form a lower layer of ECM, one or more open lumens (e.g., one or more channels), an array of organoids (e.g., pancreatic islets), and an upper layer of ECM, and perfusing the disposed biomaterials under conditions where the biomaterials self-assemble to form a pancreas model having a vascular network.
  • fabricating a 3D organ model can include depositing biomaterials on a device (e.g., a model platform) described herein.
  • a model platform can self-assemble to form an organ model.
  • organ models can be fabricated without the use of a scaffold.
  • An ECM in pancreas models can include any appropriate biomaterials (e.g., pancreatic biomaterials).
  • the cells can include any appropriate cells.
  • cells used to fabricate organ models described herein can be endothelial cells (ECs). Examples of ECs that can be used when making an organ model provided herein include, without limitation, BMECs, and MVEC.
  • ECs endothelial cells
  • cells used to fabricate organ models described herein can be stromal cells (e.g., supporting stromal cells). Examples of stromal cells that can be used when making an organ model provided herein include, without limitation, pericytes, fibroblasts, and smooth muscle cells.
  • Cells can be obtained from any appropriate mammal (e.g., a human, mouse, rat, dog, and cat). In some cases, cells can be human cells. In cases where pancreatic biomaterials include ECM components, the ECM components can include any appropriate ECM components. Examples of ECM components that can be used to make an organ model provided herein include, without limitation, thrombin, fibrinogen, fibrin, fibrin hydrogel, collagen (e.g., collagen type IV), gelatin, a gelatinous protein mixture (e.g., Matrigel®), and laminin. In some cases, ECM components can be produced by cells used to fabricate organ models described herein (e.g., ECs such as MVECs).
  • ECs such as MVECs
  • pancreatic biomaterials include other functional components
  • the other function materials can include any appropriate functional components.
  • functional components that can be used to make an organ model provided herein include, without limitation, CaCl 2 .
  • an ECM also can include one or more additional components.
  • an ECM also can include supporting stromal cells and/or supporting stromal tissues.
  • An ECM in pancreas models can be fabricated by disposing biomaterials, (e.g., pancreatic biomaterials) on a model platform in one or more layers.
  • a biomaterial layer can include multiple (e.g., 2, 3, 4, 5, 6, or more) biomaterials.
  • a biomaterial layer can include thrombin, ECs, stromal cells (e.g., pericytes), and CaCl 2 .
  • a biomaterial layer can include a single biomaterial.
  • a biomaterial layer can include fibrinogen.
  • ECM can be fabricated by alternately disposing a first biomaterial layer including thrombin, ECs, pericytes, and CaCl 2 , and a second biomaterial layer including fibrinogen. These alternating layers can be repeated any appropriate number of times (e.g., up to about 14 layers). For example, layers can be alternated until a model platform (e.g., a bottom portion and/or an upper portion of a model platform) is filled.
  • a model platform e.g., a bottom portion and/or an upper portion of a model platform
  • An array of organoids (e.g., pancreatic islets) in pancreas models can include any appropriate organoids.
  • An array of pancreatic islets can include any appropriate number of pancreatic islets (e.g., between about 20 islets per mm 3 and about 50 islets per mm 3 ).
  • An array of pancreatic islets can include pancreatic islets in any appropriate geometric arrangement (e.g., a pattern such as a ring-shaped pattern).
  • pancreatic islets used herein can be engineered pancreatic islets.
  • an engineered pancreatic islet can be an EPS.
  • An EPS can be made using any appropriate technique.
  • ⁇ cells e.g., ADSC-derived ⁇ -cells
  • ECs e.g., MVECs
  • VEGF vascular endothelial growth factor
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • ⁇ cells e.g., ADSC-derived ⁇ -cells
  • ECs e.g., MVECs
  • ⁇ cells can be co-cultured in a ratio of ⁇ cells to ECs of about 1:1 to about 10:1 (e.g., about 1.5:1 to about 10:1, about 2:1 to about 10:1, about 3:1 to about 10:1, about 4:1 to about 10:1, about 5:1 to about 10:1, about 6:1 to about 10:1, about 7:1 to about 10:1, about 8:1 to about 10:1, about 9:1 to about 10:1, about 1:1 to about 9:1, about 1:1 to about 8:1, about 1:1 to about 7:1, about 1:1 to about 6:1, about 1:1 to about 5:1, about 1:1 to about 4:1, about 1:1 to about 3:1, about 1:1 to about 2:1, about 2:1 to about 9:1, about 3:1 to about 8:1, about 4:1 to about 7:1, about 5:1 to about 6:1, about 1.1:1 to about 2:1, about 1.3:1 to about 2:1, about 1.5:1 to about 2:1,
  • the ⁇ cells and ECs can be obtained from any appropriate mammal (e.g., a human, mouse, rat, dog, and cat).
  • the ⁇ cells can be derived from any appropriate cell.
  • ⁇ cells can be ADSC-derived ⁇ -cells.
  • ADSC-derived ⁇ -cells can be obtained as described, for example, in the Examples.
  • ⁇ cells can be IPS-derived ⁇ cells.
