EP4694944A2 - Printhead and system for electroporation, and method of printing patterned tissue - Google Patents

Abstract

A method of printing patterned tissue includes flowing a bioink comprising cells and a biological cargo through a nozzle moving relative to a substrate and exposing selected voxels of the bioink to a pulsed electric field as the bioink flows through the nozzle. Consequently, a portion or all of the cells in each of the selected voxels undergoes electroporation and transfection with the biological cargo. A cell-laden filament comprising the bioink and including the selected voxels is continuously extruded from an outlet of the nozzle, and, as the nozzle moves relative to the substrate, the cell-laden filament is deposited in a predetermined pattern on the substrate. Thus, a tissue having spatial patterns of gene expression may be printed.

Description

PRINTHEAD AND SYSTEM FOR ELECTROPORATION, AND METHOD OF PRINTING PATTERNED TISSUE

RELATED APPLICATION

[0001] The present patent document claims the benefit of priority under 35 U.S.C. 119(e) to U.S. Provisional Patent Application No. 63/496,296, filed on April 14, 2023, and hereby incorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under MH123977 awarded by National Institutes of Health (NIH) and under N00014-21- 1-2958 awarded by U.S. Office of Naval Research (NAVY/ONR). The government has certain rights in this invention.

TECHNICAL FIELD

[0003] The present disclosure relates generally to genetic engineering of cells and more particularly to a method of printing patterned tissue.

BACKGROUND

[0004] Genetically engineered cells and tissues promise to revolutionize our ability to pattern human tissues. Genetic approaches to cell differentiation provide rapid and efficient methods to control cell phenotype. Transcription factor-driven differentiation can directly reprogram human induced pluripotent stem cells (hiPSCs) into specific cell types such as endothelium, neurons, fibroblasts, and cardiomyocytes. Furthermore, potent morphogens such as sonic hedgehog (SHH) have been used to induce dorsal-ventral polarization of cell types within developing brain organoids. 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. Notably, these protocols differentiate cells in a matter of days while conventional differentiation protocols may take weeks to produce the desired cell type.

[0005] Electroporation techniques, where a high intensity electric field creates pores or openings in cell membranes, are being explored for intracellular gene delivery. However, current electroporation methods are limited in scope and controllability.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1 is a schematic of an electroporation system and printhead (in two different positions) during selective transfection via electroporative printing (STEP).

[0007] FIGS. 2A and 2B show close-up schematics of the printhead during printing, where a pulsed electric field is applied in FIG. 2B.

[0008] FIGS. 3A-3C are schematics of exemplary cell-laden filaments after exposure of selected voxels to a pulsed electric field.

[0009] FIG. 4 is a cross-sectional schematic of part of an exemplary nozzle with coaxial electrodes.

[0010] FIG. 5 shows a histogram of agarose microparticle sizes (Feret diameters).

[0011] FIG. 6 shows apparent viscosity as a function of applied shear rate for 1% and

2% wt/vol jammed agarose microparticle inks.

[0012] 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.

[0013] 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 lOOxlO⁶ cells/ml; scale bar is 200 pm.

[0014] FIG. 9A shows apparent viscosity of agarose microparticle inks loaded with 0, 10, 50, 100, and 500xl0⁶ cells/ml.

[0015] FIG. 9B shows storage (closed circles) and loss moduli (open circles) of agarose microparticle inks loaded with 0, 10, 50, 100, and 500x10⁶ cells/ml.

[0016] 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).

[0017] 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.

[0018] 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.

[0019] 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.

[0020] FIG. 14 shows transfection efficiency of B JFF-hiPSCs in 1% and 2% agarose microparticle inks; BJFF-hiPSCs were transfected with 100 kV/m, 5 ms pulses, n = 3 biological replicates, **p = 0.0031.

[0021] FIGS. 15A and 15B show transfection efficiency and cell viability of HEK- 293T cells in 1% agarose microparticle inks, in particular, transfection efficiency of HEK-293T cells printed with STEP at 0-100 kV/m (FIG. 15A) and viability of HEK- 293T cells printed with STEP at different electric field strengths (FIG. 15B); n = 3 biological replicates.

[0022] FIGS. 16A and 16B show transfection efficiency and cell viability as a function of electric field strength in STEP-printed hiPSCs, in particular, transfection efficiency of hiPSCs printed with STEP at 0-160 kV/m with 5 ms pulses (FIG. 16A) and viability of hiPSCs printed with STEP at different electric field strengths (FIG. 16B); n = 3 biological replicates.

