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WO2024228814A2 - Tête d'impression et système d'électroporation, et procédé d'impression de tissu à motifs - Google Patents

Tête d'impression et système d'électroporation, et procédé d'impression de tissu à motifs Download PDF

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Publication number
WO2024228814A2
WO2024228814A2 PCT/US2024/024052 US2024024052W WO2024228814A2 WO 2024228814 A2 WO2024228814 A2 WO 2024228814A2 US 2024024052 W US2024024052 W US 2024024052W WO 2024228814 A2 WO2024228814 A2 WO 2024228814A2
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WO
WIPO (PCT)
Prior art keywords
bioink
cells
electrode
electric field
nozzle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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PCT/US2024/024052
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English (en)
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WO2024228814A3 (fr
Inventor
Jennifer A. Lewis
Jingcheng Lu (Aric)
Carlos Antonio MARQUEZ
Paul Phillip STANKEY
Jonathan R. Coppeta
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Charles Stark Draper Laboratory Inc
Harvard University
Original Assignee
Charles Stark Draper Laboratory Inc
Harvard University
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Publication of WO2024228814A2 publication Critical patent/WO2024228814A2/fr
Publication of WO2024228814A3 publication Critical patent/WO2024228814A3/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/10—Processes of additive manufacturing
    • B29C64/106—Processes of additive manufacturing using only liquids or viscous materials, e.g. depositing a continuous bead of viscous material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/20—Apparatus for additive manufacturing; Details thereof or accessories therefor
    • B29C64/205—Means for applying layers
    • B29C64/209—Heads; Nozzles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/30—Auxiliary operations or equipment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y30/00—Apparatus for additive manufacturing; Details thereof or accessories therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y40/00—Auxiliary operations or equipment, e.g. for material handling
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • B33Y70/10—Composites of different types of material, e.g. mixtures of ceramics and polymers or mixtures of metals and biomaterials

Definitions

  • the present disclosure relates generally to genetic engineering of cells and more particularly to a method of printing patterned tissue.
  • hiPSCs human induced pluripotent stem cells
  • SHH sonic hedgehog
  • Exogenous genetic elements can be activated or induced on demand by several mechanisms, including Cre-LoxP recombinase, TetOn promoters, or any number of light-inducible systems.
  • Genetically- driven differentiation can be induced in a media-agnostic manner to simultaneously derive multiple cell types in a single common co-culture.
  • Transient control over gene expression via mRNA transfection of transcription factors has been used to efficiently differentiate hiPSCs into endothelial cells via ETV2 and neurons via a transcription factor cocktail of NGN1, NGN2, NGN3, NEURODI, and NEUROD2.
  • these protocols differentiate cells in a matter of days while conventional differentiation protocols may take weeks to produce the desired cell type.
  • Electroporation techniques where a high intensity electric field creates pores or openings in cell membranes, are being explored for intracellular gene delivery.
  • current electroporation methods are limited in scope and controllability.
  • FIG. 1 is a schematic of an electroporation system and printhead (in two different positions) during selective transfection via electroporative printing (STEP).
  • FIGS. 2A and 2B show close-up schematics of the printhead during printing, where a pulsed electric field is applied in FIG. 2B.
  • FIGS. 3A-3C are schematics of exemplary cell-laden filaments after exposure of selected voxels to a pulsed electric field.
  • FIG. 4 is a cross-sectional schematic of part of an exemplary nozzle with coaxial electrodes.
  • FIG. 5 shows a histogram of agarose microparticle sizes (Feret diameters).
  • FIG. 6 shows apparent viscosity as a function of applied shear rate for 1%
  • FIG. 7 shows storage (G’) and loss moduli (G”) as a function of applied shear stress for 1% and 2% wt/vol jammed agarose microparticle inks, where closed circles indicate storage modulus and open circles indicate loss modulus.
  • FIG. 8 shows the cell distribution within an agarose microparticle inks via a maximum intensity projection of fluorescently labeled HEK-293T cells at a concentration of lOOxlO 6 cells/ml; scale bar is 200 pm.
  • FIG. 9A shows apparent viscosity of agarose microparticle inks loaded with 0, 10, 50, 100, and 500xl0 6 cells/ml.
  • FIG. 9B shows storage (closed circles) and loss moduli (open circles) of agarose microparticle inks loaded with 0, 10, 50, 100, and 500x10 6 cells/ml.
  • FIGS. 10A-10C show behavior of composite agarose-HAMA microparticle inks, including apparent viscosity (FIG. 10A), storage (closed circles) and loss moduli (open circles) of inks made from composite microparticles containing 0.5%, 0.75%, and 1% wt/vol HAMA (FIG. 10B), and evolution of storage and loss moduli of inks during UV exposure (FIG. IOC).
  • FIGS. 11 A-l 1C show simulated electric field profiles across the channel volume oriented in the xy-plane, xz-plane, and yz-plane, respectively.
  • FIG. 12 shows amplifier output during STEP pulses, in particular, measured voltage profiles during a 5 ms pulse applied during STEP using input voltages from 2 V to 7 V.
  • FIG. 13 shows modeled temperature increase due to Joule heating within printhead active regions (e.g., within the channel between the electrodes) as a function of the applied electric field during STEP.
  • FIGS. 18A and 18B show median fluorescence intensity (MFI) of STEP- printed hiPSCs as a function of the applied electric field strength and as a function of the pulse duration, respectively.
  • FIG. 19A shows efficiency of hiPSCs as a function of applied electric field strength in polymerized composite HAMA-agarose tissues.
  • FIG. 19B shows viability of hiPSCs as a function of applied electric field strength in polymerized composite HAMA-agarose tissues.
  • FIG. 20A shows single pulse resolution of eGFP expression in BJFF-hiPSCs printed in 1% agarose microparticle inks with 100 pg/ml eGFP mRNA and transfected with a singlelOO kV/m, 5 ms pulse pulse measured 24 hours after printing.
  • FIG. 20B shows diameters of filaments printed with a 1 mm 2 square outlet nozzle.
  • FIG. 22 illustrates image mapping to electroporation parameters and assignment of the print path.
  • FIG. 23 shows an immunofluorescence image of eGFP in a photo-crosslinked hiPSC tissue printed using STEP; scalebar: 1 mm.
  • FIG. 24 shows fluorescence intensity profiles of eGFP and DAPI within HEK- 293T filament, with the applied electric field profile.
  • FIG. 25 shows fluorescence images of DAPI and eGFP in hiPSC tissues transfected with eGFP using 0-140 kV/m, 5 ms pulses; scalebars: 500 pm.
  • FIG. 26 shows mean fluorescence intensity of STEP-printed tissues as a function of the applied electric field strength.
  • Described in this disclosure is a new cell patterning method that combines continuous flow electroporation and bioprinting, which may be referred to as selective transfection via electroporative printing (STEP), to manipulate gene expression in a voxel-wise manner during human tissue printing.
  • Selective application of electroporative pulses to a bioink during tissue printing can create printed filaments including patterns of cells transfected with nucleic acid vehicles that encode genes of interest.
  • the method is compatible with a broad array of cell types and gene delivery vectors, and is further capable of transfecting cells with proteins and other complex molecules, in addition to nucleic acids.
  • a new electroporative printhead designed to apply strong electric fields to a bioink during extrusion through a nozzle to carry out the STEP process.
  • the method includes flowing a bioink 102 comprising cells 104 and a biological cargo through a nozzle 108, which is moving relative to a substrate 110; that is, the nozzle 108 may be moving, the substrate 110 may be moving, or both the nozzle 108 and the substrate 110 may be moving.
