Bio-ink for 3D printing

Abstract

To provide bio-inks for 3D printing that are biocompatible and can form hydrogels rapidly via a substantially non-toxic chemical pathway.SOLUTION: The present invention provides a 3D printed hydrogel formed from a maleimide-containing polymer cross-linked using a bis-thiol containing cross-linking agent having at least two thiol functional groups.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority from Australian Provisional Patent Application No. 2018902674, entitled "Bioinks for 3D Printing", filed on July 24, 2018, the entire contents of which are incorporated herein by cross-reference.

[0002] The present technology is broadly related to bioinks for 3D bioprinting. In particular, the technology relates to 3D printed hydrogels formed from polymers crosslinked using crosslinkers, processes for preparing the hydrogels, and their uses. [Background technology]

[0003] Cells exist three-dimensionally within an extracellular matrix (ECM) in vivo. Interactions between cells and the ECM play a key role in controlling the biological properties and behavior of cells. In cancer biology, three-dimensional (3D) in vitro cell culture assays are used to culture cells in an environment that closely mimics the cells' native environment. Such assays maintain physiological cell-cell and cell-matrix interactions that are not present in two-dimensional monolayer cultures.

[0004] 3D bioprinting uses 3D printing technology to combine cells and other biomaterials to prepare synthetic structures that mimic the ECM. Generally, 3D bioprinters utilize either drop-on-demand or extrusion printing techniques to print 3D in vitro assays. Drop-on-demand bioprinting has been reported to maintain high cell viability during printing, but the low viscosity and low cell density of current 3D bioinks make this technique less commonly used compared to extrusion bioprinting.

[0005] Both synthetic and natural hydrogels have been widely used as ECM mimics. In particular, synthetic hydrogels are highly reproducible and can provide excellent control over physical and cellular responsiveness. This makes them highly desirable as ECM mimics for in vitro studies. Current approaches for manually creating 3D in vitro assays primarily utilize Matrigel® or collagen as ECM mimics. Other widely used materials are hyaluronic acid, chitosan, and alginate. Because they are sourced from natural products, natural hydrogels are usually biocompatible and, in some cases, transport biomolecules necessary for cell-matrix interactions. However, natural hydrogels have limitations as ECM mimics because they are susceptible to batch-to-batch variability, lack physical and biochemical modularity, and / or are difficult to handle.

[0006] To overcome the various limitations of natural hydrogels, various synthetic hydrogels have been prepared. In particular, covalently crosslinked synthetic hydrogels can provide systems that are more robust and mechanically precise than natural hydrogels. However, many of these synthetic hydrogels lack the biochemical properties that promote cell-matrix interactions. Furthermore, the application of covalently crosslinked synthetic hydrogels for 3D bioprinting of cells requires that the material be biocompatible so that cells can be printed in situ within the hydrogel. For example, poly(acrylamide) is a biocompatible synthetic hydrogel used in cell biology (Caliari and Burdick, 2016), but the corresponding hydrogel precursor is not biocompatible, making it unsuitable for 3D bioprinting of cells.

[0007] Current approaches to 3D bioprinting involve UV-initiated radical crosslinking reactions that allow for rapid hydrogel formation (Murphy and Atala, 2014; Donderwinkel et al., 2017: Jungst et al., 2016 Lowe et al., 2014). For example, inkjet printing of photocrosslinkable acrylated PEG and peptides was found to be suitable for generating 3D hydrogel constructs for encapsulating mesenchymal stem cells (Gao et al., 2017; Gao et al., 2015). However, while the feasibility of using this approach for 3D bioprinting of living cells has been shown, the use of UV irradiation to initiate photocrosslinking can potentially damage DNA in vivo, and the free radicals generated may also damage sensitive cells.

[0008] Advances in 3D bioprinting have been prominent in recent years and have the potential to alleviate the limitations of current approaches to 3D in vitro cell culture assays. However, while the benefits of 3D printed cell culture are well established, the utilization of 3D printed in vitro assays in cell biology is still constrained by their complexity, labor-intensive, and low-throughput nature. Thus, there is a need for alternative ECM mimics for use in 3D bioprinting. Summary of the Invention

[0009] The inventors have developed a bioink suitable for 3D printing that is capable of rapidly forming hydrogels via a biocompatible and substantially non-toxic chemical route.

[0010] In a first aspect, the present technology provides 3D printed hydrogels formed from maleimide-containing polymers crosslinked using a bis-thiol-containing crosslinker having at least two thiol functional groups.

[0011] Bisthiol containing crosslinkers may contain more than two thiol functional groups.

[0012] In one embodiment, a 3D printer is used to combine a solution containing a maleimide-containing polymer (polymer bioink) with a solution containing a bis-thiol-containing crosslinker (activator), resulting in the formation of a hydrogel.

[0013] In one embodiment, the 3D printed hydrogel is formed in about 30 minutes or less, or 10 minutes or less, or 1 minute or less, or 30 seconds or less, or 10 seconds or less, or 1 second or less from printing of the polymer bioink and activator.

[0014] In one embodiment, the polymer bio-ink is biocompatible with cells.

[0015] In one embodiment, the activator is biocompatible with the cells.

[0016] In one embodiment, when combined during 3D printing, the polymer bioink and activator form a hydrogel that is biocompatible with cells.

[0017] In one embodiment, the maleimide-containing polymer is a maleimide-containing polysaccharide, such as a polymer containing fructose, sucrose, or glucose monomers; a synthetic polymer, such as poly(ethylene glycol) (PEG) maleimide, poly(hydroxyethyl methacrylate) (PHEMA) maleimide, poly(ε-caprolactone) (PCL) maleimide, poly(vinyl alcohol) (PVA) maleimide, poly(vinylpyrrolidone) (PVP) maleimide, poly(N-isopropylacrylamide) (NIPAAM) maleimide, poly(propylene fumarate) (PPF) maleimide, poly(ethyleneimine) (PEI) maleimide, poly(3-methacrylamidopropyl)trimethylammonium (PMAETMA) maleimide, and poly( Lmaleimide, poly(arginine-methacrylate) maleimide, or derivatives thereof; maleimide-containing biopolymers, such as gelatin maleimide, cellulose maleimide, hyaluronic acid maleimide, and alginate maleimide; and maleimide-containing nucleobase polymers (i.e., maleimides comprising polymers of adenine, thiamine, guanine, and / or cytosine repeating units); and any combination thereof.

[0018] In one embodiment, the maleimide-containing polymer comprises poly(ethylene glycol) (PEG) maleimide.

[0019] In one embodiment, the maleimide-containing polymer comprises gelatin maleimide.

[0020] In one embodiment, the bis-thiol containing crosslinker is selected from a synthetic polymer, a biopolymer, a small molecule, a bioactive molecule, or any combination thereof.

[0021] In one embodiment, the synthetic polymer is selected from PEG-bis-thiol, poly(N-isopropylacrylamide)bisthiol (NIPAAM bisthiol), poly(acrylic acid)bisthiol, poly(methacrylic acid)bisthiol, poly(styrenesulfonate)bisthiol, poly(amide)bisthiol, or any combination thereof. In one embodiment, the bisthiol-containing crosslinker is PEG bisthiol.

[0022] In one embodiment, the biopolymer is selected from thiol-gelatin, thiol-cellulose, thiol-chitosan, thiol-hyaluronic acid, or any combination thereof.