[0023] FIGS. 17A and 17B show transfection efficiency and viability as a function of pulse length in STEP-printed hiPSCs, in particular, transfection efficiency of hiPSCs printed with STEP at 100 kV/m with 0-5 ms pulses (FIG. 17A), and viability of hiPSCs printed with STEP at different pulse lengths (FIG. 17B); n = 3 biological replicates.

[0024] 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. [0025] FIG. 19A shows efficiency of hiPSCs as a function of applied electric field strength in polymerized composite HAMA-agarose tissues.

[0026] FIG. 19B shows viability of hiPSCs as a function of applied electric field strength in polymerized composite HAMA-agarose tissues.

[0027] 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.

[0028] FIG. 20B shows diameters of filaments printed with a 1 mm² square outlet nozzle.

[0029] FIG. 21 shows FWHM of eGFP fluorescence profiles of a single electroporation pulse applied to agarose and HAMA-agarose inks loaded with hiPSCs; *p = 0.025.

[0030] FIG. 22 illustrates image mapping to electroporation parameters and assignment of the print path.

[0031] FIG. 23 shows an immunofluorescence image of eGFP in a photo-crosslinked hiPSC tissue printed using STEP; scalebar: 1 mm.

[0032] FIG. 24 shows fluorescence intensity profiles of eGFP and DAPI within HEK- 293T filament, with the applied electric field profile.

[0033] 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.

[0034] FIG. 26 shows mean fluorescence intensity of STEP-printed tissues as a function of the applied electric field strength.

DETAILED DESCRIPTION OF THE DRAWINGS

[0035] 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. Importantly, 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. Also described in this disclosure is a new electroporative printhead designed to apply strong electric fields to a bioink during extrusion through a nozzle to carry out the STEP process.

[0036] Referring to FIG. 1 and FIGS. 2A-2B, 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.

[0037] As the bioink 102 flows through the nozzle 108, successive voxels 112 of the bioink 102 are selectively exposed to a pulsed electric field, as illustrated in FIG. 2B. In other words, one voxel, some voxels, or all voxels 112 of the bioink 102 are exposed to the pulsed electric field during flow through the nozzle 108. The voxel(s) selectively exposed to the pulsed electric field may be referred to as “selected voxel(s).” In FIG. 1, 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. During exposure of a selected voxel 112 to the pulsed electric field, at least a portion of the cells 104 in the voxel 112 undergoes electroporation and transfection with the biological cargo. 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).

[0038] 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. Thus, 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.

[0039] Referring to FIGS. 2 A and 2B, 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. 2A and 2B, such that a faradaic current is produced upon application of the pulsed electric field. In other examples, 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.

[0040] Due to the selectivity inherent to the electroporation process, 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. For example, if all (or a consecutive grouping of) the voxels 112 of the bioink 102 are exposed to the pulsed electric field, then 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. Alternatively, 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. [0041] Experiments described below demonstrate that the number or proportion of cells that undergo electroporation and transfection in a given voxel, that is, the transfection efficiency, may be increased by increasing the strength of the pulsed electric field. An increased pulse duration may also improve transfection efficiency. More generally speaking, by controlling the strength of the pulsed electric field and/or the pulse duration, the number or proportion of cells that undergo electroporation and transfection in a given voxel may be manipulated. Accordingly, 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%). Also or alternatively, 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.

[0042] The bioink employed for the method (STEP) 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.

[0043] The polymer may be a natural or synthetic polymer. For example, 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. 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. In some examples, the polymer may undergo crosslinking during or after printing.

[0044] The cells may comprise any human cells. For example, 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. The term “germ cells” refers to any line of cells that give rise to gametes (eggs and sperm). The term “somatic 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. Examples of somatic cells 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). The term “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. Examples of stem and progenitor cells 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. The cells may also or alternatively comprise tumor or cancer cells, such as carcinoma, sarcoma, leukemia, lymphoma, melanoma, and/or multiple myeloma cells. Typically, the bioink includes the cells at a cell concentration in a range from 1 x 10⁶ cells/ml to 500 x 10⁶ cells/ml. [0045] The biological cargo, as indicated above, comprise a nucleic acid, e.g., plasmid DNA (pDNA) and/or messenger RNA (mRNA), and/or a protein. In some examples, 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. Preferably, 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. Also or alternatively, 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.