  • the nozzle 108 encloses a channel 118 extending from a channel inlet 120 to a channel outlet 122.
  • the biological cargo (not visible in the schematics) may comprise a nucleic acid, e.g., plasmid DNA (pDNA) and/or messenger RNA (mRNA), a protein, or another complex molecule to be transfected into some portion of the cells 104.
  • successive voxels 112 of the bioink 102 are selectively exposed to a pulsed electric field, as illustrated in FIG. 2B.
  • a pulsed electric field As illustrated in FIG. 2B, successive voxels 112 of the bioink 102 are selectively exposed to a pulsed electric field, as illustrated in FIG. 2B.
  • the voxel(s) selectively exposed to the pulsed electric field may be referred to as “selected voxel(s).”
  • the nozzle 108 is schematically shown in two separate positions during printing, a first position 114 where the pulsed electric field is off, and a second position 116 where the pulsed electric field is on.
  • a voxel 112 may be understood to be a volume of the bioink 102 having x and y dimensions determined by the nozzle (channel) 108 geometry, and a z dimension, or length along the flow direction 124 determined by the electrode geometry (discussed below).
  • a cell-laden filament 126 comprising the bioink 102 and including the voxels 112 selectively exposed to the pulsed electric field is continuously extruded from the outlet 122 of the nozzle 108, and, as the nozzle 108 moves relative to the substrate 110, the cell-laden filament 126 is deposited (or “printed”) in a predetermined pattern on the substrate 110.
  • printed tissue 128 having spatial patterns of gene expression may be fabricated layer-by-layer from the cell-laden filament 126, or from a number of the cellladen filaments 126 deposited in succession.
  • the pulsed electric field may be applied by a first electrode 130 and a second electrode 132 spaced apart from each other and positioned such that the electric field is applied to a predetermined volume (voxel) 112 of the bioink 102. Ideally, a majority or an entirety of the voxel 112 experiences a uniform electric field strength when the pulsed electric field is applied.
  • the pulsed electric field may be applied perpendicular to, or substantially perpendicular to (within 85-95 degrees of), the flow direction 124.
  • the first and second electrodes 130,132 may be configured to contact the bioink 102 flowing through the nozzle 108, as illustrated in FIGS.
  • first electrode and a second electrode 130,132 may be overlaid with a dielectric material so as not to contact the bioink 102 flowing through the nozzle 108, such that electroporation is induced capacitatively.
  • Various exemplary electrode configurations are discussed below. To ensure that each of the cells 104 of the bioink 102 is exposed to at most a single pulse, or at most two pulses, of the pulsed electric field, the volumetric flow rate of the bioink 102 through the nozzle 108, the print speed, and/or the pulse repetition rate may be controlled.
  • the voxels 112 of the cell-laden filament 126 may include voxels 112a exposed to the pulsed electric field (and concomitantly including transfected cells) and voxels 112b not exposed to the pulsed electric field (and concomittantly not including transfected cells) during flow through the nozzle 108, as illustrated in FIGS. 3A-3C.
  • voxels 112a including transfected cells may be immediately adjacent to each other along a flow direction 124 of the bioink 102, as illustrated in FIG. 3 A.
  • the voxels 112a of the bioink 102 exposed to the pulsed electric field and including transfected cells may be interspersed with, along a flow direction 126 of the bioink, one or more voxels 112b not exposed to the pulsed electric field, and thus not including transfected cells, as illustrated in FIGS. 3B and 3C.
  • the transfection efficiency may be increased by increasing the strength of the pulsed electric field. An increased pulse duration may also improve transfection efficiency.
  • successive voxels along the flow direction 124 may include different proportions or percentages of transfected cells, e.g., from 0% (no pulsed electric field) to 100% (pulsed electric field at high strength). More specifically, the portion or proportion of the cells that undergoes electroporation in each of the selected voxels may be greater than 0, at least 0.2 (or 20%), at least 0.4 (or 40%), at least 0.6 (or 60%), at least 0.8 (or 80%), and/or as high as 1.0 (or 100%).
  • the portion or proportion may be at most 0.9 (or 90%), at most 0.7 (or 70%), at most 0.5 (or 50%), at most 0.3 (or 30%), and/or as low as 0.01 (or 1%).
  • FIG. 3C schematically illustrates a cell-laden filament 126 including voxels containing different proportions of transfected cells (voxels 112ai and 112a2) due to the application of pulsed electric fields of different strengths during flow through the nozzle 108, and further including a single voxel 112b not exposed to the pulsed electric field.
  • the bioink employed for the method may be engineered with rheological properties suitable for direct ink writing as well as with electrochemical properties that can support efficient electroporation.
  • the bioink is preferably shearthinning and may behave as a yield stress fluid to facilitate extrusion through the nozzle followed by shape retention once deposited on the substrate.
  • the bioink may include cells, a biological cargo to be selectively transfected into at least a portion of the cells, and a polymer that may function as a matrix.
  • the polymer may be a natural or synthetic polymer.
  • the polymer may comprise agarose, hyaluronic acid methacrylate (HAMA), collagen, fibrinogen, reconstituted extracellular matrices, modified matrix-derived proteins (e.g., gelatin), modified glycosaminoglycans (e.g., hyaluronic acid, chondroitin sulfate), modified polysaccharides (e.g., alginate, dextran, chitosan), and/or functionalized polyethylene glycol.
  • HAMA hyaluronic acid methacrylate
  • collagen e.g., fibrinogen, reconstituted extracellular matrices
  • modified matrix-derived proteins e.g., gelatin
  • modified glycosaminoglycans e.g., hyaluronic acid, chondroitin sulfate
  • modified polysaccharides e.g., alginate, dextran, chitosan
  • the polymer may be formulated into microparticles for incorporation into the bioink, i.e., into particles having a nominal linear size (width or diameter) in a range from about 1 micron to about 1,000 microns, and more typically in a range from about 10 microns to 300 microns.
  • the polymer may undergo crosslinking during or after printing.
  • the cells may comprise any human cells.
  • the cells may include any mammalian cell type selected from cells that make up the mammalian body, including germ cells, somatic cells, and stem cells.
  • germ cells refers to any line of cells that give rise to gametes (eggs and sperm).
  • sematic cells refers to any biological cells forming the body of a multicellular organism; any cell other than a gamete, germ cell, gametocyte or undifferentiated stem cell.
  • somatic cells examples include fibroblasts, chondrocytes, osteoblasts, tendon cells, mast cells, wandering cells, immune cells, pericytes, inflammatory cells, endothelial cells, myocytes (cardiac, skeletal and smooth muscle cells), adipocytes (i.e., lipocytes or fat cells), parenchyma cells (neurons and glial cells, nephron cells, hepatocytes, pancreatic cells, lung parenchyma cells) and non-parenchymal cells (e.g., sinusoidal hepatic endothelial cells, Kupffer cells and hepatic stellate cells).
  • stem cells refers to cells that have the ability to divide for indefinite periods and to give rise to virtually all of the tissues of the mammalian body, including specialized cells.
  • the stem cells include pluripotent cells, which upon undergoing further specialization become multipotent progenitor cells that can give rise to functional or somatic cells.
  • stem and progenitor cells examples include hematopoietic stem cells (adult stem cells; i.e., hemocytoblasts) from the bone marrow that give rise to red blood cells, white blood cells, and platelets; mesenchymal stem cells (adult stem cells) from the bone marrow that give rise to stromal cells, fat cells, and types of bone cells; epithelial stem cells (progenitor cells) that give rise to the various types of skin cells; neural stem cells and neural progenitor cells that give rise to neuronal and glial cells; and muscle satellite cells (progenitor cells) that contribute to differentiated muscle tissue.