[0023] In one embodiment, the small molecule is dithiothreitol (DTT).

[0024] In one embodiment, the biologically active molecule is selected from short peptides having at least two cysteine ​​amino acid groups, inactive short peptides, enzyme-sensitive short peptides, matrix metalloproteinase (MMP)-sensitive peptides, or any combination thereof.

[0025] In one embodiment, the bis-thiol containing bioactive molecule is selected from MMP-sensitive bis-cysteine ​​peptides.

[0026] In one embodiment, the MMP sensitive peptide is GCIPVSLRSGCG, GCRDGPQGIWGQDRCG, GCRDPLGLDRCG, GCRDEAPLKQDRCG, or any combination thereof.

[0027] In one embodiment, the maleimide-containing polymer is a PEG maleimide and the bis-thiol-containing crosslinker is selected from the group consisting of a PEG bis-thiol, an MMP-sensitive peptide, and combinations thereof.

[0028] In one embodiment, the maleimide-containing polymer is a PEG maleimide substituted with at least one bioactive molecule selected from RGD, YIGSR, and IKVAV, and the bis-thiol-containing crosslinker is selected from PEG bis-thiol, an MMP-sensitive peptide, and combinations thereof.

[0029] In one embodiment, the MMP sensitive peptide is GCIPVSLRSGCG, GCRDPLGLDRCG, or a combination thereof.

[0030] In one embodiment, the maleimide-containing polymer is gelatinmaleimide and the bisthiol-containing crosslinker is PEG bisthiol.

[0031] In one embodiment, the molar ratio of the maleimide-containing polymer to the bis-thiol-containing polymer ranges from about 10: 1 to about 1: 10. The ratio of thiol groups in the bis-thiol-containing crosslinker(s) to maleimide groups in the maleimide-containing polymer(s) may be greater than about 60%.

[0032] In one embodiment, the hydrogel can be formed by printing a drop of the polymer bioink onto a substrate followed by printing a drop of an activator to form a hydrogel droplet, or applying a drop of an activator to the substrate followed by applying a drop of the polymer bioink to form a hydrogel. The printing process is repeated to form a hydrogel. During the printing process, cells can be included in the polymer bioink, the activator, or both the polymer bioink and the activator. Additionally or alternatively, the cells can be included in cell culture medium. The cell culture medium can be included in the polymer bioink, the activator, or can be separate from the polymer bioink and the activator. For example, the cell culture medium can be suspended between layers of a 3D printed hydrogel formed from the polymer bioink and the activator, or can be deposited on the surface of the hydrogel.

[0033] In one embodiment, the substrate is selected from any suitable container. Examples include microtiter plates with different well configurations (6, 24, 48 and 96 wells), microtiter plates with cover glass bottom with different well configurations (6, 24, 48 and 96 wells), fluoro dishes of various sizes, chamber slides with different chamber configurations (1, 2, 4, 8 and 16), cover glass or microscope slides. The container may be suitable for containing, holding or growing cells.

[0034] In one embodiment, the 3D printed hydrogel further comprises a bioactive molecule. The bioactive molecule may be attached to the maleimide-containing polymer, the bis-thiol-containing crosslinker, or both the maleimide-containing polymer and the bis-thiol-containing crosslinker. Additionally or alternatively, free bioactive molecules may be present in the 3D printed hydrogel. The free bioactive molecules may be present in the polymer bioink, the activator, or both. The bioactive molecule may be selected from peptides, MMP-sensitive peptides, proteins, polysaccharides, drugs, therapeutic agents, antibodies, small molecule inhibitors, kinase inhibitors, phosphatase inhibitors, antigens, pathogens, platelets, growth factors, cytokines, amino acids, nutrients, conditioned media, antibiotics, antivirals, RNA, and any combination thereof. Nanoparticles may also be incorporated into the 3D printed hydrogel.

[0035] In one embodiment, the bioactive molecule is selected from CRGDS, CIKVAV, CYIGSR, VEGF with a C-terminal unpaired cysteine, proteins (e.g., laminin, collagen), and MMP-sensitive peptides (e.g., GCIPVSLRSGCG, GCRDGPQGIWGQDRCG, GCRDPLGLDRCG, GCRDEAPLKQDRCG); and any combination thereof.

[0036] In one embodiment, the 3D printed hydrogel further comprises a cell culture medium. Examples of suitable culture media include Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), Media 199 Medium, Ham's F10 Medium, Ham's F12 Medium, McCoy's 5A Medium, and Roswell Park Memorial Institute (RPMI) Medium. The cell culture medium may be present in the polymer bioink, the activator, or both.

[0037] In one embodiment, the hydrogel comprises cells.

[0038] In one embodiment, the cells are present in the polymer bioink, in the activator, in both the polymer bioink and the activator, or in a separate medium prior to 3D printing, allowing for the printing of a hydrogel containing the cells.

[0039] In one embodiment, the cells are suspended substantially uniformly throughout the hydrogel.

[0040] The concentration of printed cells was approximately 1 × 10 5 ~Approx. 5×10 8 The concentration of cells may range from 0.1 to 1.0 cells / mL. It will be appreciated that lower or higher concentrations of cells can be printed to form a printed hydrogel containing cells.

[0041] In one embodiment, the cell can be selected from a hepatic cell, a gastrointestinal cell, a pancreatic cell, a kidney cell, a lung cell, a tracheal cell, a vascular cell, a skeletal muscle cell, a cardiac cell, a skin cell, a smooth muscle cell, a connective tissue cell, a corneal cell, a urogenital cell, a breast cell, a germ cell, an endothelial cell, an epithelial cell, a fibroblast, a neuronal cell, a Schwann cell, an adipocyte, a bone cell, a bone marrow cell, a chondrocyte, a pericyte, a mesothelial cell, an endocrine tissue-derived cell, a stromal cell, a stem cell, a progenitor cell, a lymphatic cell, a blood cell, an endoderm-derived cell, an ectoderm-derived cell, and a mesoderm-derived cell, and any combination thereof.

[0042] In a second aspect, the present technology provides a method of preparing a 3D printed hydrogel, the method comprising the steps of: providing a polymer bioink comprising a maleimide-containing polymer; providing an activating agent comprising a bis-thiol-containing crosslinker having at least two thiol functional groups; and Printing the polymer bioink and activator to form a 3D printed hydrogel.

[0043] In one embodiment, the method further comprises providing cells to form a 3D printed hydrogel containing cells. The cells may be present in the polymer bio-ink, in the activator, in both the polymer bio-ink and the activator, or in a separate medium prior to 3D printing.

[0044] In one embodiment, the 3D printed hydrogel is formed within 30 minutes, or within 10 minutes, or within 1 minute, or within 30 seconds, or within 10 seconds, or within 1 second of printing the polymer bioink and activator.

[0045] In one embodiment, the polymer bio-ink, the activator, or both are adjusted to a pH of about 7.4 prior to printing.

[0046] In one embodiment, the pH is adjusted using NaOH.

[0047] The method can be used to prepare a 3D printed hydrogel according to the first aspect.

[0048] In a third aspect, the present technology provides a kit for 3D printing, the kit comprising: A polymer bioink comprising a maleimide-containing polymer; and An activator comprising a bis-thiol-containing crosslinker having at least two thiol functional groups.