[0046] The method may further include, after printing the tissue, casting a pre-gel solution over the tissue and curing the pre-gel solution. For example, 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. By casting a pre-gel solution containing fibrinogen and thrombin over the printed tissue, it is possible to circumvent fibrinogen-induced cell death and to protect the printed filaments for extended culture. 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.

[0047] Now that the method has been explained, a printhead designed for electroporating cells during extrusion-based printing, or direct ink writing, is described. Referring to FIGS. 1 and FIGS. 2A-2B, 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. For example, the channel 118 may have a rectangular and/or square transverse cross-section.

[0048] 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. For example, as shown in FIGS. 2 A and 2B, 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). Alternatively, the first electrode and the second electrode may be positioned adjacent to each other on adjacent channel walls or on one channel wall. In another example, the first electrode 130 and the second electrode 132 may be positioned in a coaxial or concentric arrangement, as shown in FIG. 4. Also or alternatively, the 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.

[0049] 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.

[0050] Referring again to FIG. 1, 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. The ink dispenser 134 may be a syringe extruder, for example, which may be controlled by an Arduino microcontroller to ensure that the bioink 102 is being dispensed into the channel inlet 120 at the desired flow rate. For the purposes of this disclosure, 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. Accordingly, 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.

[0051] In the examples below, the nozzle has a channel outlet with a cross-sectional area of 1 mm² and a square cross-section. Generally speaking, the cross-sectional area of the channel outlet may lie in a range from about 0.25 mm² to about 9 mm². To carry out the selective transfection described above, 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. As indicated above, 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.

[0052] EXAMPLES

[0053] In the examples described below, 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.

[0054] Methods

[0055] Electroporation buffer formulation

[0056] Electroporation buffer (EP 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)-! -piperazineethanesulfonic acid (HEPES, MilliporeSigma, #H4034), 150 mM sucrose, and 1% vol/vol dimethyl sulfoxide in deionized water. The EP buffer is filter sterilized through a 0.2 pm filter and stored at 4°C before use. [0057] Cell culture

[0058] 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). For passaging, cells were rinsed with Dulbecco’s phosphate buffered saline without calcium or magnesium (DPBS-/-, Corning #20-031-CV), dissociated with ReLeSR (STEMCELL Technologies, #100- 0483), and replated in mTeSRl supplemented with 5 pM Y27632 (Biogems, #1293823) for 1 day, then subsequently cultured in mTeSRl with daily media changes.

[0059] 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.

[0060] Microparticle generation

[0061] 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.

[0062] 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. 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.

[0063] mRNA synthesis

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Bioinks

[0068] 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 survival¹⁴⁴: 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 lOOxlO⁶ 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. 500 pg/ml 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.

[0069] 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.

[0070] 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.

[0071] Rheological measurements

[0072] 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.

[0073] 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.

[0074] Fluorescent labeling of cell-laden inks

[0075] To evaluate the distribution of cells within an ink, 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. [0076] Electroporative printheads

[0077] Printheads are designed using SOLIDWORKS™ (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.

[0078] 3D bioprinting

[0079] 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 Arduino 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.

[0080] 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. [0081] Gel encapsulation of printed human tissues

[0082] 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. 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.

[0083] Flow cytometry

[0084] 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. Cells are rinsed once in Dulbecco’s phosphate buffered saline with calcium and magnesium (DPBS+/+, Corning, #20-030- CV), then stained with a far-red cell viability stain (Invitrogen, #L34973) protected from light for 15 min at room temperature. Negative viability controls are generated by killing non-transfected cells with a 5-15 min heat treatment at 65°C. Cells are then fixed in BD Cytofix (BD Biosciences, #554714) for 15 min at 4°C and rinsed three times with DPBS+/+ with 3% wt/vol bovine serum albumin (BSA, MilliporeSigma, #10735078001). Flow cytometry was performed on a Cytek Aurora for all experiments. Single stain controls were run to set detector voltages and gating criteria. [0085] Immunostaining

[0086] 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.

Table 2: Secondary antibodies for immuno staining. [0087] Image analysis of STEP resolution

[0088] 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.

[0089] Results and Discussion

[0090] STEP Bioinks

[0091] First, an 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.