  • IPS-derived ⁇ cells can be obtained as described elsewhere (see, e.g., Pagliuca et al., 2014 Cell 158:428-439; and Millman et al., 2016 Nat. Comm. 7:11463).
  • ⁇ cells can be fibroblast-derived ⁇ cells.
  • Fibroblast-derived ⁇ cells can be obtained as described elsewhere (see, e.g., Zhu et al., 2016 Nat. Comm. 7:10080).
  • derived ⁇ -cells can be derived from human cells.
  • ADSC-derived ⁇ -cells can be human ADSC-derived ⁇ -cells.
  • an organoid can be a vascularized organoid (e.g., a pre-vascularized organoid such as a pre-vascularized islet).
  • an organoid e.g., an EPS
  • an EPS also can include one or more additional types of cells.
  • an EPS also can include ⁇ -cells, ⁇ -cells, ⁇ cells, ⁇ -cells, and/or ECs.
  • An open lumen (e.g., a channel) in pancreas models can be made using any appropriate method.
  • one or more strands of temporary support material e.g., a matrix such as a sacrificial matrix
  • the one or more strands of temporary support material can be disposed on a layer of ECM in any appropriate shape and/or pattern (e.g., a line or a network).
  • a strand of temporary support material can be disposed on a first layer (e.g., a lower layer) of ECM, and a second layer (e.g., an upper layer) of ECM can be disposed on the strand of temporary support material.
  • a temporary support material can be any material that can be removed such that an open lumen is left in its place.
  • a strand of temporary support material can be disposed along a middle region of a first layer of ECM, and a second layer of ECM can be disposed on the strand of temporary support material such that the open lumen runs through the center of the pancreas model and is surrounded (e.g., completely surrounded) by ECM.
  • materials that can be used as a temporary support e.g., a sacrificial matrix
  • examples of materials that can be used as a temporary support include, without limitation, alginate, agarose, gelatin, sugar, and poloxamer (e.g., Pluronic® such as Pluronic® F-127).
  • the one or more strands of temporary support material can be any appropriate size.
  • the size of the one or more strands of temporary support material can reflect the diameter(s) (e.g., internal diameter(s)) of blood vessels in a vascular network.
  • an alginate strand can have a width of from about 200 ⁇ m to about 800 ⁇ m (e.g., from about 300 ⁇ m to about 800 ⁇ m, from about 400 ⁇ m to about 800 ⁇ m, from about 500 ⁇ m to about 800 ⁇ m, from about 600 ⁇ m to about 800 ⁇ m, from about 200 ⁇ m to about 700 ⁇ m, from about 200 ⁇ m to about 600 ⁇ m, from about 200 ⁇ m to about 500 ⁇ m, from about 200 ⁇ m to about 400 ⁇ m, from about 300 ⁇ m to about 700 ⁇ m or from about 400 ⁇ m to about 500 ⁇ m).
  • an alginate strand can have a width of from about 400 ⁇ m to about 500 ⁇ m.
  • Strands of temporary support material can be removed using any appropriate method.
  • strands of temporary support material can be removed using a manual removal process or an aspiration removal process.
  • the alginate can be removed using a solution (e.g., sodium citrate) to decrosslink the alginate strand.
  • a solution e.g., sodium citrate
  • an organ model having one or more strands of alginate can be maintained in a sodium citrate solution to decrosslink the alginate strands.
  • Removal of the one or more strands of temporary support material can be used to generate open lumens (e.g., of the same diameter(s) as the one or more alginate strands) within the organ model.
  • methods of removing alginate strands can be performed as described elsewhere (see, e.g., Yu et al., Scientific Reports, 6:28714 (2016)).
  • vascular networks in pancreas models can be made using any appropriate method.
  • ECs e.g., MVECs
  • the pancreas model can be perfused (e.g., under conditions where the biomaterials self-assemble to establish a vascular network).
  • a pancreas model can be perfused with any appropriate perfusate (e.g., media such as serum-free media).
  • a pancreas model can be perfused with any appropriate technique (e.g., laminar flow).
  • a pancreas model can be perfused for any appropriate amount of time.
  • an organ model can be perfused for about 3 days to about 60 days (e.g., for about 3 days to about 50 days, for about 3 days to about 45 days, for about 3 days to about 40 days, for about 3 days to about 35 days, for about 3 days to about 30 days, for about 3 days to about 25 days, for about 3 days to about 20 days, for about 3 days to about 15 days, for about 3 days to about 14 days, for about 3 days to about 10 days, for about 3 days to about 5 days, for about 5 days to about 60 days, for about 10 days to about 60 days, for about 15 days to about 60 days, for about 20 days to about 60 days, for about 25 days to about 60 days, for about 30 days to about 60 days, for about 35 days to about 60 days, for about 40 days to about 60 days, for about 45 days to about 60 days, or for about 50 days to about 60 days).
  • 3 days to about 60 days e.g., for about 3 days to about 50 days, for about 3 days to about 45 days, for about 3 days to about 40 days, for
  • an organ model can be perfused to establish neovascularization.
  • a pancreas model can be perfused with any appropriate rate and/or pressure.