  • hematopoietic stem cells adult stem cells; i.e., hemocytoblasts
  • mesenchymal stem cells adult stem cells
  • epithelial stem cells progenitor cells
  • neural stem cells and neural progenitor cells that give rise to neuronal and glial cells
  • muscle satellite cells progenitor cells
  • the cells may also or alternatively comprise tumor or cancer cells, such as carcinoma, sarcoma, leukemia, lymphoma, melanoma, and/or multiple myeloma cells.
  • the bioink includes the cells at a cell concentration in a range from 1 x 10 6 cells/ml to 500 x 10 6 cells/ml.
  • the biological cargo as indicated above, comprise a nucleic acid, e.g., plasmid DNA (pDNA) and/or messenger RNA (mRNA), and/or a protein.
  • the biological cargo may include at least two cargo species that are controllably introduced into the bioink so as to be present in different voxels during flow through the nozzle, or the at least two cargo species may be controllably introduced into the bioink so as to be present in the same voxel(s) during flow through the nozzle.
  • the bioink includes only negatively charged macromolecules (e.g., the polymer(s)) to prevent nucleic acid binding with the nucleic acids, which may be highly negatively charged.
  • the charge of the polymer may be controlled via the addition of functional groups and/or manipulation of pH value.
  • the bioink may include an electroporation buffer such as Biorad® Gene Pulser Buffer or Gibco® Opti-MEM, which may be different from conventional cell culture media and may promote electroporation.
  • the bioink may in some examples include a crosslinking initiator, e.g., lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), to promote crosslinking of the polymer after extrusion of the bioink through the nozzle.
  • the bioink may include an apoptosis inhibitor, such as Y27632 or Chroman 1, to inhibit or prevent cell death. It may be beneficial for the ink to have a conductivity low enough to prevent Joule heating, which can negatively impact cell viability.
  • the method may further include, after printing the tissue, casting a pre-gel solution over the tissue and curing the pre-gel solution.
  • gel-encapsulated printed tissue may be incubated at 37°C for a suitable time duration (e.g., 15-45 min) to fully cure the gel, which may be a gelatin-fibrin hydrogel.
  • a suitable time duration e.g. 15-45 min
  • the method may also or alternatively include, after extruding the bioink, crosslinking the polymer, e.g., by exposing the tissue to ultraviolet light. The crosslinking may occur before or after deposition of the filament(s) onto the substrate.
  • the printhead 100 includes (a) a nozzle 108 enclosing a channel 118 extending from a channel inlet 120 to a channel outlet 122, and (b) a first electrode 130 and a second electrode 132 spaced apart from each other and configured to apply a pulsed electric field to a bioink 102 flowing through the channel 118.
  • the channel 118 may have a polygonal, circular, oval, or irregular transverse crosssection, which is defined by one or more channel walls.
  • the channel 118 may have a rectangular and/or square transverse cross-section.
  • the first and second electrodes 130, 132 are electrically isolated from each other and positioned between the channel inlet 120 and the channel outlet 122.
  • the first electrode 130 and the second electrode 132 may be positioned in opposition to each other on opposing channel walls e.g., in the case of a square or rectangular cross-section) or on one channel wall e.g., in the case of a circular or oval cross-section).
  • the first electrode and the second electrode may be positioned adjacent to each other on adjacent channel walls or on one channel wall.
  • the first electrode 130 and the second electrode 132 may be positioned in a coaxial or concentric arrangement, as shown in FIG. 4.
  • first electrode 130 and the second electrode 132 may be positioned in parallel with each other, as illustrated in FIGS. 2 A and 2B. As indicated above, the first electrode 130 and the second electrode 132 may be positioned to come into direct contact with the bioink 102 flowing through the channel 118, or the electrodes may be positioned to avoid direct contact with the bioink, e.g., a dielectric material may overlie the first and second electrodes, such that transfection may occur capacitatively.
  • the electrodes 130,132 may be constructed from an electrically conductive material, such as a metal having the form of a foil or strip of suitable size and shape to be positioned within the nozzle 108.
  • the nozzle 108 may be 3D printed from a suitable material, such as a photocurable resin, or fabricated using another method, such as molding.
  • the channel dimensions e.g., the width or diameter of the channel outlet, may be constructed to be large enough to accommodate constituents of the bioink, in particular the cells and/or polymeric microparticles, which may have respective linear sizes (widths or diameters) in a range from 1 pm to 100 pm and from 10 pm to 300 pm, typically. It is preferred that the channel outlet is at least two to three times larger than the mean linear size of the microparticles to avoid clogging and discontinuous printing. However, a smaller channel size and electrode geometry may allow for the use of lower voltages to the requisite similar electric field strengths. Accordingly, the width or diameter of the channel, and in particular the width or diameter of the channel outlet, typically lies in a range from 0.5 mm to 3 mm.
  • the printhead 100 described above may be part of an electroporation system that includes an ink dispenser 134 in fluid communication with the channel inlet 120, a substrate 110 facing the channel outlet 122 for deposition of the printed filament(s) 126, a three-axis motion controller 136 configured to move the printhead 100 and/or the substrate 110 in the x-, y-, and/or z-directions, and a voltage source 138 electrically connected to the first and second electrodes 130,132 to provide the pulsed electric field.
  • an ink dispenser 134 in fluid communication with the channel inlet 120
  • a substrate 110 facing the channel outlet 122 for deposition of the printed filament(s) 126 for deposition of the printed filament(s) 126
  • a three-axis motion controller 136 configured to move the printhead 100 and/or the substrate 110 in the x-, y-, and/or z-directions
  • a voltage source 138 electrically connected to the first and second electrodes 130,132 to provide the pulsed electric field.
  • the ink dispenser 134 may be a syringe extruder, for example, which may be controlled by an PC microcontroller to ensure that the bioink 102 is being dispensed into the channel inlet 120 at the desired flow rate.
  • two components described as being in “in fluid communication with” each other may be understood to be configured (e.g., connected directly or indirectly) such that fluid may flow between and/or through the components in one or both directions.
  • the substrate 110 may comprise a solid surface onto which the cell-laden filament(s) 126 may be deposited, or the substrate 110 may comprise a liquid or gel into which the cell-laden filament(s) 126 may be deposited.
  • references to deposition on a substrate 110 in this disclosure may be understood to encompass deposition onto a solid substrate and/or deposition into a liquid or gel substrate.
  • the voltage source 138 may include, for example, one or more power amplifiers (two are employed in the examples below).
  • An arbitrary waveform generator may be used to provide the pulse waveforms that drive the voltage source, such that a pulsed electric field is generated for electroporation.
  • Custom C drivers and libraries may be used to control the arbitrary waveform generator, which may be configured to send pulses only upon receiving a trigger signal from a machine controller. This set-up facilitates real-time synchronous extrusion and electroporation to facilitate producing accurate, high- resolution patterns of transfected cells.
  • the nozzle has a channel outlet with a cross-sectional area of 1 mm 2 and a square cross-section.
  • the cross-sectional area of the channel outlet may lie in a range from about 0.25 mm 2 to about 9 mm 2 .
  • the process parameters may generally be selected as follows.
  • the print speed may be at least 1 mm/s, at least 2 mm/s, or at least 5 mm/s, and/or as high as 8 mm/s, or as high as 10 mm/s.