[0092] The rheological behavior of these agarose microparticle-based inks was measured to identify the appropriate agarose concentrations for further use. Both inks exhibited similar shear-thinning behavior; however, inks made with 2% agarose microparticles were stiffer and possessed a lower yield stress (FIGS. 6 and 7). Next, human cells were incorporated into microparticle-based inks. HEK-293T cells were combined with agarose microparticles at concentrations of 0 (acellular), 10, 50, and lOOxlO⁶ cells/ml by jamming microparticles and cells together through centrifugation. HEK293T cells have a diameter of approximately 14 pm in normal culture conditions, which is much smaller than the average size of agarose microparticles. At lOOxlO⁶ cells/ml, 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. Notably, these values are unaffected by high cell concentrations up to lOOxlO⁶ cells/ml. By contrast, the purely cell-based ink, which contains approximately 500x10⁶ cells/ml, exhibits lower G’, G”, r_y, and | than agarose microparticle inks (FIGS. 9A and 9B). It is believed that particle- particle interactions still dominate rheological behavior at cell concentrations below lOOxlO⁶ cells/ml.

[0093] A second set of bioinks containing hyaluronic acid methacrylate (HAMA), which is photocros slinkable and is negatively charged at physiological pH, i.e., it repels negatively charged nucleic acids, was generated. Lithium phenyl-(2,4,6- Trimethylbenzoyl) phosphinate (LAP) is used as a photoinitiator due to its relatively high solubility in water and biocompatibility. Directly adding HAMA to the solution phase of the bioinks led to the non-uniform distribution of these species and subsequent differences in tissue stiffness upon photopolymerization. To mitigate this, the focus was on incorporating HAMA within agarose microparticles themselves. Specifically, 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.

[0094] STEP printheads

[0095] A customized nozzle with embedded electrodes capable of applying the high intensity electric field pulses needed to drive electroporation was designed and fabricated. During printing, 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. Using 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. It was verified that the STEP amplifiers produce accurate pulses with minimal sag during 5 ms pulses across the range of input voltages tested (FIG. 12). The amplifiers used to drive electroporation have a bandwidth of 16 kHz, which imposes a minimum pulse duration of 62.5 ps. The pulse repetition rate (PRR) was set so that each voxel experiences a single electroporative pulse; assuming plug flow, the PRR is calculated as:

[0097] where 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 and 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. [0098] 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:

[0099] E = V/d Eq. 2

[00100] where E is the electric field strength, V is the voltage applied across the electrodes, and d is the distance between the electrodes. However, Eq. 2 does not account for edge effects and fringing fields that may change the electric field strength. To account for these effects, 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. 2 within the volume defined by the electrode area and channel walls. The electric field is well-confined to the voxel volume across the full range of applied voltages. Edge effects produce extremely high electric field strengths at the electrode edges, but these are confined to a small proportion of the total voxel volume. Taken together, these results show that the majority of the voxel volume experiences an even electric field strength during an electroporative pulse.

[00101] During STEP, Joule heating effects are limited within the nozzle to minimize cell death. Assuming a cubic voxel volume and adiabatic conditions, where the width of the channel is equal to the electrode width, the increase in temperature within the electroporated voxel is given by:

[00103] where 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, and 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. For typical electroporation pulse parameters (V/d = 100 kV/m, r = 5 ms, 1 pulse per cell) applied to a 1 mm wide nozzle, a modest temperature increase of 2.8 K is observed (FIG. 13). Using smaller channel and electrode geometries allows the use of lower voltages to generate similar electric field strengths.

[00104] 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:

[00105] I = Vad Eq. 4

[00106] Current is proportional to the distance between electrodes, indicating that larger nozzle electrodes result in a higher rate of electrolysis. However, electrolysis only occurs at the electrode surfaces, indicating that a lower proportion of the total voxel volume is affected by electrolysis in larger electrodes, as the surface area to volume ratio is lower for larger nozzle geometries. Using Faraday’s law of electrolysis, it is found that a 100 kV/m, 5 ms electroporative pulse in 1 mm-wide electrodes (voxel volume: 1 pl) produces 1.73xl0'³ pl of total gas which occupies 0.17% of the total voxel volume, while the same pulse parameters in 0.5 mm-wide electrodes (voxel volume: 0.25 pl) produces 1.19xl0'³ pl of total gas which occupies 0.48% of the total voxel volume. Therefore, in larger electrodes, electrolysis affects a lower proportion of cells within a given voxel.