  • an organ model can be perfused at a rate of from about 0.1 dyne/cm 2 to about 10 dyne/cm 2 (e.g., about 1 dyne/cm 2 ).
  • an organ model can be perfused at a steady rate.
  • an organ model can be perfused at a rate (e.g., a shear rate) that changes (e.g., increases, decreases, or oscillates) during a period of perfusion.
  • a pancreas organ model can be perfused with increasing shear rates during day 1 (e.g., from 0 hours to about 24 hours) and at a steady shear rate from days 2-14.
  • Biomaterials e.g., pancreatic biomaterials
  • arrays of organoids e.g., pancreatic islets
  • strands of temporary support material can be disposed (e.g., on a model platform) using any appropriate technique.
  • techniques that can be used to dispose pancreatic biomaterials, arrays of pancreatic islets, and/or strands of temporary support material on a model platform include, without limitation, hanging drop, microwell, micropatterned matrix, microfluidic, acoustic force, and magnetic force based techniques.
  • Methods of fabricating a 3D organ model can be manual, automated, or a combination thereof. In some cases, automated fabrication of a 3D organ model can include bioprinting (e.g., 3D bioprinting).
  • Bioprinting can be done using any appropriate bioprinter (e.g., an inkjet bioprinter). Bioprinting can include droplet-, extrusion-, and/or laser-based bioprinting. In some cases, methods of fabricating organ models can be as described elsewhere (see, e.g., Peng et al., Trends Biotechnol. 34:722-32 (2016); and Yu et al., Scientific Reports, 6:28714 (2016)).
  • a pancreas-on-a-chip model can be made by depositing layers of pancreatic biomaterials (e.g., thrombin, MVECs, pericytes, CaCl 2 , fibrinogen, alginate strands, fibrin hydrogel, and pre-vascularized islets) on a model platform having a bottom portion and an upper portion.
  • pancreatic biomaterials e.g., thrombin, MVECs, pericytes, CaCl 2 , fibrinogen, alginate strands, fibrin hydrogel, and pre-vascularized islets
  • a temporary support material can be deposited using extrusion-based bioprinting.
  • a first layer of thrombin, ECs, pericytes, CaCl 2 can be bioprinted in the bottom portion of the model platform, followed by a second layer of fibrinogen bioprinted on the first layer.
  • the first and second layers can be alternated any appropriate number of times (e.g., up to about 14 layers) to fill the bottom portion of the model platform.
  • Alginate strands can be extrusion-printed on the alternating first and second layers (e.g., on the filled bottom portion of a model platform).
  • Pre-vascularized islets e.g., EPSs
  • EPSs Pre-vascularized islets
  • the upper portion of the model platform can be assembled onto the bottom portion, and the first and second layers can be alternately bioprinted until the model platform is full.
  • the model platform can be maintained in a sodium citrate solution to decrosslink the alginate strand and flushed to generate open lumen, followed by seeding MVECs in the open lumen.
  • the model platform can be perfused for about 14 days at a rate of 0.1 dyne/cm 2 during the first 6 hours, a rate of 1 dyne/cm 2 thereafter during the first day, followed by a rate of 10 dyne/cm 2 after the first day to establish a vascular network.
  • An exemplary method of making a functional, 3D, human pancreas model is shown in FIG. 8 and is described in Example 3.
  • organ models provided herein can be used for screening drugs (e.g., therapeutic agents).
  • organ models provided herein can be used to evaluate pharmacokinetics (e.g., absorption, distribution, metabolism, and clearance) of a drug.
  • organ models provided herein can be used to evaluate pharmacodynamics (e.g., mechanism of action, toxicity, and dose-response relationship (such as efficacy and/or potency)) of a drug.
  • organ models provided herein can be used to identify and/or optimize a drug.
  • methods of screening drugs can be high-throughput screening methods.
  • methods of screening drugs can include real-time observation.
  • pancreas model can be used to screen drugs (e.g., candidate drugs) for treating any appropriate pancreatic disease.
  • pancreatic diseases include, without limitation, diabetes mellitus (e.g., T1D and type 2 diabetes), pancreatitis, exocrine pancreatic insufficiency, and cystic fibrosis.
  • a functional pancreas model can be used to screen drugs for treating T1D.
  • organ models provided herein can be used for personalized (e.g., patient-specific) drug screening.
  • a patient-specific pancreas model can be fabricated using ADSC-derived ⁇ -cells and ECs (e.g., MVECs) obtained from that patient to engineer patient specific EPSs.
  • a patient can be any appropriate mammal (e.g., a human, mouse, rat, dog, and cat).
  • a patient can be a human, and a patient-specific pancreas model can be fabricated using ADSC-derived ⁇ -cells derived from that human's adipose tissue.
  • Example 1 Vascularization of Engineered Pancreatic Spheroids
  • EPSs engineered pancreatic spheroids
  • RVECs rat heart microvascular endothelial cells
  • EPSs cultured in hydrogel constructs maintained their viability and functionality over time, while non-vascularized EPSs, without the presence of RHMVECs, could not retain their viability nor functionality.
  • Micro-vascularization of engineered islets is demonstrated, where patient-specific stem cell-derived human beta cells can be combined with micro-vascular endothelial cells for an effective treatment of T1D.