  • the volumetric flow rate may be in a range from 1 pl/s to 10 pl/s.
  • the pulse repetition rate may be in a range from 1 Hz to 100 Hz.
  • the print speed, volumetric flow rate and pulse repetition rate may be balanced to ensure that each cell in the bioink is exposed to the pulsed electric field once or at most twice during printing.
  • the strength of the pulsed electric field may be in a range from greater than 0 kV/m to 160 kV/m, such as 10 kV/m to 160 kV/m, and more typically from 40 kV/m to 160 kV/m. Above 160 kV/m cell lysis may occur, and below 40 kV/m, the field strength may not be sufficient to induce electroporation.
  • the pulsed electric field typically includes pulses having a duration from 50 ps to 50 ms.
  • agarose microparticle inks having suitable properties for direct ink writing while permitting electroporation to occur within the printhead are prepared and utilized. It is demonstrated that STEP is compatible with hiPSCs, which can transfect mRNA at high transfection efficiencies (>90%) while maintaining high cell viability. It is shown that the proportion of transfected cells within a given voxel may be selectively controlled by simply adjusting the applied electric field used during electroporation. STEP is used to produce human tissues with different spatially patterned regions and proportions of transfected hiPSCs.
  • Electroporation buffer is composed of 2.5 mM ethylene glycol- bis(P-aminoethyl ether)-N,N,N’,N’- tetraacetic acid (EGTA, MilliporeSigma, #E3889), 1.4 mM potassium phosphate monobasic (MilliporeSigma, #P5655), 3.6 mM potassium phosphate dibasic (MilliporeSigma, #P3786), 5 mM magnesium chloride, 25 mM 4-(2- hydroxyethyl)-!
  • EGTA ethylene glycol- bis(P-aminoethyl ether)-N,N,N’,N’- tetraacetic acid
  • EGTA ethylene glycol- bis(P-aminoethyl ether)-N,N,N’,N’- tetraacetic acid
  • EGTA ethylene glycol- bis(P-aminoethyl ether)-N,N,N’,
  • BJFF hiPSCs were karyotyped, verified for pluripotency via flow cytometry, and are maintained between passages 30 and 60.
  • BJFF-hiPSCs are passaged and cultured in mTeSRl (STEMCELL Technologies, #85850) on tissue culture-treated flasks coated in growth factor-reduced Matrigel (Corning, #354320).
  • HEK-293T cells are purchased from ATCC (#CRL-3216) and were maintained between passages 1 and 15. HEK-293T cells are passaged and cultured in DMEM + 10% FBS, consisting of Dulbecco’s Modified Eagle Medium (DMEM, Corning #10-013-CV) supplemented with 10% vol/vol fetal bovine serum (Gibco, #16140071). For passaging, cells with rinsed with DPBS-/-, dissociated with 0.05% trypsin-EDTA (Gibco, #25300054), and replated in DMEM + 10% FBS for 1 d, prior to culturing them in DMEM + 10% FBS. Media is changed every 48 h.
  • DMEM + 10% FBS consisting of Dulbecco’s Modified Eagle Medium (DMEM, Corning #10-013-CV) supplemented with 10% vol/vol fetal bovine serum (Gibco, #16140071).
  • DMEM + 10% FBS consisting
  • Agarose microparticles are made according to the following protocol. Briefly, low melting point agarose (IBI Scientific, #IB70051) is dissolved in EP buffer at 80°C, then autoclaved for 30 minutes to sterilize solutions. Warm agarose solutions are then transferred to 3 ml syringes and cooled to 4°C. After cooling, bulk agarose gels are fragmented into microparticles by sequentially extruding gels through a 0.84 mm, 0.33 mm, 0.2 mm, and 0.1 mm-diameter luer-lock nozzles (Nordson EFD, #7018122, #7018314, #7005008, and #7018462, respectively).
  • Agarose microparticles are collected in a 15 ml conical tube and centrifuged at 2000g for 5 min to compact microparticles. After centrifuging, the supernatant is removed and the compacted microparticles are stored at room temperature until further use.
  • a modified protocol is used to make composite agarose-methacrylated hyaluronic acid microparticles.
  • 50 kDa methacrylated hyaluronic acid (HAMA, Nanosoft Polymers, #13204) is dissolved in EP buffer and stored at 4°C protected from light until use.
  • Mixed microparticles are prepared immediately before use by combining HAMA in EP buffer 1 : 1 with sterile agarose in EP buffer at 80°C. Mixed solutions are then transferred to 1 ml or 3 ml syringes and gelled at 4 °C.
  • microparticles After cooling, bulk composite gels are fragmented into microparticles by sequentially extruding gels through a 0.84 mm, 0.33 mm, and 0.2 mm-diameter luer-lock nozzles. Microparticles are collected by centrifuging solutions at 700g for 5 min and discarding the supernatant.
  • eGFP mRNA is purchased from Trilink Biotechnologies (#L-7601).
  • DNA vectors containing a T7 RNA polymerase promoter sequence, a proprietary 5’ untranslated region (UTR), an ETV2 open reading frame, a proprietary 3’ UTR, and an encoded poly(A) tail were purchased from Trilink Biotechnologies and VectorBuilder.
  • VB Ultrastable e. coli transfected with the ETV2 vector are streaked onto LB agar plates with 50 pg/ml kanamycin and cultured at 37°C overnight.
  • E. coli cultures are expanded overnight in 100 ml LB broth with 50 pg/ml kanamycin at 37°C.
  • Vectors are purified from cultures using the Macherey-Nagel Xtra Midi Plus plasmid kit (#740412). Briefly, e. coli cultures are centrifuged at 3000g for 15 min at 4°C. Bacterial cell kits are resuspended in RES buffer at 4°C, then lysed by adding LYS buffer for 5 min at room temperature. Lysis is stopped by adding NEU buffer and mixing. Crude cell lysate is passed through an equilibrated DNA binding column before washing the DNA with WASH buffer and eluting using ELU buffer. DNA is then washed in 100% isopropyl alcohol and 70% ethanol before resuspending in 1 mM Tris-HCl, pH 7.0. DNA concentrations are measured by spectrophotometry.
  • DNA vectors are linearized using 10 U/ug SapI restriction enzyme, then purified using the Monarch® PCR and DNA Cleanup Kit (New England Biolabs, #T1030S). mRNA is synthesized from DNA vectors using the HiScribe® T7 mRNA Kit with CleanCap® Reagent AG (New England Biolabs, #E2080S).
  • 1 pg of template DNA is incubated with 6 mM adenosine triphosphate, 5 mM N ⁇ methylpsuedouridine triphosphate, 5 mM cytidine triphosphate, 5 mM guanosine triphosphate, 4 mM cap analog, and 5000 U/ml T7 RNA polymerase in T7 CleanCap Reagent AG Reaction buffer for at least 2 h at 37°C.
  • Template DNA is then digested using 80 U/ml DNase I (New England Biolabs, #M0303S) for 15 min at 37°C.
  • mRNA is purified from the reaction buffer using the Monarch® RNA Cleanup Kit (New England Biolabs, #T2050S). mRNA concentrations are measured by spectrophotometry.
  • mRNA is stored in 1 mM sodium citrate, pH 6.4 at -80 °C until use.