[00107] Taking into account pattern resolution, power supply requirements, and manufactur ability, a decision was made to proceed with nozzles with 1 mm² square crosssections with 1 mm-wide electrodes, which could be robustly assembled without electrical short circuits or fluidic leaks. The resolution of STEP-produced patterns can be increased by reducing the nozzle outlet area and electrode dimensions to shrink the voxel. The microparticle-based ink formulations limit the minimum nozzle area, as the outlet must be several times larger than the particle diameter for consistent extrusion. Nozzles with outlets of 500 pm or larger were designed, as nozzle diameters are preferably 2-3 times larger than the mean diameter of the microparticles to avoid clogging and discontinuous printing. From FIG. 5, it can be seen a minority of particles that are much larger than average, which may require even larger nozzle diameters for extrusion. [00108] Transfection efficiency and cell viability in STEP

[00109] After identifying an ink formulation and nozzle design, the next step was characterizing the performance of STEP in transfecting cell-laden filaments. 1% and 2% wt/vol agarose-microparticle inks with lOOxlO⁶ BJFF-hiPSCs/ml and 100 pg/ml eGFP- mRNA were formulated so that eGFP expressing cells could be quantified using flow cytometry. Printing both ink formulations and transfecting cells with 100 kV/m, 5 ms pulses, it was found that BJFF-hiPSCs transfected significantly more efficiently in 1% agarose microparticle inks (FIG. 14). It is hypothesized that the higher agarose content in 2% microparticle inks inhibits mRNA electrophoresis and leads to lower transfection efficiency. Thus, 1% agarose microparticle formulations were used.

[00110] To optimize transfection protocols, HEK-293T cells were transfected with mRNA encoding enhanced green fluorescent protein (eGFP-mRNA). In the initial experiments with HEK-293T cells, the pulse duration was held constant to focus on optimizing the electric field strength. At 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.

[00111] Next, STEP is demonstrated to be compatible with printing and transfecting hiPSC-based bioinks. Initially, it was found that hiPSCs experienced near total cell death during STEP. To mitigate this, an apoptosis inhibitor, e.g., Y27632 or Chroman 1, was added to prevent cell death during transfection. The presence of an inhibitor dramatically improved cell survival during STEP, allowing hiPSCs to be transfected with 95% efficiency while maintaining cell viabilities in excess of 80% (FIGS. 16A and 16B). Akin to HEK-293T cells, the transfection efficiency of hiPSCs increased with the applied electric field strength, while viability remained consistently high across all field strengths tested. 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. Importantly, it was found that the transfection efficiencies and cell viability of hiPSCs in STEP are comparable to those of hiPSCs observed for conventional electroporation protocols. These findings suggest that STEP is likely compatible with a broad range of cell types for which electroporation protocols have been reported.

[00112] To explore the effect of pulse duration on gene expression in STEP-printed hiPSCs, the electric field strength was held at 100 kV/m and the transfection efficiency and cell viability of hiPSCs electroporated at pulse durations between 0 and 5 ms was systemically investigated. As expected, cell viability was consistently high ( >80%) across the range of pulse durations tested (FIGS. 17A and 17B). Interestingly, transfection efficiency appears to linearly increase within the range of pulse durations tested, as compared to its exponential scaling with electric field strength.

[00113] 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.

[00114] From the flow cytometry data, it is found that STEP produces both transfected and non-transfected cells. These populations mix to control the proportion of transfected cells within a printed voxel. The median fluorescence intensity (MFI) of transfected hiPSCs increases with both electric field strength and pulse duration (FIGS. 18A and 18B). Using regression analysis to fit both parameters to MFI and transfection efficiency, it was found that both electric field strength and pulse duration are weakly predictive of MFI (r² = 0.41 for electric field strength, r² = 0.47 for pulse duration) but strongly predictive of transfection efficiency (r² = 0.70 for electric field strength, r² = 0.77 for pulse duration). Hence, it was concluded that dithered gradients composed of transfected and non-transfected cells are produced by STEP, rather than smooth gradients. Gradients were generated by tuning the electric field strength, since the linear relationship between pulse duration and transfection efficiency is expected to fail at pulse durations shorter than 1 ms. Exceptionally high electric fields are expected to be required for successful electroporation under those conditions, which make producing lower ranges of a gradient difficult. The results show that STEP can transfect multiple cell-laden filaments at high efficiency and cell viability and demonstrate the potential of STEP to create spatial patterns of gene expression within printed tissues. This has interesting applications in controlling cell phenotypes within a voxel, and in creating gradients of gene expression across an entire printed tissue.