  • ⁇ TC3s Mouse insulinoma ⁇ TC3 cells ( ⁇ TC3s) were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Corning Cellgro, Manassas, Va.) supplemented with 20% fetal bovine serum (Life Technologies, Grand Island, N.Y.), 1 mM sodium pyruvate (Life Technologies), 2 mM Glutamax (Life Technologies), and 100 U/mL penicillin G, 100 ⁇ g/mL streptomycin (Life Technologies).
  • DMEM Dulbecco's Modified Eagle's Medium
  • fetal bovine serum Life Technologies, Grand Island, N.Y.
  • 1 mM sodium pyruvate Life Technologies
  • 2 mM Glutamax Life Technologies
  • penicillin G 100 ⁇ g/mL streptomycin
  • Rat heart microvessel endothelial cells (RHMVEC) (VEC Technologies, Rensselaer, N.Y.) were cultured in MCDB 131 medium (Corning Cellgro) supplemented with 5% fetal bovine serum, 2 mM Glutamax, 1 ⁇ g/mL hydrocortisone (Sigma-Aldrich, St. Louis, Mo.), 1 ⁇ g/mL human epidermal growth factor (Sigma-Aldrich), 12 ⁇ g/mL bovine brain extract (Lonza, Walkersville, Md.), and 100 U/mL penicillin G, 100 ⁇ g/mL streptomycin. Cells were maintained at 37° C. in a 5% CO 2 humidified atmosphere.
  • Negative patterns were designed with cylindrical micro-pillars of different diameters on top of the mold surface.
  • PTC Creo software (Parametric Technology Corporation, Exton, Pa.) was used to create a 3D computer aided design (CAD) model.
  • the CAD model was then converted to a stereolithography (STL) file to fabricate the mold using a Perfactory® Micro Hi-Res 3D printer (EnvisionTec, Detroit, Mich.).
  • a high-resolution material, HTM140M, (EnvisionTec) was used to manufacture the mold.
  • the cylindrical micro-pillars were 300 ⁇ m in diameter with a total number of 124 wells.
  • ⁇ TC3 and RHMVEC were detached from cell culture flasks using trypsin; cell media was added to deactivate trypsin, and suspension was centrifuged for 5 minutes at 1,600 rpm. Cells were counted using a hemocytometer. ⁇ TC3 and RHMVEC, respectively, were combined in ratios of 1:1 or 2:1. Also a third ratio was created by ⁇ TC3-only cells. A total of 2 million cells were suspended in 100 ⁇ l of medium and carefully pipetted on the top chamber of the agarose mold.
  • Gravity acts to pull cells down into the agarose wells where cells aggregate as described elsewhere (see, e.g., Napolitano et al., Biotechniques, 43:494-500 (2007)). Over the next 9 hours, at 3 hour intervals, a small amount ( ⁇ 150 ⁇ L) of fresh medium was gently added to the top of the mold to provide nutrition for the developing EPSs. The petri dish containing the molds was also filled with cell culture media to ensure proper hydration of agarose molds. After 12 hours, EPSs compacted and media was changed every 24 hours.
  • EPSs formed using ⁇ TC3-only, a 1:1 ratio of ⁇ TC3 cells to RHMVEC cells, or a 2:1 ratio of ⁇ TC3 cells to RHMVEC cells were assayed for size and proliferation at three time points: 1, 5, and 10 days.
  • the size of 15 random EPSs for three cell ratios in concentrations of 1, 2, and 3 ⁇ 10 6 cells/mold was measured using an EVOS FL Auto inverted microscope (ThermoFisher, Pittsburgh, Pa.) and software in bright field mode.
  • the relative difference in average diameter between 1 st and 10 th day was determined by the equation:
  • EPSs The morphology of EPSs was determined by razor cutting of the agarose mold containing EPSs. EPSs were observed and imaged on the EVOS FL Auto (Thermofisher) inverted light microscope to visualize mold characteristics and EPS morphology vertically dimension.
  • EVOS FL Auto Thermofisher
  • EPSs Field emission scanning electron microscopy (SEM) (Zeiss SIGMA VP-FESEM) was used to investigate the surface topography of EPSs.
  • SEM Field emission scanning electron microscopy
  • EPSs were harvested after three days of culture in the agarose mold and fixed in 4% paraformaldehyde (Sigma Aldrich) overnight. EPSs were then carefully washed in PBS and dehydrated using graded ethanol solutions (25% to 100%). To ensure complete removal of water, EPSs were then further dried in a critical point dryer (CPD300, Leica EM). On complete dehydration, EPSs were sputter coated with gold using the Bal-tec SCD-050 Sputter Coater (Leica, Wetzlar, Germany) and observed at an accelerating voltage of 3 kV.
  • CPD300 critical point dryer
  • TEM Transmission electron microscopy
  • EPSs were fixed in 4% paraformaldehyde (Sigma Aldrich, USA) for 30 minutes and then centrifuged shortly to form a pellet and quickly washed in 0.1 M cacodylate buffer. The pellet was then subjected to 1% OsO 4 treatment for 60 minutes. After the OsO 4 treatment, the pellet was carefully washed in the cacodylate buffer again for 10 minutes. Following this, En Bloc staining was carried out using 2% uranyl acetate diluted in 50% ethanol for 30 minutes.