[0049] In one embodiment, the kit further comprises cells.

[0050] In one embodiment, the kit further comprises a printing substrate.

[0051] In a fourth aspect, the present technology provides a cellular assay comprising a 3D printed hydrogel containing cells according to the first aspect.

[0052] In a fifth aspect, the present technology provides use of a 3D printed hydrogel containing cells according to the first aspect for a cellular assay.

[0053] definition Below are definitions of some terms used in the art that may be helpful in understanding this specification. These are intended as general definitions and are not intended to limit the scope of the invention to only those terms, but are presented for a better understanding of the following description.

[0054] Unless the context requires otherwise or to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps, or elements explicitly encompass both the singular and plural forms of the recited integer, step, or element.

[0055] As used herein, the term "about" means ±10% of the stated value.

[0056] As used herein, the terms "cross-linker" and "cross-linking agent" are used interchangeably to refer to a compound that can covalently cross-link maleimide-containing polymers. The cross-linker of the present technology contains at least two thiol functional groups and is referred to herein as a "bis-thiol containing cross-linker" or "bis-thiol containing cross-linking agent". The bis-thiol containing cross-linker may contain more than two thiol functional groups, for example, three, four, five, six, or more thiol functional groups.

[0057] As used herein, the term "polymeric bioink" refers to a solution, preferably an aqueous solution, that includes a maleimide-containing polymer or a combination of two or more maleimide-containing polymers. As used herein, the term "activator" refers to a solution, preferably an aqueous solution, that includes at least one bis-thiol-containing crosslinker. Upon mixing the polymeric bioink with the activator of the present technology, i.e., during 3D printing, a thiol-maleimide crosslinking reaction occurs to form a hydrogel.

[0058] As used herein, the term "biocompatible" in reference to a material means that the material is substantially non-toxic to living cells or tissues.

[0059] As used herein, the term "bioactive molecule" refers to a molecule that has a biological activity that can be used on cultured cells.

[0060] As used herein, the term "cellular polymer bioink" refers to cells mixed with a polymer bioink that includes at least one maleimide-containing polymer. As used herein, the term "cellular activator" refers to cells mixed with an activator that includes at least one bis-thiol-containing crosslinker. The cellular polymer bioink and cellular activator of the present technology may be aqueous solutions of one or more types of bioactive molecules in which cells are suspended and may remain suspended throughout the 3D bioprinting process.

[0061] The term "cell culture medium" refers to a liquid that can be used to store, maintain, grow, or cultivate cells.

[0062] As used herein, the term "drop-on-demand" with respect to 3D bioprinting refers to a printing process in which the delivery of bioink is based on the generation of discrete droplets. Drop-on-demand bioprinting includes, but is not limited to, thermal inkjet, piezo inkjet, and microvalve-based bioprinting.

[0063] Where ranges of values ​​are given throughout the specification and claims, the recited range is intended to include every value within that range.

[0064] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. Not to be construed as an admission that any or all of such matters form part of the prior art or were common general knowledge in the art relevant to the present invention prior to the priority date of each claim herein.

[0065] Throughout this specification, unless the context dictates otherwise, the word "comprise", or variations such as "comprises" or "comprising", means the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of other elements, integers, or steps, or group of elements, integers, or steps.

[0066] The term "consisting of" means "consisting only of," that is, including and limited to the specified elements, integers, or steps, and excluding other elements, integers, or steps. The term "consisting essentially of" means including the recited elements, integers, or steps, but may also include other elements, integers, or steps that do not materially alter or contribute to the operation of the invention.

[0067] In order that the present technology may be more clearly understood, preferred embodiments will now be described with reference to the following drawings and examples. [Brief description of the drawings]

[0068] [Figure 1] FIG. 1 is a representative schematic of the 3D bioprinting process. [Diagram 2]FIG. 2 shows (a) UV analysis; and (b) quantification of CRGDS conjugation analyses by 1H NMR of the modification of 4-arm PEG-Mal with CRGDS. [Diagram 3] Figure 3 shows the (a) frequency; (b) strain; (c) stiffness; and (d) viscosity results of rheological studies of hydrogels prepared from 4-arm PEG-Mal hydrogels containing 2.5 mM RGD (10 wt%) and crosslinked with PEG bis-thiol and MMP2-responsive bis-cysteine ​​peptides (mixed at a thiol molar concentration of 50:50). [Figure 4] Figure 4 shows the results of a stiffness-rheological study of (a) a 7.5 wt % blank 4-arm PEG-Mal hydrogel crosslinked with PEG bisthiol and (b) a PEG tripeptide hydrogel crosslinked with an MMP-sensitive bis-cysteine ​​peptide activator. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] This technology involves 3D-printed hydrogels that have the potential to maintain cell viability over extended periods of time. The hydrogels in this technology are prepared by 3D printing of polymeric bioinks and activators that are biocompatible and rapidly form hydrogels upon printing, enabling high-throughput generation of 3D cellular structures and assays.

[0070] In particular, the inventors have found that maleimide-containing polymers covalently crosslinked using bis-thiol-containing crosslinkers allow for the rapid formation of homogenous hydrogels under physiological conditions, without the aid of current reactants, via a biocompatible crosslinking route, and without the production of toxic by-products. Thus, the 3D printed hydrogels of the present technology are formed by combining a polymer bioink comprising a maleimide-containing polymer with an activating agent comprising a bis-thiol-containing crosslinker having at least two thiol functional groups.

[0071] Polymer Bioink The polymer bioinks of the present technology include a maleimide-containing polymer, or a combination or two or more maleimide-containing polymers. The maleimide-containing polymer may be any polymer that contains a maleimide reactive group for the formation of a hydrogel. Polymers that contain free maleimide groups may also be present in the hydrogel.

[0072] Maleimide containing polymers of various molecular weights, molecular architectures, molecular distributions, and functionalities can be used in the polymer bioink. For example, the molecular weight of the maleimide-containing polymers can range from about 1 kDa to about 10,000 kDa, or about 5 kDa to about 10,000 kDa, or about 5 kDa to about 5,000 kDa, or about 5 kDa to about 1,000 kDa, or about 10 kDa to about 500 kDa. The maleimide-containing polymers may have any suitable molecular architecture, such as linear, block, branched, star, cyclic, comb, or brush polymers, or any combination thereof, among others. Molecular distribution, which refers to the distribution of monomer units along the polymer backbone, may include homo, block, statistical, random, or multiblock distributions, or any combination thereof, among others. Functionalities (i.e., types of monomers) can include, but are not limited to, hydroxyl, amine, thiol carboxylic acid, alkene, alkyne, and multi-membered carbon rings, among others. The polymer may contain one functionality, or two or more functionality (ie, a copolymer).

[0073] In one embodiment, the maleimide-containing polymer is biocompatible.

[0074] Maleimide-containing polymers are synthetically prepared by incorporating two or more maleimide reactive groups into a polymer. The two or more maleimide groups can be incorporated into the polymer by any suitable method known in the art, such as carbodiimide coupling, Stecklich esterification, and thiol-maleimide coupling with an excess of bismaleimide molecules. Additionally or alternatively, commercially available maleimide-containing polymers can be used.