[00115] While eGFP mRNA enables rapid and efficient characterization of transfection efficiency in STEP, its compatibility with other mRNA-encoded proteins is central to this platform technology. Bioinks were therefore formulated with mRNA encoding ETV2, a transcription factor that has been used to direct hiPSC differentiation into endothelial cells. After STEP, hiPSCs were replated onto Matrigel coated plates for 2D differentiation and robust ETV2 expression after 24 h that increases with the applied electric field strength was observed. In the highest field conditions (100 and 120 kV/m), there is strong co-localization between DAPI-labeled cell nuclei and ETV2 protein, suggesting an extremely high transfection efficiency with ETV2 mRNA. However, cells stop expressing ETV2 roughly 48 hours after printing, consistent with the short half-life of mRNA expression. Furthermore, only a low relative fraction of these cells expressed the endothelial cell marker CD31, suggesting that the mRNA-directed differentiation is inefficient or is missing crucial cofactors.

[00116] The performance of agarose-HAMA composite microparticles in STEP was then characterized. A maximum transfection efficiency of 77.2 ± 6.81 % was achieved using 140 kV/m, 5 ms pulses to transfect cells within the HAMA ink (FIGS. 19A and 19B). However, cell viability is considerably lower for the HAMA inks across the entire range of electric field strengths tested, and decreases slightly as the electric field strength increases - a trend not seen in our data with neat agarose inks. Neither UV exposure nor the mere presence of HAMA in the ink formulation causes the same decrease in cell viability, suggesting that the decreased cell viability could be a result of mechanical confinement within the polymerized ink, which is stiffer than extracellular matrices used in other hiPSC-derived tissues. Cell viability could be potentially improved by adding diluents, such as non-methacrylated hyaluronic acid, or by reducing the number of reactive methacrylate groups on the polymer; both of these approaches would result in a softer polymerized tissue that may result in less cell death. hiPSCs may also be particularly sensitive to mechanical confinement, and it is possible other cell types may tolerate the stiff matrix conditions in the bioink. However, lower cell viabilities can be overcome by increasing the cell concentration in the bioink such that the final viable cell yield in the tissue is satisfactory. These results demonstrate that multiple cell types can be printed with STEP with high transfection efficiency and viability.

[00117] Human tissue printing via STEP

[00118] Next, the fidelity of patterns printed with STEP was characterized. Using the neat agarose microparticle formulations, cell-laden filaments printed and then encapsulated in a gelatin-fibrin gelbrin hydrogel. This separates the crosslinkable proteins from the electroporation process, where they could interfere with transfection by binding nucleic acids. In particular, the presence of fibrinogen at typical concentrations used in tissue engineering (1-10 mg/ml) causes total cell death during electroporation. By casting a pre-gel solution containing fibrinogen and thrombin over the printed filaments, it was possible to circumvent fibrinogen- induced cell death and protect printed filaments for extended culture.

[00119] The resolution of a single electroporative pulse applied to hiPSC laden filaments printed with a nozzle containing 1 mm-wide electrodes and a 1 mm² square outlet was measured while controlling the volumetric flow rates and print speeds to ensure that each voxel has a total volume of 1 pl. Measuring the full width half-maximum (FWHM) of the eGFP fluorescence intensity profile 24 hours after printing, it was found that the minimum length of a single pulse is 1242 ± 45.85 pm, while the mean filament diameter is 1110 + 24.42 pm (FIGS. 20A and 20B). Given that the electric field profile extends beyond the electrode, it is not surprising that the actual voxel dimensions exceed the target value of 1 mm. 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.

[00120] The ability of STEP to pattern two distinct voxels within a single filament was evaluated. Applying pulses to two adjacent voxels produces consistent eGFP expression across both voxels, with no apparent drop in intensity. Inserting a single non-transfected (E - kV/m) voxel between two transfected voxels yields a cleanly switching pattern along the printing direction. As previously stated, the resolution is primarily determined by the size of the filament and electrodes and can be adjusted to print patterns at arbitrary resolutions. Finer patterns can be printed with smaller nozzles and electrode geometries but must be printed at lower feed rates and longer print times.