  • Bioinks containing B JFF-hiPSCs are prepared by dissociating B JFF-hiPSCs with TrypLE Express (Gibco, #12605010) for 7 min at 37°C, 5% CO2. Dissociation is quenched by adding DMEM/F12 with HEPES (Gibco, #11330032), centrifuging cells at 250g for 5 min, and resuspending cells in EP buffer, supplemented with the CEPT cocktail to promote stem cell survival 144 : 50 nM chroman 1, 5 pM emricasan, 700 nM trans-ISRIB, and 52.5 ng/ml polyamine solution (GLP Bio, #GK1004).
  • Live cells are counted using trypan blue exclusion on a Countess 3 (Invitrogen). Cells are rinsed once in EP buffer with CEPT by centrifuging at 300g for 3 min and discarding the supernatant. Agarose or composite agarose-HAMA microparticles are added to the cell pellet to bring the concentration of cells in the ink to lOOxlO 6 cells/ml. In some prints, the full volume of microparticles is instead replaced by a 3:1 mix of agarose microparticles to 20 mg/ml HAMA in EP buffer. 100 pg/ml mRNA and 10 pM Y27632 are added to the ink and mixed by careful pipetting.
  • lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, MilliporeSigma, #900889) is added to inks containing HAMA to enable photocrosslinking after printing. Inks are then transferred to 1 ml syringes and centrifuged at 700g for 5 min, with syringe tips facing upwards. After centrifugation, the supernatant is aspirated off and the loaded syringe is transferred to the printer for STEP.
  • Bioinks containing HEK-293T cells are prepared in a similar manner as described above. However, HEK-293T cells are dissociated with 0.05% trypsin-EDTA for 3 min at 37°C, 5% CO2. Trypsin is quenched by adding DMEM + 10% FBS, centrifuging cells at 250g for 5 min, and resuspending cells in EP buffer with no additional supplements. Inks are then prepared in the same manner as above, with the omission of Y27632, which is not needed for HEK-293T cell survival.
  • An Omnicure S2000 (Excelitas Technologies) is used to photo-crosslink inks containing HAMA. Immediately after printing, tissues are transferred into an ultraviolet (UV) light curing chamber and crosslinked at 66 mW cm' 2 for 90 s. After crosslinking, mTeSR Plus (STEMCELL Technologies, #100-0276) supplemented with CEPT cocktail is added to tissues which are transferred to a 37°C, 5% CO2 incubator for cell recovery. 30 min after crosslinking, 100 U/ml penicillin- streptomycin is added to media to prevent bacterial contamination. One day after printing, the media is changed to mTeSR Plus with 100 U/ml penicillin- streptomycin. Tissues were maintained for up to 4 days with daily media changes.
  • Rheological measurements are conducted on a stress-controlled Discovery HR- 3 rheometer (TA Instruments). A 20-mm parallel plate geometry with a gap height of 1 mm is used for all experiments. All experiments are conducted at room temperature. Inks are extruded directly onto the bottom rheometer plate, and excess ink is trimmed with a plastic spatula before measurements. First, oscillatory amplitude sweeps were performed from 0.01 to 1000 Pa at a frequency of 0.5 Hz. Next, oscillatory frequency sweeps were performed from 0.01 to 100 rad s' 1 with an applied shear stress of 0.5 Pa. Yield stresses are calculated as the crossover point between the measured storage and loss modulus.
  • Opto-rheological measurements are conducted using a UV curing attachment (TA Instruments). Ink polymerization via UV crosslinking is assessed by loading inks onto a 20 mm parallel plate geometry and 1 mm gap size. An Omnicure S2000 is connected to the rheometer and calibrated to provide a power flux of 66 mW cm' 2 . A 5 min time sweep with constant oscillatory stress at a frequency of 1 Hz was used to take measurements before and during UV exposure. Measurements were taken every 6 s for 2 min prior to UV exposure to establish baseline rheological characteristics. At 2 min, samples were continuously exposed to UV light for 3 min, taking measurements every 6 seconds.
  • HEK-293T cells are incubated DMEM + 10% FBS with 5 pM CellTracker Red CMPTX (Invitrogen, #C34552) for 30 minutes. Cells are then incorporated into agarose inks as described above. Inks are extruded into custom-built imaged chambers and imaged on a Zeiss LSM710 confocal microscope. Images are processed in ImageJ using the 3Dscript plugin.
  • Printheads are designed using SOLIDWORKSTM (Dassault Systemes). Individual nozzle components are printed on a Form 3B (Formlabs) using Clear v4 resin (Formlabs, #RS-F2-GPCL- 04) and then rinsed in isopropyl alcohol and dried completely before final assembly. Electrodes are laser-cut (Oxford Lasers) from 25 pm-thick platinum foil (Surepure Chemetals). Electrodes are secured to printed nozzle components using conductive screws. Superglue (Loctite) is used to bond the printed nozzle components together and seal the printhead assembly to prevent ink leaks. 22 AWG wire is used to connect the nozzle to the power amplifier. Before each use, assembled printheads are sequentially rinsed in 70% ethanol and then EP buffer to displace the ethanol.
  • a 3-axis gantry (Minitech) controlled by a Nstep stepper controller (NSTEP-4, Aerotech) is used to control stage motion for 3D printing.
  • a custom syringe extruder controlled by an iOS microcontroller is used for ink deposition through the electroporative printhead.
  • Custom C drivers and libraries are used to control an arbitrary waveform generator (AWG, Keysight, #35522A) and generate pulse waveforms for electroporation.
  • Custom Geode commands are used to control the AWG, which provides the pulse waveforms that drive the amplifiers.
  • the machine controller uses a separate digital signal line to trigger the AWG, which only sends pulses upon receiving a trigger signal from the machine controller. This facilitates the necessary real-time, synchronous extrusion and electroporation needed to produce accurate, high-resolution patterns of transfected cells.
  • the AWG provides monopolar square pulses to modulate two serially connected inverting power amplifiers (Kepco, #BOP-100-4D) that drive the printhead by delivering electric pulses as the ink flows through the nozzle.
  • the amplifiers are connected to the embedded electrodes with 22 AWG wire through conductive steel screws.
  • the STEP electroporative printhead is aligned 1 mm above a substrate prior to bioprinting.
  • Custom Geode commands are used to configure electroporation and volumetric flow rates of each ink during the printing process.
  • Human tissues are printed at print speeds between 2 mm s' 1 and 8 mm s’ 1 . Pulse repetition rates are set such that cells printed with these bioinks experience a single electroporative pulse, and while their volumetric flow rates are calculated to produce filaments of a specified diameter.
  • a gelatin-fibrin (gelbrin) hydrogel mixture is prepared prior to printing follow a published protocol.
  • a 15 wt/vol% gelatin solution was produced by adding gelatin powder (MilliporeSigma, #G2500) to DPBS-/- and stirring for 12 h at 70°C and adjusting the pH to 7.5 using 1 M NaOH.
  • Part 1 of the gel solution is made by diluting the 15 wt/vol% gelatin 1:1 with mTeSRl and adding 2.5 mM calcium chloride, 10 pM Y-27632 and 1 U/ml thrombin (MilliporeSigma, #T4648) for a final 7.5 wt/vol% gelatin mix.
  • Part 2 of the gel solution is produced by dissolving lyophilized bovine blood plasma fibrinogen (MilliporeSigma, #341576) at 37°C in DPBS-/- at 50 mg/ml. Both parts of the pre-gel solution are maintained in separate tubes at 37°C prior to use. Immediately after STEP printing, both parts of the pre-gel solution are mixed and quickly cast over the printed tissues, the gel-encapsulated prints were incubated at 37°C for 30 min to allow the gel to fully cure. mTeSRl supplemented with 10 pM Y27632 and 100 U/ml penicillinstreptomycin (Gibco, #15140122) is then added to gel-encapsulated tissues to prevent bacterial contamination.