[0075] Non-limiting examples of suitable maleimide-containing polymers for use in the polymer bioinks of the present technology include maleimide-containing polysaccharides, such as polymers containing fructose, sucrose, or glucose monomers; poly(ethylene glycol) (PEG) maleimide, poly(hydroxyethyl methacrylate (PHEMA) maleimide, poly(ε-caprolactone) (PCL) maleimide, poly(vinyl alcohol) (PVA) maleimide, poly(vinylpyrrolidone) (PVP) maleimide, poly(N-isopropylacrylamide) (NIPAAM) maleimide, poly(propylene fumarate) (PPF) maleimide, poly(ethyleneimine) (PEI) maleimide, poly(3-methacrylamidopropyl)trimethylammonium (PMAETMA) maleimide, poly( L maleimide-containing biopolymers such as gelatin maleimide, cellulose maleimide, hyaluronic acid maleimide, and alginate maleimide; and maleimide-containing nucleobase polymers (i.e., maleimide-containing polymers of adenine, thiamine, guanine, and / or cytosine repeat units); or any combination thereof.

[0076] PEG is a particularly attractive material for the preparation of synthetic hydrogels as ECM mimics because it is commercially available, inert, non-toxic in biological applications, and versatile for various chemical modifications. In addition, PEG-based polymer bioinks may have low relative viscosities due to the low molecular weight of the PEG polymer precursor, making them particularly suitable for drop-on-demand bioprinting. Thus, in one embodiment, the maleimide-containing polymer is a poly(ethylene glycol) (PEG) polymer containing at least two maleimide reactive groups (PEG-maleimide). The PEG-maleimide may have any suitable molecular structure, including, but not limited to, a 3-arm PEG maleimide, a 4-arm PEG maleimide, a block copolymer of PEG with at least two maleimide groups, or any combination thereof. The PEG group may have any suitable molecular weight. For example, the molecular weight of the PEG group may range from about 1 kDa to about 50 kDa, or from about 1 kDa to about 40 kDa, or from about 1 kDa to about 30 kDa, or from about 5 kDa to about 30 kDa, or from about 5 kDa to about 20 kDa, or from about 5 kDa to about 10 kDa, or from about 10 kDa to about 20 kDa. In one embodiment, the molecular weight of the PEG group is about 5 kDa, or about 10 kDa, or about 20 kDa.

[0077] The term "3-arm PEG maleimide" refers to the following structure (n=38-3, 788):

[0078] [ka]

[0079] It refers to a polymer compound having the following structure:

[0080] The term "4-arm PEG maleimide" refers to the following structure (n=28-2, 841):

[0081] [ka]

[0082] It refers to a polymer compound having the following structure:

[0083] The properties of maleimide-containing polymers can be tailored for specific applications by partially modifying the maleimide groups with one or more functional groups or other moieties. Partial modification means that at least one, preferably at least two, maleimide groups remain intact for crosslinking with a bisthiol-containing crosslinker. Thus, when a bioactive molecule is incorporated into the maleimide-containing polymer, the maleimide-containing polymer contains at least two, typically at least three, maleimide groups. This approach allows the introduction of relevant biological properties into 3D-printed hydrogels while maintaining the reproducibility and modularity of the polymer. Thus, in one embodiment, the maleimide groups of the maleimide-containing polymer may be partially modified with one or more bioactive molecules.

[0084] The polymer bioink of the present technology can be prepared as an aqueous solution containing maleimide-containing polymers in any suitable formulation known in the art. For example, the polymer bioink can be prepared as an aqueous formulation containing at least one maleimide-containing polymer and, optionally, one or more bioactive molecules. For example, the aqueous formulation can be a solution containing the maleimide-containing polymer in a pH buffer or cell culture medium. In some embodiments, the polymer bioink can include two maleimide-containing polymers in any ratio, for example, a ratio between about 10:1 and about 1:10, for example, about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0085] In one embodiment, the polymer bioink is provided in the form of an aqueous solution comprising a maleimide-containing polymer. Typically, the polymer bioink is prepared by dissolving the maleimide-containing polymer in a solution, preferably a culture medium. In some embodiments, the polymer bioink may comprise a mixture of two or more maleimide-containing polymers. The polymer bioink may comprise any suitable concentration of the maleimide-containing polymer in solution. For example, the concentration of the maleimide-containing polymer in the polymer bioink solution may range from about 1% to 50% by weight, or from about 1% to 40% by weight, or from about 1% to about 30% by weight, from about 5% to 20% by weight, from about 5% to 15% by weight, or from about 10% to 20% by weight. The polymer bioink may comprise one or more bioactive molecules covalently bonded to the maleimide-containing polymer. Additionally or alternatively, the polymer bioink may comprise one or more free bioactive molecules in solution. The concentration of the bioactive molecule in the polymer bioink may range from about 0.1 mM to 10 mM, about 0.5 mM to 10 mM, about 1 mM to 10 mM, about 1 mM to 5 mM, about 2 mM to 5 mM, or about 2 mM to 3 mM.

[0086] In one embodiment, the polymer bio-ink is prepared as a sterile solution. Suitable methods for preparing a sterile solution or for sterilizing a polymer bio-ink will be known to those of skill in the art.

[0087] In one embodiment, the polymer bio-ink is biocompatible.

[0088] Activator The activator of the present technology comprises a bisthiol-containing crosslinker.The bisthiol-containing crosslinker may be any compound that contains at least two thiol groups, referred to herein as bisthiol.Preferably, the thiol group is a terminal thiol group.

[0089] In one embodiment, the bis-thiol containing crosslinker is biocompatible.

[0090] In one embodiment, the bis-thiol-containing crosslinker may be a bis-thiol-containing polymer, preferably a terminal bis-thiol-containing synthetic polymer. Suitable bis-thiol-containing synthetic polymers include, but are not limited to, PEG bis-thiol, poly(N-isopropylacrylamide) bis-thiol (NIPAAM bis-thiol), poly(acrylic acid) bis-thiol, poly(methacrylic acid) bis-thiol, poly(styrenesulfonate) bis-thiol, poly(amide) bis-thiol, or any combination thereof. Alternatively, the bis-thiol-containing crosslinker may be a bis-thiol-containing biopolymer, preferably a terminal bis-thiol-containing biopolymer. Suitable bis-thiol-containing biopolymers include, but are not limited to, thiol-gelatin (e.g., from bovine, porcine, and cold fish), thiol-hyaluronic acid (e.g., from streptococcus equi, bovine, and human umbilical cord), and any combination thereof.

[0091] In one embodiment, the bisthiol-containing crosslinker is a PEG bisthiol. The PEG bisthiol may have any suitable molecular structure, including, but not limited to, linear, branched, and star-shaped PEG structures. In one embodiment, the PEG bisthiol is a linear PEG bisthiol. The molecular weight of the PEG group may vary, for example, the molecular weight of the PEG group may range from about 1 kDa to about 50 kDa, or from about 1 kDa to about 40 kDa, or from about 1 kDa to about 30 kDa, or from about 5 kDa to about 30 kDa, or from about 5 kDa to about 20 kDa, or from about 5 kDa to about 10 kDa, or from about 10 kDa to about 20 kDa. In one embodiment, the molecular weight of the PEG group is about 5 kDa, or about 10 kDa, or about 20 kDa.

[0092] An example of a linear PEG bisthiol has the following structure (n=11-1, 136):

[0093] [ka]

[0094] has.