  • Fibrin hydrogel was prepared by blending fibrinogen protein isolated from bovine plasma (Sigma-Aldrich) and bovine thrombin from plasma (Sigma-Aldrich). Both solutions were dissolved separately in DPBS in the following concentration: 10 mg/mL fibrinogen and 3 U/mL thrombin at the 37° C. Both components were combined in equal amounts yielding a final concentration of 5 mg/mL fibrinogen and 1.5 U/mL thrombin. EPSs were gently suspended in thrombin, which is blended with fibrinogen. After gentle, thorough pipetting, the pre-crosslinked suspension was deposited on 12 mm round cover slips placed in a 24-well plate. After 15 minutes of crosslinking in the incubator, cell media was deposited on top of fibrin.
  • the constructs culture medium was formulated in 1:1 ratio of ⁇ TC3 media and EGM-2V media (Lonza). Fabricated fibrin constructs were installed into a live cell imaging chamber and observed using a Keyence BZ-9000E microscope (Keyence Corp, Boston, Mass.). Images were captured every hour for a 65-hour period.
  • EPSs formed using ⁇ TC3-only, a 1:1 ratio of ⁇ TC3 cells to RHMVEC cells, or a 2:1 ratio of ⁇ TC3 cells to RHMVEC cells), alone and embedded in fibrin were stained to determine the viability at three time points, 1, 5, and 10 days.
  • EPSs were removed from the agarose well and rinsed three times with DPBS (Life Technologies).
  • EPSs and those embedded in fibrin were transferred to the glass-bottom dishes for imaging on confocal laser scanning microscopy (Olympus FV10i, Olympus, America Inc., Center Valley, Pa.) to detect calcein (excitation 499, emission 520) and ethidium homodimer (excitation 577, emission 603).
  • calcein excitation 499, emission 520
  • ethidium homodimer excitation 577, emission 603
  • Ten representative areas of each sample type, ratio, and time point were randomly selected for imaging. Viability was quantified using ImageJ (National Institutes of Health). A minimum of 180 EPSs from three separate runs were quantified.
  • EPSs from one, full mold were used per each time point for each cell ratio. The same amount of cells served for the proliferation measurement, since cells were seeded in each mold in equal quantity. EPSs were flushed from agarose wells, washed twice with Dulbecco's phosphate buffered saline (DPBS; Life Technologies), and suspended in 100 ⁇ L of the media without phenol red. To each sample, 10 ⁇ L of 12 mM MTT solution was added; a negative control consisted of cell medium without cells.
  • DPBS Dulbecco's phosphate buffered saline
  • the EPSs embedded within the fibrin were cultured in a 24-well plate for 3 days and then fixed overnight with 4% paraformaldehyde (Sigma-Aldrich) at 4° C. Constructs were then washed 3 times (10 minutes each in DPBS) at room temperature. Permeabilization was performed with 0.25% Triton X-100 (Sigma-Aldrich) and 5% normal goat serum (Abcam) diluted in DPBS and incubated for 1 hour. Primary antibodies were diluted in blocking solution (1:50 for PECAM-1 and 1:200 for insulin) applied to constructs. After overnight incubation at 4° C., samples were washed 3 times (10 minutes each in DPBS).
  • PECAM secondary goat anti-rabbit antibody Alexa Fluor 647, Life Technologies diluted 1:500 was incubated within the constructs for 1 hour at room temperature in dark and then washed for 10 minutes in DPBS. Then, insulin goat anti-guinea pig secondary antibody (Alexa Fluor 568, Life Technologies) diluted 1:500, was incubated within the constructs for 1 hour at room temperature in dark. Staining was followed by 10 minutes DPBS wash and incubated for 30 minutes in 5 ⁇ g/mL Hoechst.
  • Samples were washed three times per 10 minutes in DPBS, followed by quick rinse in distilled water and then placed on a droplet of Vectashield (Vector Laboratories) on glass-bottom dishes for imaging on a confocal laser scanning microscope (Olympus FV10i) by lasers Alexa Fluor 568 (excitation 577, emission 603) and Alexa Fluor 647 (excitation 653, emission 668). Images were pseudo-colored, green for PECAM and red for insulin.
  • EPSs formed from the three cell mixing ratios were grown for 72 hours prior to the insulin secretion analysis.
  • the two-dimensional (2D) culture was performed as a comparison control to EPSs. Two million cells for each control were seeded per tissue culture dish, which is as an equivalent of the cell amount in a single mold of EPSs.
  • the insulin secretion analysis was conducted at 3 time points (1, 5, and 10 days) of ⁇ TC3-only, 1:1 ratio of ⁇ TC3 to RHMVEC, and 2:1 ratio of ⁇ TC3 to RHMVEC, and one-day, 2D culture of ⁇ TC3-only, 1:1 ratio of ⁇ TC3 to RHMVEC, and 2:1 ratio of ⁇ TC3 and RHMVEC, respectively.