[00121] To pattern tissues with increased complexity, STEP was used to print spacefilling curves that reduce the need for repeated starts and stops. Third-order Hilbert curves were first printed using 1% agarose microparticles, and transfecting portions of the filament with eGFP mRNA. Immediately after printing, the printed tissue was encapsulated with a gelatin-fibrin gel to support extended culture. After 24 hours, robust eGFP expression was observed in transfected regions of the STEP tissue. However, the transition between transfected and non-transfected segments of the filament is disrupted by cells which were displaced during the gel encapsulation process. After 4 days of culture, printed hiPSC filaments show lower and uneven eGFP expression through the printed filament, a hallmark of mRNA-mediated gene expression. Since agarose does not support cell attachment or adhesion, the STEP tissue is extremely fragile. Hence, the focus shifted to developing photo-crosslinkable bioinks to overcome these limitations.

[00122] The resolution of single electroporative pulses applied to cells within the composite HAMA-agarose ink, which can be photo-polymerized to form solid tissues, was measured. It was found that single pulses in the agarose-HAMA composite ink produce voxel lengths of 1083 ± 37.8 pm (FIG. 21), which are significantly lower than the voxel lengths in native agarose inks (*p = 0.025). The removal of additional processing steps, such as gel encapsulation, eliminates capillary forces which could displace cells and negatively impact resolution and improves pattern fidelity.

[00123] To demonstrate the patterning capabilities of STEP with photo-polymerizable inks, an H-pattern was designed and printed within an hiPSC tissue (FIG. 22). To simplify print path planning, 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.

[00124] Finally, to demonstrate the gradient printing capabilities of STEP, gradient HEK-293T filaments were printed. Within a gradient filament, the eGFP fluorescence intensity profile tracks well with the electric field strength prescribed to each voxel (FIG. 24). The change in fluorescence intensity within a single voxel arises from a variable number of transfected cells within a voxel, which are evenly distributed throughout the voxel with non-transfected cells. At the scale of individual voxels, the variable number of transfected cells results in an apparent change in fluorescence intensity. Furthermore, clean switching from non-transfected to transfected voxels at the start of the filament is observed, indicating that STEP-produced gradients have no switching delays or defects. 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.

[00125] 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). As expected, fluorescence intensity increases with the electric field strength, which correlates with the proportion of eGFP expressing cells. Altogether, these results demonstrate the ability of STEP to control transfection efficiency on a voxel-by-voxel basis, which can be used to control gene expression across entire tissues.

[00126] In conclusion, this disclosure describes a new printing technology, STEP, that combines electroporation and direct ink writing to transfect cell-laden bioinks in a voxelwise manner. In the above examples, 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. Furthermore, 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. As such, STEP can be used to rapidly produce mixed tissue compositions from a single homogenous bioink. By linking genetic engineering tools with biomanufacturing technologies, 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.

[00127] The subject matter of this disclosure may also relate to the following aspects: [00128] 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.

[00129] 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.

[00130] 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.

[00131] 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.

[00132] 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.

[00133] 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.

[00134] 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.

[00135] 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.

[00136] 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.

[00137] 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. [00138] 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.

[00139] A twelfth aspect relates to the method of any preceding aspect, wherein the cells comprise any human cells.

[00140] 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 10⁶ cells/ml to 500 x 10⁶ cells/ml.

[00141] A fourteenth aspect relates to the method of any preceding aspect, wherein the bioink is shear- thinning.

[00142] A fifteenth aspect relates to the method of any preceding aspect, wherein the bioink further comprises a polymer.

[00143] 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.

[00144] A seventeenth aspect relates to the method of any preceding aspect, wherein the polymer has the form of microparticles in the bioink.

[00145] 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.

[00146] 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.

[00147] 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.

[00148] A twenty-first aspect relates to the method of any preceding aspect, wherein the bioink further comprises a crosslinking initiator. [00149] A twenty-second aspect relates to the method of any preceding aspect, wherein the bioink further comprises an apoptosis inhibitor.

[00150] A twenty-third aspect relates to the method of any preceding aspect, wherein the bioink further comprises an electroporation buffer.

[00151] 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.

[00152] 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.

[00153] 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.

[00154] 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.

[00155] 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.

[00156] 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.

[00157] 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.

[00158] 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.

[00159] 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.

[00160] 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.

[00161] A thirty-fourth aspect relates to the printhead of any preceding aspect, wherein the channel has a rectangular and/or square transverse cross-section.

[00162] A thirty-fifth aspect relates to the printhead of any preceding aspect, wherein the nozzle is formed by 3D printing.

[00163] A thirty-sixth aspect wherein the electrodes are constructed from a metal foil or strip.

[00164] 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.

[00165] A thirty-eighth aspect relates to the electroporation system of any preceding aspect, wherein the voltage source comprises one or more power amplifiers.