  • mTeSRl supplemented with 10 pM Y27632 and 100 U/ml penicillinstreptomycin (Gibco, #15140122) is then added to gel-encapsulated tissues to prevent bacterial contamination.
  • STEP printing One day after STEP printing, the media is changed to mTeSRl with 100 U/ml penicillin- streptomycin and 58.5 iU/ml aprotinin (MP Biomedicals, #191158). STEP tissues were maintained for up to 4 days with daily media changes.
  • Gel encapsulated and photo-crosslinked tissues are dissociated using 1 mg/ml hyaluronidase (MilliporeSigma, #H3884) in mTeSR Plus with 10 pM Y27632 for 1 h, while non- crosslinked tissue controls are dissociated with 0.05% trypsin-EDTA for 3 minutes. Dissociated tissues are passed through a 40 pm filter to remove aggregates and large microparticles from the cell suspension.
  • Tissues are fixed in 4% vol/vol paraformaldehyde for at least 1 h at room temperature. Tissues are permeabilized in 0.2% vol/vol Triton-X in DPBS+/+ for 10 min at RT, then blocked in DPBS+/+ with 2% vol/vol donkey serum for at least 2 h. Next, tissues are incubated in primary antibodies diluted in DPBS+/+ with 2% donkey serum on an orbital shaker overnight at room temperature. Antibodies are washed out by rinsing tissues three times with DPBS+/+ containing 0.2% vol/vol Tween-20.
  • Tissues are incubated in secondary antibodies diluted in DPBS+/+ with 2% donkey serum and 600 nM of 4’,6-diamidino-2-phenylindole (DAPI) to label cell nuclei on an orbital shaker overnight at room temperature. Secondary antibodies and DAPI are washed out by rinsing tissues again in DPBS+/+ with 0.2% Tween-20. Tissues are immersed in Easylndex (LifeCanvas Technologies) overnight to improve light penetration within tissues. Tissues are imaged in Easylndex on a Zeiss Axiozoom VI 6. A list of primary antibodies used is given in Table 1, and a list of secondary antibodies used is given in Table 2.
  • Table 1 Primary antibodies for immuno staining.
  • Measurements of individual voxels produced by STEP are taken using filaments electroporated with a single, 100 kV/m, 5 ms pulse. Measurements of gradients of eGFP expression in STEP-printed filaments are taken by printing single filaments and applying electroporative pulses to each voxel within the filament, increasing the electric field strength with each printed voxel. Filaments are fixed 24 hours after printing, counterstained with DAPI, index-matched with Easylndex, and imaged on a Zeiss Axiozoom VI 6. The voxel resolution is calculated from the full- width half maximum of the fluorescence intensity profile of eGFP using a custom MATLAB script.
  • Images are aligned horizontally, then DAPI and eGFP channels are reduced to 1- dimensional fluorescence intensity profiles by averaging the fluorescence intensity across the width of the filament. Fluorescence intensity profiles are smoothed using a 100-pixel averaging filter before aligning the fluorescence profiles with the applied electric field intensity profile.
  • agarose-based microparticle ink that satisfies the requirements for STEP was developed.
  • Agarose is a polysaccharide material which is extensively used in DNA electrophoresis, as and tissue engineering. Agarose does not bind nucleic acids and is biocompatible and behaves as an elastic solid and fractures when sufficient shear stress is applied. To leverage these properties, extrusion fragmentation was used to produce agarose microparticles from 1% and 2% wt/vol agarose gels, both of which are solid at 37°C.
  • Agarose microparticles produced by extrusion fragmentation are highly irregular in shape and are widely distributed in diameter, with a mean Feret diameter of 116 pm for 2% wt/vol gels, as can be seen in FIG. 5.
  • the Feret diameter is a measure of an object’s size along a specified direction. In microscopy, it is applied to projections of a three- dimensional (3D) particle on a 2D plane.
  • the fragmentation process yields a large fraction of microparticles which are below this characteristic size, while a smaller population of particles as large as 700 pm are observed.
  • the irregular size and shape of the particles may increase the effective solids loading the ink, possibly resulting in a stiffer ink and a more mechanically robust tissue.
  • the size of agarose microparticles can be adjusted by changing the parameters of the extrusion fragmentation, or by changing the concentration of agarose used to form the bulk gel. Finally, alternative methods of producing microparticles can be used to alter particle geometry or reduce the variation in particle size.
  • HEK293T cells have a diameter of approximately 14 pm in normal culture conditions, which is much smaller than the average size of agarose microparticles.
  • cells occupy about 20% of the total volume of ink and are evenly mixed with agarose microparticles (FIG. 8); higher concentrations of cells may disrupt particleparticle interactions to a greater extent. It is believed the cells reside within the solution phase, i.e., the interstitial space between jammed agarose microparticles.
  • the apparent viscosities and yield stresses of cell-laden inks were measured to understand how microparticle-based inks behave when cells are mixed into the interstitial space.
  • HAMA hyaluronic acid methacrylate
  • LAP Lithium phenyl-(2,4,6- Trimethylbenzoyl) phosphinate
  • HAMA was combined with molten agarose prior to microparticle generation to produce agarose- HAMA composite microparticles which are photopolymerizable after extrusion fragmentation. Varying HAMA concentrations in these bioinks were evaluated on their initial rheological and cured properties. Bioinks made with HAMA concentrations of 0.5%, 0.75%, and 1% wt/vol exhibit similar shear yield stress and shear-thinning behavior (FIGS. 10A-10C). However, upon photopolymerization, bioinks containing 1% HAMA produced a stiffer tissue with a plateau G’ of 16 kPa. Therefore, 1% HAMA was employed in the microparticle formulations moving forward.
  • a customized nozzle with embedded electrodes capable of applying the high intensity electric field pulses needed to drive electroporation was designed and fabricated.
  • cube-shaped voxels are generated in a square channel with two parallel electrodes placed at the outlet of the nozzle to limit pattern distortion by Taylor dispersion.
  • Platinum was chosen as the electrode material due to its electrochemical stability, biocompatibility, and corrosion resistance.
  • the electrodes are driven by two serially connected power amplifiers, which provide a voltage gain of 20 and were chosen to support the peak instantaneous power draw of electroporation.
  • Eq. 1 it is found that the power required to sustain an electroporative pulse at an electric field strength of 100 kV/m is 2.38 W.
  • the pulse repetition rate (PRR) was set so that each voxel experiences a single electroporative pulse; assuming plug flow, the PRR is calculated as:
  • Q(v) is the volumetric extrusion rate
  • U is the volume of the active region defined by the channel profile and electrodes
  • v is the translation speed of the printer
  • I is the width of the electrodes.
  • Typical pulse repetition rates are around 10 Hz, and in practice are limited by the maximum extrusion rate of the syringe extruders and the maximum translation speed of the printer.
  • the channel size and electrode design determine the voxel dimensions. In this electrode geometry, the electric field intensity within the square channel can be approximated by:
  • E the electric field strength
  • V the voltage applied across the electrodes
  • d the distance between the electrodes.
  • Eq. 2 does not account for edge effects and fringing fields that may change the electric field strength.
  • COMSOL simulations were developed to model the electric field strength through the full volume of the nozzle channel, sweeping the applied voltage. It was found that the models agree with Eq. 2 in planes perpendicular to the direction of flow, where the modeled electric field strength deviates from Eq. 2 by less than 5% (evaluated at the median plane) (FIGS. 11 A-l 1C)). Parallel to the direction of flow, the electric field strength drops by up to 30% from Eq.