  • 1 ⁇ Krebs buffer was freshly prepared (25 mM HEPES, 115 mM NaCl, 24 mM NaHCO 3 , 5 mM KCl, 1 mM MgCl 2 , 0.2 g 0.1% BSA dissolved MQ H 2 O).
  • To the final solution was added 2.5 mM CaCl 2 and the pH was adjusted to 7.4 with 1 M NaOH. This solution was filtered through 0.22 ⁇ m filter and stored at 4° C.
  • EPSs e.g., EPSs formed using ⁇ TC3-only, 1:1, and 2:1 ratio of ⁇ TC3 and RHMVEC, respectively
  • 2D cultures of the same cell ratios were shortly rinsed in a glucose solution and then incubated in a fresh solution for 1 hour in 37° C. and 5% CO 2 . After incubation, 100 ⁇ L of solution of the supernatant was kept in ⁇ 20° C. for the insulin enzyme-linked immunosorbent assay (ELISA) analysis.
  • ELISA insulin enzyme-linked immunosorbent assay
  • Mouse ultrasensitive insulin ELISA kit (Alpco, Salem, N.H.) was used to detect insulin secretion of EPSs and 2D cultures after glucose simulation test at 1 st , 5 th , and 10 th day of experiments. Samples were gently mixed, and centrifuged at 2,000 rpm for 5 minutes to remove cell debris. ELISA protocol was performed according to manufacturer's instruction. Briefly, 5 ⁇ L of standard solution or sample was applied in duplicate wells of the pre-coated ELISA microplate and then, 75 ⁇ L of HRP conjugate was added. Plate was incubated for 2 hours on a shaker (300 rpm) and then rinsed thoroughly 6 times with wash buffer.
  • TMB substrate was pipetted into each well; plate was incubated for 30 minutes on a platform shaker protected from light. Afterward, 100 ⁇ L of stop solution was added into each well. Absorbance readings were performed at 450 nm on a Powerwave X-340 spectrophotometer (BioTek) and insulin concentrations were calculated by KCjunior software.
  • EPSs encapsulated in fibrin were taken at each at day 1, 3 and 5 on the EVOS FL Auto (Thermofisher). These images were then processed on ImageJ software (NIH) using the Angiogenesis Analyzer plugin to generate a skeleton of the sprouts. The generated skeleton was further analyzed using the Analyze Skeleton plugin to quantify the sprout length and number. The average sprouting length and the average sprout number was calculated for a set of 10 spheroids for each ratio (e.g., EPSs formed using a 1:1 ratio of ⁇ TC3 to RHMVEC, and a 2:1 ratio of ⁇ TC3 to RHMVEC) at each time point (day 1, 3, and 5).
  • NIH ImageJ software
  • EPSs in three ratios e.g., EPSs formed using ⁇ TC3-only, a 1:1 ratio of ⁇ TC3 to RHMVEC, and a 2:1 of ⁇ TC3 to RHMVEC
  • the EPSs-laden fibrin was stabilized by adding 300 ⁇ L of 1.5% agarose (RPI Corp.) on the top, prior fixation in 4% (v/v) paraformaldehyde overnight.
  • the constructs were gradually dehydrated in alcohol and sectioned at 8 ⁇ m. Sections were then stained by Leica Autostainer XL (Leica).
  • FIG. 1A The printed plastic mold ( FIG. 1B ) was characterized by high resolution of fine details and a superior surface finish.
  • the mold When filled with liquefied agarose, the mold formed a reproducible uniform hydrogel having 124 microwells and a 300 ⁇ m diameter, as shown in FIG. 2A (left).
  • EPSs After 24 hours of incubation in the agarose mold, EPSs exhibited a compact and rigid morphology (see FIG. 2A , right) that capable of being flushed out of the mold with a gentle pipetting.
  • ⁇ TC3 and RHMVEC were co-cultured for EPSs formation in three ratios, ⁇ TC3-only, a 1:1 ration of ⁇ TC3 to RHMVEC, and a 2:1 ratio of ⁇ TC3 to RHMVEC.
  • ⁇ TC3s provided the insulin-secreting component
  • RHMVEC served to strengthen EPS formation and generate internal and external neovascularization.
  • Cells seeded in biocompatible, non-toxic, mechanically stable, and non-adhesive agarose molds aggregated successfully (see FIG. 2A ), and cell distribution was uniform throughout the microwell.
  • EPS diameters were measured at 1, 5, and 10 days ( FIG. 2B ).
  • the average diameter of ⁇ TC3-only spheroids seeded at 1 ⁇ 10 6 cells/mold measured 202.2 ⁇ 8.88 ⁇ m on day 1, 238.8 ⁇ 8.93 ⁇ m on day 5, and 269.3 ⁇ 8.45 ⁇ m on day 10.
  • the average size increase was over 33% over a ten-day period.
  • average EPS diameter ranged from 228.9 ⁇ 6.77 ⁇ m on day 1, 269.8 ⁇ 15.2 ⁇ m on day 5, and 285.8 ⁇ 11.4 ⁇ m on day 10, increasing over 24%.