[00166] 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.

[00167] To clarify the use of and to hereby provide notice to the public, the phrases "at least one of <A>, <B>, ... and <N>" or "at least one of <A>, <B>, ... or <N>" or "at least one of <A>, <B>, ... <N>, or combinations thereof" or "<A>, <B>, ... and/or <N>" are defined by the Applicant in the broadest sense, superseding any other implied definitions hereinbefore or hereinafter unless expressly asserted by the Applicant to the contrary, to mean one or more elements selected from the group comprising A, B, ... and N. In other words, 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. Unless otherwise indicated or the context suggests otherwise, as used herein, "a" or "an" means "at least one" or "one or more." [00168] While various embodiments have been described, it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible. Accordingly, the embodiments described herein are examples, not the only possible embodiments and implementations.

[00169] In addition to the features mentioned in each of the independent aspects enumerated above, some examples may show, alone or in combination, the optional features mentioned in the dependent aspects and/or as disclosed in the description above and shown in the figures.

Claims

1. 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 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 cellladen 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.

2. The method of claim 1, 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.

3. The method of claim 2, 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.

4. The method of claim 2, wherein the pulse duration is selected to be from 50 ps to 50 ms.

5. The method of claim 1, 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 100%.

6. The method of claim 1, wherein the selected voxels of the bioink are immediately adjacent to each other along a flow direction of the bioink.

7. The method of claim 1, 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.

8. The method of claim 1, wherein the pulsed electric field is applied substantially perpendicular to a flow direction of the bioink.

9. The method of claim 1, 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.

10. The method of claim 1, 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.

11. The method of claim 1, 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.

12. The method of claim 1, wherein the cells comprise human cells.

13. The method of claim 1, wherein a cell concentration in the bioink is in a range from 1 x 10⁶ cells/ml to 500 x 10⁶ cells/ml.

14. The method of claim 1, wherein the bioink is shear- thinning.

15. The method of claim 1, wherein the bioink further comprises a polymer.

16. The method of claim 15, 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.

17. The method of claim 15, wherein the polymer has a form of microparticles in the bioink.

18. The method of claim 1, wherein the biological cargo comprises a nucleic acid, a protein, or another complex molecule.

19. The method of claim 1, wherein the biological cargo includes at least two cargo species controllably introduced into the bioink so as to be present in different voxels.

20. The method of claim 1, wherein the biological cargo includes at least two cargo species controllably introduced into the bioink so as to be present in the same voxels.

21. The method of claim 1, wherein the bioink further comprises a crosslinking initiator.

22. The method of claim 1, wherein the bioink further comprises an apoptosis inhibitor.

23. The method of claim 1, wherein the bioink further comprises an electroporation buffer.

24. The method of claim 1, 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.

25. The method of claim 1, wherein the bioink further comprises a polymer, and further comprising, after printing the tissue, exposing the tissue to ultraviolet light, thereby crosslinking the polymer.

26. 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.

27. The printhead of claim 26, wherein the first electrode and the second electrode are positioned in opposition to each other on a channel wall or on opposing channel walls.

28. The printhead of claim 26, wherein the first electrode and the second electrode are positioned adjacent to each other on a channel wall or on adjacent channel walls.

29. The printhead of claim 26, wherein the first electrode and the second electrode are positioned in parallel with each other.

30. The printhead of claim 26, wherein the first electrode and the second electrode are positioned in a coaxial or concentric arrangement.

31. The printhead of claim 26, wherein the first electrode and the second electrode are positioned to come into direct contact with the bioink flowing through the channel.

32. The printhead of claim 26, 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.

33. The printhead of claim 26, wherein the channel has a circular, oval, polygonal or irregular transverse cross-section.

34. The printhead of claim 26, wherein the channel has a rectangular and/or square transverse cross-section.

35. The printhead of claim 26, wherein the nozzle is formed by 3D printing.

36. The printhead of claim 26, wherein the electrodes are constructed from a metal foil or strip.

37. An electroporation system comprising: the printhead of claim 26; an ink dispenser in fluid communication with the channel inlet; a substrate facing the channel outlet; a three-axis motion controller configured to move the printhead and/or the substrate; and a voltage source electrically connected to the first and second electrodes.

38. The electroporation system of claim 37, wherein the voltage source comprises one or more power amplifiers.

39. The electroporation system of claim 37, wherein an arbitrary waveform generator is configured to drive the voltage source.