[0095] In some embodiments, the bis-thiol-containing crosslinker may be a bis-thiol-containing small molecule. Suitable bis-thiol-containing molecules include, but are not limited to, dithiothreitol (DTT) (used with or without sodium borate or borax), or any combination thereof. In another embodiment, the bis-thiol-containing crosslinker may be a short peptide containing at least two cysteine ​​amino acid groups, preferably a terminal cysteine ​​group (i.e., a thiol-adjacent amino acid sequence). Suitable short peptides containing bis-thiol flanked by two cysteine ​​amino acid groups include, but are not limited to, an inactive short peptide sequence, an enzyme-sensitive short peptide (e.g., an MMP-sensitive, serine-sensitive, cysteine-sensitive, aspartic-sensitive, glutamic-sensitive, caspase-sensitive, or trypsin-sensitive peptide sequence), or any combination thereof.

[0096] In one embodiment, the bis-thiol-containing crosslinker is a bis-thiol-containing bioactive molecule. For example, the bis-thiol-containing short peptide can be an MMP-sensitive bis-cysteine ​​peptide. For example, the MMP-sensitive peptide can be an MMP2-sensitive bis-cysteine ​​peptide (e.g., GCIPVSLRSGCG), an MMP1, MMP, MMP3, MMP7, MMP8, and MMP9-sensitive bis-cysteine ​​peptide (e.g., GCRDGPQGIWGQDRCG), an MMP15-sensitive thiolated peptide (e.g., GCRDEAPLKQDRCG), or an MMP1, MMP, MMP3, MMP7, MMP8, MMP9, MMP12, MMP13, and MMP14-sensitive bis-cysteine ​​peptide (e.g., GCRDPLGLDRCG).

[0097] The activator may include a combination of two or more types of bis-thiol containing crosslinkers in any ratio. For example, the activator may include a combination of two types of bis-thiol containing crosslinkers in a ratio between about 10:1 and about 1:10, such as about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0098] In one embodiment, the activator may be in the form of an aqueous solution containing the bis-thiol-containing crosslinker. The aqueous solution may be a cell culture medium. In some embodiments, the activator may include a mixture of two or more bis-thiol-containing crosslinkers. The activator may include any suitable concentration of the bis-thiol-containing crosslinker in solution. In a preferred embodiment, the concentration of the bis-thiol-containing crosslinker in the activator is equal to or less than the concentration of the maleimide-containing polymer in the complementing polymer bio-ink. The molar ratio of the maleimide-containing polymer to the bis-thiol-containing crosslinker may range from about 10:1 to 1:1, about 9:1 to 1:1, about 8:1 to 1:1, about 7:1 to 1:1, about 6:1 to 1:1, about 5:1 to 1:1, about 4:1 to 1:1, about 3:1 to 1:1, or about 2:1 to 1:1. The bis-thiol-containing crosslinker may include a bioactive molecule or may include one or more bioactive molecules covalently attached thereto. Additionally or alternatively, the activator may include one or more free bioactive molecules in solution. The concentration of the bioactive molecule in the activator may range from about 0.1 mM to 10 mM, about 0.5 mM to 10 mM, about 1 mM to 10 mM, about 1 mM to 5 mM, about 2 mM to 5 mM, or about 2 mM to 3 mM.

[0099] In one embodiment, the activator is a sterile solution. Suitable methods for preparing a sterile solution or for sterilizing an activator solution will be known to those of skill in the art.

[0100] The formulation of the activator may be the same as the formulation of the polymer bioink, or it may be different. In a preferred embodiment, the formulations used for the polymer bioink and the activator are the same.

[0101] In one embodiment, the activator is biocompatible.

[0102] 3D printed hydrogels The 3D printed hydrogels of the present technology comprise maleimide-containing polymers crosslinked using a bis-thiol-containing crosslinker. In one embodiment, the 3D printed hydrogels comprise cells.

[0103] In one embodiment, the 3D printed hydrogel is prepared by 3D printing of a polymer bioink comprising a maleimide-containing polymer and an activator comprising a bis-thiol-containing crosslinker.

[0104] To be suitable for use in 3D bioprinting, hydrogel formation should occur rapidly. However, the rapid formation of hydrogels used in prior art has typically resulted in the formation of irregular hydrogels. Current approaches to control crosslinking rates and generate uniform gels in 3D bioprinting include the use of external driving forces such as UV irradiation and co-reactants or catalysts. These external driving forces do not participate in the formation of the hydrogel, but rather assist in its formation and may affect the cellular and biological properties of cells encapsulated in the hydrogel. In particular, photochemical crosslinking reactions, including the use of UV irradiation, may involve the use of free radical sources that must be removed from the product so that they do not interact with the cells in the hydrogel.

[0105] The inventors have surprisingly found that by reacting maleimide-containing polymers with bis-thiol-containing crosslinkers, rapid formation of uniform hydrogels capable of supporting cells at high density and viability can be achieved. Maleimide groups react rapidly and spontaneously with thiols under physiological conditions. This reaction does not require an external driving force, making it particularly attractive for use in 3D bioprinting of in vitro assays, as it eliminates the need for potentially cytotoxic co-reactants, radical initiators, or UV irradiation. Furthermore, no unwanted or cytotoxic by-products are produced, allowing for in situ printing of cells without the generation of unwanted by-products.

[0106] The crosslinking reaction between maleimide-containing polymers using bis-thiol-containing crosslinkers occurs rapidly when the polymer bioink comes into contact with the activator, allowing for the rapid formation of 3D printed hydrogels. Thus, in some embodiments, the hydrogel is formed within 30 minutes of printing the polymer bioink and activator. For example, the hydrogel may be formed within 30 minutes, or within 20 minutes, or within 10 minutes, or within 1 minute, or within 30 seconds, or within 10 seconds, or within 1 second of printing the polymer bioink and activator. Various factors, such as size, shape, and composition, can affect the rate of formation of the 3D printed hydrogel.

[0107] An exemplary reaction between a maleimide-containing polymer and a bisthiol-containing crosslinker is shown below: Scheme 1The reaction involves a nucleophilic (Michael) addition between a thiol group on the bis-thiol-containing crosslinker (1) and an alkene functional group of a maleimide moiety on the maleimide-containing polymer (2), generating a 3-substituted succinimide group under aqueous conditions (e.g., in cell culture medium). This reaction is particularly useful for 3D bioprinting in vitro assays because it eliminates the need for potentially cytotoxic co-reactants, radical initiators, or UV irradiation. Furthermore, covalent crosslinking occurs rapidly and spontaneously when the maleimide-containing polymer and the bis-thiol crosslinker are mixed under physiological conditions (e.g., a pH of about 6.5-7.5, preferably pH 7.4) to generate the crosslinked polymer (3).

[0108] [ka] Scheme 1

[0109] In one embodiment, the pH of the polymer bioink, the activator, or both may be adjusted to a physiological pH (e.g., about 6.5-7.5, preferably about pH 7.4) prior to 3D printing using a suitable base. Suitable bases will be apparent to one of skill in the art.

[0110] In one embodiment, the base is NaOH.

[0111] The inventors found that by adjusting the pH of the polymer bioink using NaOH, the activator accelerates gelation during 3D printing, allowing for rapid formation of 3D printed hydrogels (e.g., in less than about 1 second). Additionally, the inventors found that the presence of NaOH reduces cell aggregation in the polymer bioink, the activator, or both, allowing for high concentrations of cells to be dispersed in solution for 3D printing through small nozzle openings.