  • a T is the change in temperature
  • cr is the electrical conductivity of the buffer solution
  • V is the applied voltage
  • r is the electroporation pulse duration
  • p is the density of the media
  • c is the heat capacity of the media
  • d is the width of the channel and electrodes.
  • Our electroporation buffer which has a conductivity of 0.238 ⁇ 0.0381 S/m at 25 °C, is assumed to have the same density and heat capacity as water.
  • a secondary consideration in STEP is the electrolysis of water during the electroporation pulse, which causes pH shifts near the electrodes and produces gas bubbles that may interfere with flow and damage electroporated cells.
  • the rate of electrolysis during the electroporation process is sought to be minimized.
  • the rate of electrolysis is proportional to the current I flowing through the voxel during an electroporative pulse, which is given by:
  • HEK-293T cells were transfected with mRNA encoding enhanced green fluorescent protein (eGFP-mRNA).
  • the pulse duration was held constant to focus on optimizing the electric field strength.
  • the peak condition 100 kV/m applied electric field strength, 5 ms pulse duration
  • greater than 95% of cells were positive for eGFP 24 hours after printing while cell viability was over 80% (FIGS. 15A and 15B).
  • the transfection efficiency is proportional to the strength of the electric field; lower electric field strengths produce lower transfection efficiencies. Cell viability was consistently high across the range of electric field intensities tested. Together, the high transfection efficiency and viability of HEK-293T cells printed with STEP demonstrate its potential for transfecting cells during the tissue fabrication process.
  • hiPSCs requried higher electric field strengths for successful transfection, which arises due to their smaller diameters as compared to HEK-293T cells.
  • Higher electric field strengths are needed to generate equal transmembrane voltages for hiPSCs.
  • 160 kV/m appears to be an upper limit to the applied electric field strength, above which excessive cell lysis prevents accurate measurement of transfection efficiency and viability.
  • the transfection efficiencies and cell viability of hiPSCs in STEP are comparable to those of hiPSCs observed for conventional electroporation protocols.
  • the near-unity transfection efficiency achievable with STEP ensures the transfection of cells within entire voxels during printing. While the transfection efficiency can be optimized simply by tuning the applied electric field strength, it is possible to vary the relative fraction of transfected cells within a given voxel on-the-fly.
  • the nature of STEP-produced gradients was investigated. Two possibilities were hypothesized: first, gene expression could be directly controllable through electric field strength or pulse duration, and cells electroporated at weaker conditions have uniformly lower gene expression than cells electroporated at stronger conditions, forming a true gradient. Alternatively, transfection in STEP could exhibit threshold-like behavior where each voxel is composed of a mix of transfected and non-transfected cells, and the resulting gradient resembles a dithered or halftone grayscale gradient.
  • the increased width of the filament relative to the nozzle dimensions is likely due to the wetting and spreading of the aqueous bioinks on the underlying substrate. Importantly, volumetric flow rate and print speed can be adjusted to achieve precise features. There was evidence of wicking effects along the printed filament, where cells are pulled away during the gel encapsulation process.
  • an H-pattern was designed and printed within an hiPSC tissue (FIG. 22).
  • algorithms were developed that take in a bitmap of the desired pattern and automatically generate printer and electroporator commands.
  • the algorithm uses transfection efficiency data to assign electroporation parameters for each voxel based on the grayscale value for each pixel in the bitmap. 24 hours after printing, robust eGFP expression is seen in the prescribed H-pattern (FIG. 23).
  • the pluripotency markers Oct4 and Sox2 are consistently expressed throughout the tissue in transfected and non-transfected regions, showing that STEP does not affect hiPSC pluripotency.
  • STEP can therefore specify fluorescence intensity in a voxel-wise manner and does not need to produce continuous gradients. This is a major advantage of STEP as compared to other methods of producing mixtures or gradients in direct ink writing, which must extrude continuous gradients between two materials or cell types.
  • STEP’S ability to precisely control a voxel’s intensity profile can be used to produce tissues with mixed cellular populations. Rather than applying different electroporation conditions on a voxel-by-voxel basis, these same electroporation conditions can be applied on a tissue scale to control the fluorescence on a tissue scale. Furthermore, it is possible to accurately predict the proportion of transfected cells within the tissue. As a demonstration, individual tissues composed of a mixture of eGFP expressing hiPSCs and non-fluorescent hiPSCs were printed using electric field strengths between 0 and 140 kV/m (FIG. 25 and FIG. 26).
  • this disclosure describes a new printing technology, STEP, that combines electroporation and direct ink writing to transfect cell-laden bioinks in a voxelwise manner.
  • STEP utilized agarose and hyaluronic acid-based ink formulations that allowed electroporation at high efficiencies. It was shown that HEK-293T cells and hiPSCs can be transfected with mRNA within these ink formulations at extremely high efficiencies. The high transfection efficiency of STEP may enable the fabrication of sophisticated patterns of gene expression within printed tissues. STEP-produced patterns can be improved by incorporating photo-crosslinkable elements within the ink formulation, which lock transfected cells into the desired pattern.
  • the intensity of gene expression within each voxel can be controlled by changing the applied electric field strength, which enables the design and fabrication of graded mixtures of transfected cells throughout a tissue.
  • STEP can be used to rapidly produce mixed tissue compositions from a single homogenous bioink.
  • STEP may enable new genetic approaches to controlling cell and tissue composition within synthetic tissues that can produce the structured, multicellular tissues required for organ repair and replacement.
  • a first aspect relates to a method of printing patterned tissue, the method comprising: flowing a bioink comprising cells and a biological cargo through a nozzle moving relative to a substrate; during the flow of the bioink, exposing selected voxels of the bioink to a pulsed electric field, whereby a portion or all of the cells in each of the selected voxels undergoes electroporation and transfection with the biological cargo; continuously extruding a cell-laden filament from an outlet of the nozzle, the cell-laden filament comprising the bioink and including the selected voxels; and as the nozzle moves relative to the substrate, depositing the cell-laden filament in a predetermined pattern on the substrate, thereby printing a tissue having spatial patterns of gene expression.
  • a second aspect relates to the method of the first aspect, further comprising selecting a strength of the pulsed electric field and/or a pulse duration to control the portion of the cells that undergoes electroporation and transfection in each of the selected voxels.
  • a third aspect relates to the method of any preceding aspect, wherein, for each of the selected voxels, the strength of the pulsed electric field is selected to be from 10 kV/m to 160 kV/m.
  • a fourth aspect relates to the method of any preceding aspect, wherein the pulse duration is selected to be from 50 ps to 50 ms.
  • a fifth aspect relates to the method of any preceding aspect, wherein the portion of the cells that undergoes electroporation and transfection in each of the selected voxels is in a range from greater than 0 to 1.0.
  • a sixth aspect relates to the method of any preceding aspect, where the selected voxels of the bioink are immediately adjacent to each other along a flow direction of the bioink.
  • a seventh aspect relates to the method of any preceding aspect, wherein the selected voxels are interspersed with, along a flow direction of the bioink, one or more voxels not exposed to the pulsed electric field.
  • An eighth aspect relates to the method of any preceding aspect, wherein the pulsed electric field is applied substantially perpendicular to a flow direction of the bioink.
  • a ninth aspect relates to the method of any preceding aspect, wherein the pulsed electric field is applied by a first electrode and a second electrode spaced apart from each other and positioned to contact the bioink flowing through the nozzle.