  • ⁇ TC3-only group seeded at 3 ⁇ 10 6 cells/mold, average EPS diameter measured 278.4 ⁇ 5.98 ⁇ m on the 1st day, 314.2 ⁇ 10 ⁇ m on the 5th day, and 321.95 ⁇ 7.32 ⁇ m on the 10th day.
  • the average size increase was only 15.6% over a ten-day period suggesting that the microwell size limits the maximum diameter of the EPS.
  • Similar results were seen in EPSs formed from 1:1 and 2:1 ratios of ⁇ TC3 to RHMVEC.
  • EPS diameters were 186.7 ⁇ 20.5 ⁇ m (day 1), 215.8 ⁇ 15.88 ⁇ m (day 5) and 233.5 ⁇ 10 ⁇ m (day 10) while the diameter for 2:1 ratio was measured 201.3 ⁇ 6.89 ⁇ m (day 1), 218.9 ⁇ 10.7 ⁇ m (day 5) and 231.2 ⁇ 14.6 ⁇ m (day 10). Average diameter increase was 25.1% and 14.8% for 1:1 and 2:1 ratios of ⁇ TC3 to RHMVEC, respectively.
  • EPS diameter was 251.6 ⁇ 17.76 ⁇ m (day 1), 267.5 ⁇ 12.6 ⁇ m (day 5), and 289.08 ⁇ 9.42 ⁇ m (day 10) for 1:1 ratio.
  • EPS measured 247.3 ⁇ 8.98 ⁇ m (day 1), 276.3 ⁇ 5 ⁇ m (day 5) and 285.5 ⁇ 6.87 ⁇ m (day 10).
  • the average size increased by 14.9% (1:1) and 15.4% (2:1) over the 10-day period.
  • EPSs exhibited expansion in their diameter under all seeding conditions, suggesting cell proliferation, proper growth conditions and a synergistic relationship between the pancreatic and vascular cell lines. Based on growth rate and the necessity to maintain an average diameter below 300 ⁇ m, 2 ⁇ 10 6 cells/mold was used for the rest of the study.
  • the ECM was deposited by cells and is presented as slightly irregular crown on the surface, which was magnified in the bottom row of FIG. 2C .
  • Endothelial cells contribute directly to synthesizing the ECM, which includes collagen type IV and laminin and serves as physical barrier for immune system cells. Therefore, in EPSs formed from a 1:1 ration of ⁇ TC3 to RHMVEC and EPSs formed from a 2:1 ratio of ⁇ TC3 to RHMVEC the higher amount of ECM is deposited and EPS in this ratios are characterized by smoother surface topology.
  • the average viability of free-standing ⁇ TC3-only EPSs was 87.41 ⁇ 4.24% on day 1, 73.25 ⁇ 1.02% on day 5, and 56.44 ⁇ 5.09% on day 10.
  • the average viability decreases significantly (by over 35%) over the 10-day period. This trend was not observed for EPSs formed from a 1:1 ratio of ⁇ TC3 to RHMVEC, where the average viability was 91.62 ⁇ 4.3% on day 1, 89.59 ⁇ 5.78% on day 5, and 87.51 ⁇ 7.4% on day 10.
  • EPSs formed from a 2:1 ratio of ⁇ TC3 to RHMVEC cell viability was 88.56 ⁇ 3.43% on the 1 st day, 88.21 ⁇ 3.94% on the 5th day, and 83.7 ⁇ 5.97% on the 10 th day.
  • the MTT results revealed that proliferation of ⁇ TC3-only significantly decreased over time, by 61.12% between 1 and 10 days.
  • the 1:1 and 2:1 ratio EPS exhibited a stable proliferation rate, which did not differ significantly over time. It indicated that 3D co-culture of ⁇ TC3 and RMHVEC supports the proliferation rate and confirmed the live/dead staining results.
  • Free-standing ⁇ TC3-only group were very fragile after couple days in culture. Spheroids disaggregated easily, also during the extraction out of the mold. However, during culture, EPSs were surrounded with fresh media within immediate contact among 10 days. The situation looked differently with ⁇ TC3-only group embedded in fibrin, where the media was added onto of the fibrin construct and, therefore, ⁇ TC3-only group had limited exposure to media. Decrease in the viability after 10 days was similar for free-standing and embedded in fibrin ⁇ TC3-only.
  • the representative pictures from live/dead staining were presented in the FIG. 4B , for both free-standing (left panel) and embedded in fibrin EPS (right panel) for each time point and cell ratio. Viability in fibrin could be maintain in a high level due to expansive character of RMHVEC.
  • the insulin granules were visible in 70 ⁇ m thick sections of EPSs. These unique granules for beta-cells, were visible in different stages of maturation and were surrounded by characteristic halo and membrane (as described elsewhere; see, e.g., Fava et al., Diabetologia, 55:1013-1023 (2012)).
  • the granules were present in EPSs formed from a 1:1 ratio of ⁇ TC3 to RHMVEC and in EPSs formed from a 2:1 ratio of ⁇ TC3 to RHMVEC ( FIG. 5A ) in both immature and mature state.
  • Early insulin granules had larger, sparse granule with faded membrane ( FIG.