[0112] In some embodiments, the molar ratio of the maleimide-containing polymer is greater than that of the bisthiol-containing crosslinker, so that the bisthiol-containing crosslinker is consumed. The resulting hydrogel can contain the crosslinked product and excess maleimide-containing polymer (if any) without additional undesirable by-products. For example, the molar ratio of the maleimide-containing polymer to the bisthiol-containing crosslinker can range from about 10:1 to 2:1, about 9:1 to 2:1, about 8:1 to 2:1, about 7:1 to 2:1, about 6:1 to 2:1, about 5:1 to 2:1, about 4:1 to 2:1, about 3:1 to 2:1.

[0113] Preferably, for hydrogel formation, the number of thiol groups in the bisthiol-containing crosslinker is greater than about 60% of the number of maleimide groups in the maleimide-containing polymer. For example, the number of thiol groups in the bisthiol-containing crosslinker to the maleimide groups in the maleimide-containing polymer may be about 60%, or 70%, or 80%, or 90%, or 100%, or 200% or more. In some embodiments, the number of thiol groups in the bisthiol-containing crosslinker is the same as the number of maleimide groups in the maleimide-containing polymer (i.e., 100%).

[0114] Because the polymeric bioinks and activators disclosed herein can be used to generate large quantities of assays, the materials used in the preparation of the polymeric bioinks and activators are preferably readily commercially available in bulk or readily prepared from such commercially available materials.

[0115] A typical process for the preparation of 3D printed hydrogels of the present technology may include the following steps: providing a polymer bioink comprising a maleimide-containing polymer; providing an activating agent comprising a bis-thiol-containing crosslinker; and 3D printing the polymer bioink and activator to form a 3D printed hydrogel.

[0116] In one embodiment, the process further includes providing cells such that 3D printing of the polymer bioink, the activator, and the cells forms a 3D printed hydrogel comprising the cells.

[0117] In one embodiment, the cells are suspended in the hydrogel. The cells may be provided with a polymer bioink, an activator, or both, or another medium. The cells may be suspended in a portion (or portions) of the hydrogel, or may be substantially uniformly suspended throughout the hydrogel.

[0118] In one embodiment, the cells are provided in cell culture medium. The cell culture medium may be included within the polymer bioink, the activator, or may be provided separately from the polymer bioink and the activator. For example, the cell culture medium may be suspended between layers of a 3D printed hydrogel formed from the polymer bioink and the activator, or may be deposited on the surface of the hydrogel.

[0119] In one embodiment, the 3D printed hydrogel further comprises a bioactive molecule. The bioactive molecule may be provided in the polymer bioink, the activator, or both. The bioactive molecule may be freely suspended in the polymer bioink, the activator, or both, or may be bound to the maleimide-containing polymer and / or the bis-thiol-containing crosslinker.

[0120] In one embodiment, the 3D printing process is carried out in a substantially sterile environment.

[0121] bioactive molecules The 3D printed hydrogels of the present technology may be modified with one or more bioactive molecules to introduce specific biological properties. For example, the biological properties of the 3D printed hydrogels may be modified by incorporating proteins, peptides (e.g., MMP-responsive peptides), chemoattractants, growth factors, organic dyes, fluorescent dyes, drugs, therapeutic agents, antibodies, small molecule inhibitors, kinase inhibitors, phosphatase inhibitors, antigens, pathogens, platelets, cytokines, nutrients (e.g., monosaccharides and polysaccharides), conditioned media, antibiotics, antivirals, RNA and related variants (e.g., siRNA, mRNA), and the like. One or more bioactive molecules can be incorporated into the 3D printed hydrogels by covalently linking them to maleimide-containing polymers, bis-thiol-containing crosslinkers, or any combination thereof. Nanoparticles can also be incorporated into the 3D printed hydrogels.

[0122] In one embodiment, the bioactive molecule is a short peptide (e.g., Arg-Gly-Asp (RGD) containing a cysteine ​​(C) amino acid at either the N- or C-terminus), a growth factor (e.g., VEGF), a protein (e.g., laminin, collagen), or a chemoattractant (e.g., stromal cell-derived factor-1 (CXCL12)), or any combination thereof. Other suitable bioactive molecules will be known to those of skill in the art.

[0123] In some embodiments, one or more bioactive molecules may be incorporated into the 3D printed hydrogel by incorporating free bioactive molecules into the polymer bioink, the activator, or both. The polymer bioink, the activator, or both may include cell culture media.

[0124] In one embodiment, bioactive molecules can be attached to the maleimide-containing polymer. Bioactive molecules can be introduced into the maleimide-containing polymer by reacting the maleimide group with the bioactive molecule to form a covalent bond. For example, the covalent bond can be formed by a Michael-type addition reaction between a thiol and a vinyl-carrying bioactive molecule (e.g., in an aqueous medium), a Schiff base reaction between an amino group and an aldehyde group, a Diels-Alder reaction, click chemistry, an aminolysis reaction to an active ester group, or enzymatic crosslinking. Because thiol moieties are abundant in many bioactive molecules, incorporation of bioactive molecules into the maleimide-containing polymer occurs by nucleophilic (Michael) addition of the thiol group to the alkene moiety of the maleimide group to generate a 3-substituted succinimide moiety. Depending on the conditions and molar ratios of the reactants used, the resulting compound converts at least one maleimide group into a bioactive molecule bearing a 3-substituted succinimide group and leaves at least one, and preferably at least two, maleimide groups intact. One of skill in the art would be able to determine appropriate conditions (e.g., molar ratios of reactants) to control the reaction to ensure that at least one, or at least two or more, maleimide groups remain intact.

[0125] In a typical process for incorporating a thiol-containing bioactive molecule into a maleimide-containing polymer, the bioactive molecule and the maleimide-containing polymer are mixed at room temperature in an aqueous solution (e.g., buffer or cell culture medium) in a molar ratio where the maleimide-containing polymer exceeds the ratio of the bioactive molecule (e.g., 3:1, or 4:1, or 5:1). Completion of the reaction can be monitored, for example, via an Ellman assay to ensure that no free thiol groups remain. A typical reaction time is about 30 minutes. After completion of the reaction, the pH of the solution can be adjusted to physiological pH (about 7.4) by adding a suitable base (e.g., NaOH) as needed. Suitable bases for adjusting the pH to physiological pH will be apparent to one of skill in the art. The resulting modified maleimide-containing polymer solution can be used without the need for isolation or purification.

[0126] In one embodiment, the pH of the biomolecule-containing polymeric bioink or activator solution is adjusted to physiological pH (e.g., about 6.5-7.5, preferably pH 7.4) using NaOH. The use of NaOH to adjust the pH of the polymeric bioink or activator allows for rapid polymerization during subsequent 3D printing, thereby accelerating hydrogel formation (e.g., less than 1 second). Additionally, the use of NaOH reduces cell aggregation in the polymeric bioink, activator, or both, allowing for dispersion of high concentrations of cells into the solution for 3D printing through small nozzle openings.