  • a tenth aspect relates to the method of any preceding aspect, wherein the pulsed electric field is applied by a first electrode and a second electrode spaced apart from each other and overlaid with a dielectric material so as not to contact the bioink flowing through the nozzle.
  • An eleventh aspect relates to the method of any preceding aspect further comprising controlling: a volumetric flow rate of the bioink through the nozzle, the movement of the nozzle relative to the substrate, and/or a pulse repetition rate, such that each of the cells of the bioink is exposed to at most a single pulse of the pulsed electric field.
  • a twelfth aspect relates to the method of any preceding aspect, wherein the cells comprise any human cells.
  • a thirteenth aspect relates to the method of any preceding aspect wherein a cell concentration in the bioink is in a range from 1 x 10 6 cells/ml to 500 x 10 6 cells/ml.
  • a fourteenth aspect relates to the method of any preceding aspect, wherein the bioink is shear- thinning.
  • a fifteenth aspect relates to the method of any preceding aspect, wherein the bioink further comprises a polymer.
  • a sixteenth aspect relates to the method of the preceding aspect, wherein the polymer comprises a natural or synthetic polymer selected from the group consisting of: agarose, hyaluronic acid methacrylate (HAMA), collagen, fibrinogen, reconstituted extracellular matrix, a modified matrix-derived protein, gelatin, a modified glycosaminoglycan, hyaluronic acid, chondroitin sulfate, a modified polysaccharide, alginate, dextran, chitosan, and polyethylene glycol.
  • HAMA hyaluronic acid methacrylate
  • a seventeenth aspect relates to the method of any preceding aspect, wherein the polymer has the form of microparticles in the bioink.
  • An eighteenth aspect relates to the method of any preceding aspect, wherein the biological cargo comprises a nucleic acid, a protein, or another complex molecule.
  • a nineteenth aspect relates to the method of any preceding aspect, wherein the biological cargo includes at least two cargo species controllably introduced into the bioink so as to be present in different voxels.
  • a twentieth aspect relates to the method of any preceding aspect, wherein the biological cargo includes at least two cargo species controllably introduced into the bioink so as to be present in the same voxels.
  • a twenty-first aspect relates to the method of any preceding aspect, wherein the bioink further comprises a crosslinking initiator.
  • a twenty-second aspect relates to the method of any preceding aspect, wherein the bioink further comprises an apoptosis inhibitor.
  • a twenty-third aspect relates to the method of any preceding aspect, wherein the bioink further comprises an electroporation buffer.
  • a twenty-fourth aspect relates to the method of any preceding aspect, further comprising, after printing the tissue, casting a pre-gel solution over the tissue and curing the pre-gel solution, thereby forming a gel-encapsulated tissue.
  • a twenty-fifth aspect relates to the method of any preceding aspect, wherein the bioink further comprises a polymer, and further comprising, after printing the tissue, exposing the tissue to ultraviolet light, thereby crosslinking the polymer.
  • a twenty-sixth aspect relates to a printhead for electroporating cells, the printhead comprising: a nozzle enclosing a channel extending from a channel inlet to a channel outlet; and a first electrode and a second electrode configured to apply a pulsed electric field to a bioink flowing through the channel, the first and second electrodes being spaced apart from each other between the nozzle inlet and the nozzle outlet.
  • a twenty-seventh aspect relates to the printhead of the preceding aspect, wherein the first electrode and the second electrode are positioned in opposition to each other on a channel wall or on opposing channel walls.
  • a twenty-eighth aspect relates to the printhead of any preceding aspect, wherein the first electrode and the second electrode are positioned adjacent to each other on a channel wall or on adjacent channel walls.
  • a twenty-ninth aspect relates to the printhead of any preceding aspect, wherein the first electrode and the second electrode are positioned in parallel with each other.
  • a thirtieth aspect relates to the printhead of any preceding aspect, wherein the first electrode and the second electrode are positioned in a coaxial or concentric arrangement.
  • a thirty-first aspect relates to the printhead of any preceding aspect, wherein the first electrode and the second electrode are positioned to come into direct contact with the bioink flowing through the channel.
  • a thirty-second aspect relates to the printhead of any preceding aspect, wherein a dielectric material overlies the first and second electrodes, the first electrode and the second electrode being positioned to avoid direct contact with the bioink flowing through the channel.
  • a thirty-third aspect relates to the printhead of any preceding aspect, wherein the channel has a circular, oval, polygonal or irregular transverse cross-section.
  • a thirty-fourth aspect relates to the printhead of any preceding aspect, wherein the channel has a rectangular and/or square transverse cross-section.
  • a thirty-fifth aspect relates to the printhead of any preceding aspect, wherein the nozzle is formed by 3D printing.
  • Electrodes are constructed from a metal foil or strip.
  • a thirty-seventh aspect relates to an electroporation system comprising: the printhead of any preceding aspect; an ink dispenser in fluid communication with the channel inlet; a substrate facing the channel outlet; a multi-axis motion controller configured to move the printhead and/or the substrate; and a voltage source electrically connected to the first and second electrodes.
  • a thirty-eighth aspect relates to the electroporation system of any preceding aspect, wherein the voltage source comprises one or more power amplifiers.
  • a thirty-ninth aspect relates to the electroporation system of any preceding aspect, wherein an arbitrary waveform generator is configured to drive the voltage source.
  • the phrases mean any combination of one or more of the elements A, B, ... or N including any one element alone or the one element in combination with one or more of the other elements which may also include, in combination, additional elements not listed.
  • "a” or “an” means “at least one” or “one or more.”

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  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Un procédé d'impression de tissu à motifs consiste à faire couler une bio-encre comprenant des cellules et un chargement biologique à travers une buse se déplaçant par rapport à un substrat et exposer des voxels sélectionnés de la bio-encre à un champ électrique pulsé lorsque la bio-encre s'écoule à travers la buse. Par conséquent, une partie ou la totalité des cellules dans chacun des voxels sélectionnés subit une électroporation et une transfection avec le chargement biologique. Un filament chargé de cellules comprenant la bio-encre et comprenant les voxels sélectionnés est extrudé en continu à partir d'une sortie de la buse, et, lorsque la buse se déplace par rapport au substrat, le filament chargé de cellules est déposé selon un motif prédéterminé sur le substrat. Ainsi, un tissu ayant des motifs spatiaux d'expression génique peut être imprimé.
PCT/US2024/024052 2023-04-14 2024-04-11 Tête d'impression et système d'électroporation, et procédé d'impression de tissu à motifs Pending WO2024228814A2 (fr)

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WO2009011709A1 (fr) * 2007-07-19 2009-01-22 The Board Of Trustees Of The University Of Illinois Impression par jet électrohydrodynamique à haute résolution pour des systèmes de fabrication
KR20160125530A (ko) * 2010-10-21 2016-10-31 오가노보, 인크. 조직의 제작을 위한 디바이스, 시스템 및 방법
CN111826262B (zh) * 2019-04-18 2023-06-20 深圳先进技术研究院 生物3d打印系统与方法
EP3739609B1 (fr) * 2019-05-14 2024-09-18 Hitachi Energy Ltd Buse pour un disjoncteur, disjoncteur et procédé d'impression 3d d'une buse d'un disjoncteur
KR102308151B1 (ko) * 2020-06-04 2021-10-06 주식회사 로킷헬스케어 바이오 3차원 프린터의 시린지 출력 제어 장치 및 방법
US11715866B2 (en) * 2021-05-18 2023-08-01 GM Global Technology Operations LLC Method of forming edge materials on electrochemical cell component

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