  • the insulin granules of ⁇ TC3 cells a mouse insulinoma cell line, had a diameter not exceeding 250 nm that corresponds to the size of rat insulin granules (243 nm) as described elsewhere (see, e.g., Fava et al., Diabetologia, 55:1013-1023 (2012)).
  • EPSs were seeded within fibrin hydrogel to induce vascularization. Over 65 hours of culture, EPSs were observed to form an extensive vascularization (see FIG. 6A ). Endothelial sprouts developed approximately 20 hours after seeding and formed a more complex vasculature over time. Complete time lapse images were obtained. EPSs were able to fuse together, contract, and create a void in fibrin as shown in the FIG. 5A (top right). The void created in fibrin might indicate that the mechanical contraction of fusing EPSs was stronger than the strength of the fibrin hydrogel. Fibrin microstructure allowed migration of RHMVECs, while ⁇ TC3 cells maintained their position within the EPSs.
  • RHMVECs exhibited typical endothelial sprouting within the fibrin, while ⁇ TC3 cells stained with insulin antibody were maintained within the EPS over the culture period. Endothelial cells also formed vascularization within EPSs after two days in culture ( FIG. 6B , top), thicker vessels were formed. When EPSs were placed close together, sprouts between them tended to merge as what appears to be a nascent vascular network ( FIG. 6B bottom).
  • the morphology of EPS in both 1:1 and 2:1 ratios showed the duct-like lumens.
  • the 1:1 ratio was characterized by more lose structure with larger voids and vascularization inside the EPS. Contraction of EPS in 1:1 ratio was large and caused a void in fibrin hydrogel, as shown on FIG. 6C (red arrows).
  • the morphology was more compact, with smaller gaps. However, the duct-like lumens were present.
  • Sprouting length and number was determined in EPSs formed from a 1:1 ratio of ⁇ TC3 to RHMVEC and in EPSs formed from a 2:1 ratio of ⁇ TC3 to RHMVEC at three time points (1, 3, and 5 days) as shown in the FIG. 6D .
  • the average sprouting length increased by about 65% from 1 st day to 3 rd day and by about 83% to 5 th day of culture for EPSs containing 1:1 ratio of ⁇ TC3 to RHMVEC.
  • a similar trend in increase of sprout length was observed for the 2:1 ratio of ⁇ TC3 to RHMVEC which underwent an increase of about 28% to 64% over a period of five days.
  • EPSs viable and functional clusters
  • Functional EPSs can act as building blocks to form larger, viable tissue constructs for various purposes such as engineering functional organ (e.g., human pancreas) models.
  • adipose tissue removal e.g. panniculectomy
  • excised adipose tissue was minced and rinsed to remove residual blood and digested with collagenase at 37° C., and subsequently centrifuged to isolate the stromal vascular fraction (SVF) pellet.
  • SVF stromal vascular fraction
  • the SVF was further washed in buffer and underwent magnetic activated cell sorting (MACS) with CD45+ cells (leukocytes) being extracted and discarded.
  • MACS magnetic activated cell sorting
  • the leukocyte depleted SVF fraction underwent two rounds of MACS isolation of ECs (CD31) and pericytes (CD146 and NG2).
  • the remaining depleted SVF fraction consisted primarily of adipocyte-derived stem cells (ADSCs) (CD73+ and CD90+). Cellular isolates were verified by flow cytometry for yield and purity. Microvascular endothelial cells (MVECs) and pericytes were maintained and expanded in appropriate maintenance media and conditions.
  • ADSCs adipocyte-derived stem cells
  • MVECs Microvascular endothelial cells
  • pericytes were maintained and expanded in appropriate maintenance media and conditions.
  • a hybrid bioprinting technology was developed to build a pancreas-on-a-chip model using fabricated pre-vascularized islets.
  • a two-part device was used where a cover slide was attached to the bottom face of the device for imaging purposes.
  • the detailed steps undertaken are as shown in FIG. 8 (see, e.g., Steps 1 - 6 ).
  • MVECs were seeded into the channel to allow them to settle and attach on both sides of the channels (see, e.g., FIG. 8 , Step 7 ).
  • the device After seeding MVECs, the device was perfused. To train MVECs to better attach and make tight junctions in 3 days, the device was perfused with a shear rate of 0.1 dyne/cm 2 during the first 6 hours, 1 dyne/cm 2 thereafter during the first day, followed by 10 dyne/cm 2 after the first day (see, e.g., FIG. 9B ). The device was perfused for 14 days. Around Days 5-7, capillary formation in the gel and robust capillary sprouts from islets was obtained (see, e.g., FIG.
  • Step 8 followed by sprouting of capillaries from the main channel and anastomosis of these capillaries with the capillaries growing in the gel around Days 10-12 ( FIG. 8 , Step 9 , and FIG. 9C , D1, and D2).
  • bioprint pancreatic islets In order to bioprint pancreatic islets, a custom-made bioprinter was developed.
  • the bioprinter which runs using the aspiration principle, enables bioprinting of pancreatic islets (or any other organoids) that is more precise and more accurate than is achievable by manual deposition.

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