[0127] An example of a 4-arm PEG-maleimide partially modified with RGD (4-arm PEG-maleimide-RGD) was prepared by reacting 4-arm PEG-maleimide with CRGDS in a 4:1 molar ratio, resulting in the following structure (n=28-2, 841):

[0128] [ka]

[0129] has.

[0130] An example of a 4-arm PEG-maleimide partially modified with VEGF (4-arm PEG-maleimide-VEGF) was prepared by reacting 4-arm PEG-maleimide with VEGF in a 4:1 molar ratio, and has the following structure (n=28-2, 841):

[0131] [ka]

[0132] has.

[0133] In some embodiments, bioactive molecules, such as CRGDS, CIKVAV, VEGF with a C-terminal unpaired cysteine, and / or MMP-sensitive peptides (e.g., GCIPVSLRSGCG, GCRDGPQGIWGQDRCG.GCRDPLGLDRCG, or GCRDEAPLKQDRCG), are incorporated into the 3D printed hydrogel to introduce biological functionality. For example, cell adhesion can be introduced by incorporation of CRGDS or CIKVAV cell adhesion motifs, angiogenesis can be promoted by incorporation of VEGF, and cell motility can be promoted by incorporation of MMP-sensitive peptide crosslinkers. GCIPVSLRSGCG is particularly suitable for use as a bis-thiol-containing crosslinker in the crosslinker, but can additionally or alternatively be covalently attached to the maleimide-containing polymer, the bis-thiol-containing crosslinker, or both, or added as a free bioactive molecule to the polymer bioink, the active agent, or both.

[0134] The bioactive molecule may be present in the 3D printed hydrogel at any suitable concentration to achieve the desired biological properties. For example, the bioactive molecule may be present in the 3D printed hydrogel at a concentration ranging from about 1 nM to 100 μM, about 1 nM to 50 μM, about 5 nM to 50 μM, about 10 nM to 50 μM, about 50 nM to 50 μM, about 100 nM to 50 μM, about 100 nM to 1 μM, or about 500 nM to 1 μM.

[0135] cell Cells can be incorporated into the 3D printed hydrogels of this technology. Cell culture media can also be incorporated into the hydrogel to maintain cell viability or promote proliferation and cell division. Cells and / or cell culture media can be incorporated into the hydrogel by incorporating them into the polymer bioink or activator, or both.

[0136] In one embodiment, the cells are provided with the polymer bio-ink, the activator, both the polymer bio-ink and the activator, or in separate media.

[0137] The polymeric bioink and / or activator may include a cell culture medium. The cell culture medium may be selected from any suitable medium that is compatible with the cells and maintains cell viability during the 3D printing process. For example, the cell culture medium may be a solution that includes water (e.g., sterile MilliQ water), a buffer, and a cell culture medium. Suitable buffers include, but are not limited to, phosphate buffered saline (PBS), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), and 2-(cyclohexylamino)ethanesulfonic acid (CHES), or any combination thereof. Non-limiting examples of suitable cell culture media include Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), Media 199 Medium, Ham's F10 Medium, Ham's F12 Medium, McCoy's 5A Medium, and Roswell Park Memorial Institute (RPMI) Medium.

[0138] In some embodiments, the cell culture medium may further include a reducing agent (e.g., 2-mercaptoethanol or tris(2-carboxyethyl)phosphine (TCEP)), a stabilizing agent (e.g., Ficoll® or PVP), and / or a growth supplement (e.g., L-glutamine or hydrocortisone). Non-limiting examples of suitable growth supplements include fetal calf serum (FCS), epidermal growth factor (EGF), basic fibroblast growth factor (bFBF), fibroblast growth factor (FBF), endothelial growth factor (ECGF), insulin-like growth factor 1 (IGF-1), and platelet-derived growth factor (PDGF). The reducing agent can, for example, prevent the formation of disulfide bridges between free thiol groups in the crosslinker. The stabilizing agent can, for example, be used to prevent cells from settling out of the cell culture medium. If necessary, the pH of the cell culture medium can be adjusted to a physiological pH of about 7.4 using an appropriate base. In one embodiment, sodium hydroxide (NaOH) is used as a base to neutralize or control the pH of the cell culture medium because it does not promote cell aggregation.

[0139] The harvested cells can be resuspended at a selected cell concentration in the polymer bioink, activator, or both. The concentration of cells in the polymer bioink, and / or activator can be about 1×10 5 From about 5 × 10 8 cells / mL, or approximately 1 x 10 5 From about 1×10 8 cells / mL, or approximately 1 x 10 5 From about 1×10 7 cells / mL, or approximately 5 x 10 5 From about 1×10 7 cells / mL, or approximately 5 x 10 5 From about 5 × 10 6 cells / mL, or approximately 5 x 10 6 From about 5 × 10 6In one embodiment, the cells are in the range of about 4×10 cells / mL of the polymer bioink, and / or activator, or cell culture medium. 6 cells / mL, or 5 x 10 6 Suspended in cells / mL.

[0140] Any suitable cells can be used with the polymer bioinks and activators of the present technology. For example, suitable cells may include, but are not limited to, mammalian hepatic cells, gastrointestinal cells, pancreatic cells, kidney cells, lung cells, tracheal cells, vascular cells, skeletal muscle cells, cardiac cells, skin cells, smooth muscle cells, connective tissue cells, corneal cells, urogenital cells, breast cells, germ cells, endothelial cells, epithelial cells, fibroblasts, neural cells, Schwann cells, adipocytes, bone cells, bone marrow cells, chondrocytes, pericytes, mesothelial cells, endocrine tissue-derived cells, stromal cells, stem cells, progenitor cells, lymphatic cells, blood cells, endoderm-derived cells, ectoderm-derived cells, mesoderm-derived cells, or any combination thereof.

[0141] Other suitable cell types may include other eukaryotic cells (e.g., Chinese hamster ovary cells), bacteria (e.g., Helicobacter pylori), fungi (e.g., Penicillium chrysogenum), and yeast (e.g., Saccharomyces cerevisiae).

[0142] The 3D printed hydrogel may comprise at least about 100, or about 1,000, or about 5,000, or about 10,000, or about 50,000, or about 100,000, or about 150,000, or about 200,000, or about 250,000, or about 300,000, or about 350,000, or about 400,000, or about 450,000, or about 500,000 or more cells. It will be understood by those of skill in the art that other numbers of cells can be used.

[0143] In some embodiments, the process further comprises incubating the cells suspended in the hydrogel under conditions that allow for cell growth or maintenance. In some embodiments, the cells can be suspended in the hydrogel under conditions that allow for stem cell differentiation. The incubation can be performed at any suitable temperature and conditions. For example, incubation suitable for cell growth, maintenance, or differentiation can be performed at about 37° C. with about 5% CO2 for at least 24 hours. It will be understood that the incubation can be performed at any temperature and duration that allows for cell growth, maintenance, or differentiation of the cell type in the hydrogel.

[0144] In other embodiments, the process further comprises incubating the cells suspended in the hydrogel under conditions that may not be suitable for cell growth. For example, incubation can be performed under conditions lacking oxygen, in a serum-free environment to starve the cells, in an acidic or basic environment, under applied external pressure, in the presence of an electric field, under constant shaking, or any combination thereof. Other suitable conditions for mimicking specific biological conditions will be known to those of skill in the art.

[0145] 3D Bioprinting The polymeric bioinks and activators of the present technology are suitable for drop-on-demand (DOD) bioprinting, whereby bioprinting is based on the generation of discrete droplets of the polymeric bioink and activator, which may include thermal inkjet, piezo inkjet, and microvalve-based bioprinting, among others. As with other drop-on-demand printing techniques, the polymeric bioink and activator must be liquid within the bioprinter cartridge. Furthermore, the polymeric bioink and activator must be fabricated from biocompatible materials to maintain high cell viability when stored in the bioprinting cartridge.

[0146] DOD bioprinting of 3D assays involves repeated deposition of droplets of polymer bioink and activator at a size and location on a substrate determined by the bioprinter software. Thus, in one embodiment, 3D bioprinting is performed using a 3D bioprinter capable of forming and depositing droplets. The bioprinter may have fluid reservoirs for at least the polymer bioink, activator, and wash solution, a three-axis motion control stage, a drop-on-demand droplet dispensing system, and a pressure regulator for controlling the pressure in the fluid reservoirs. The droplet dispensing system and the three-axis stage may be housed in a sterile chamber, such as a laminar flow cabinet. The printing platform may include adapters for printing on various types of materials, such as microwell plates and Petri dishes. The printing platform may be heated to about 37° C. to support cell growth.

[0147] The 3D bioprinter can be controlled by a computer having software to define the printing format.

[0148] The 3D bioprinter can be configured to print (deposit) droplets of polymer bioink and activator in a manner that allows for cellular integrity, viability or functionality. It will be understood that the deposition of the polymer bioink and activator can be performed in any order. Droplets of the polymer bioink can be applied to the substrate followed by droplets of the activator to form hydrogel droplets, or droplets of the activator can be applied to the substrate followed by droplets of the polymer bioink to form hydrogel droplets.

[0149] The hydrogel formed by the process of the present technology comprises a substantially uniform 3D structure. When the hydrogel comprises cells, the cells may be substantially uniformly distributed throughout the hydrogel, or they may be suspended within a portion of the hydrogel. The cells may be deposited at one or more selected locations within the hydrogel.

[0150] The printing substrate is a biocompatible consumable used to encapsulate and culture the printed hydrogel structures. The substrate can be selected from any suitable container for containing, holding, or growing cells. Examples include microtiter plates with different well configurations (6, 24, 48, and 96 wells), microtiter plates with cover glass bottoms with different well configurations (6, 24, 48, and 96 wells), fluoro dishes of various sizes, chamber slides with different chamber configurations (1, 2, 4, 8, and 16), cover glasses, or microscope slides, or petri dishes. In some embodiments, the 3D bioprinting process is performed to form multiple 3D tissue culture models on the substrate. For example, a 96-well microtiter plate is a suitable substrate and can be used for multiple cell assays. Other suitable containers will be known to those skilled in the art.

[0151] The 3D structure of the assay can be designed using bioprinter software.Suitable bioprinters and bioprinter software will be known to those skilled in the art.For example, in the case of bioprinting in a 96-well plate, the bioprinter software can be configured to repeatedly deposit droplets in the wells in layers of selected diameters.

[0152] The polymeric bioink and activator react rapidly upon printing to form a hydrogel, enabling rapid high-throughput generation of 3D structures and assays. In one embodiment, the polymeric bioink and activator of the present technology form a hydrogel within seconds of mixing (i.e., upon printing). For example, the hydrogel can be formed within 5 seconds of mixing, preferably within 4, 3, 2, or 1 second of mixing.

[0153] The polymer bioink and activator of the present technology can be used with any suitable DOD bioprinting system. The inventors have recently developed a DOD bioprinting system specifically designed for bioprinting of 3D in vitro assays. More specifically, the bioprinter can print the polymer bioink and activator at up to 200 cP and up to 500 million cells / mL with over 95% cell viability. The developed system has been successfully used to bioprint 3D spheroid assays in a high-throughput manner. Thus, the bioink of the present technology is particularly useful for 3D cell printing using the 3D bioprinting system developed by the inventors.

[0154] The fast gelling properties of the hydrogels of the present technology, combined with the novel printing logic of the developed bioprinter, enable the bioprinter to rapidly print complex 3D assays in a 96-well format. For example, bioprinting of a 3D assay in a 96-well plate can be accomplished in less than about 1 hour, preferably less than about 30 minutes, for example, about 25 minutes, or about 20 minutes, or about 15 minutes, or less.

[0155] The polymer bioink and activator can be loaded into separate sterile cartridges immediately prior to printing, or they can be stored in the cartridge. If the cell bioink is stored in the cartridge, the cartridge is preferably stored at 4° C. until further use is required. The polymer bioink and activator of the present technology may support cell viability for an extended period of time prior to printing. For example, cells can remain viable in the polymer bioink and activator prior to printing for at least about 2 hours. In some embodiments, the polymer bioink and activator of the present technology can be stored at −20° C. for at least 6 months without substantially affecting cell viability. Similarly, the 3D printed hydrogels of the present technology may support cell viability for an extended period of time after printing. For example, cells can remain viable in the 3D printed hydrogels for at least about 21 days.

[0156] When ready for use, the cartridge can be connected to a clean bioprinter. Cleaning of the cartridge and / or bioprinter (including fluid lines, valves and nozzles) can be accomplished by any suitable method known in the art, for example, by wiping with ethanol (70% v / v in water), air drying, and placing in a biosafety cabinet. Preferably, the bioprinter is purged with the tissue culture medium being used after cleaning and before printing.

[0157] Purpose The 3D printed hydrogels of the present technology may have a variety of in vitro applications. In one example, the 3D printed hydrogels of the present technology may be used to assess cell motility, cell migration, cell invasion, transendothelial migration, epithelial-mesenchymal transition, mesenchymal-epithelial transition, spheroid formation and proliferation, cell differentiation (e.g., stem cell differentiation, monitoring of cell differentiation markers), cell death (e.g., cell apoptosis, cell necrosis), cell autophagy, cell proliferation, cell metabolism, protein turnover, protein distribution and location, cell signaling and downstream events, drug efficacy, pharmacodynamics, drug mechanism of action, drug receptor-mediated transport, drug internalization mechanism, biomarker evaluation, cell-cell junctions, cell-cell signaling and downstream events, cell morphology, cell adhesion, gene expression, protein expression, cell homing, cell cycle regulation and control, cytokine release, insulin production, protein secretion and intracellular trafficking. Assays can be prepared for the examination of biological phenotypes including, but not limited to, cellular signaling and transport, receptor-ligand binding, antibody binding, antibody specificity, protein phosphorylation, proteosome function, enzyme function (e.g., enzyme inhibition), immunomodulation, clonogenicity, oxidative stress, protein folding, cytoskeleton, organelle morphology and function (e.g., mitochondria, chloroplasts, peroxisomes, secretory vesicles, vacuoles, ribosomes, nuclei, lysosomes, cilia, endoplasmic reticulum, Golgi), membrane trafficking, hypoxia, angiogenesis, wound healing, neurites (growth or formation), kinase function, phosphatase function, lamellipodial formation and dynamics, focal contact / adhesion formation, dynamics and signaling, cell sensing, and mechanical signaling.

[0158] The 3D printed hydrogels of the present technology may also be suitable for in vivo applications (e.g., animal studies). For example, the 3D printed hydrogels of the present technology can be used in orthotopic and subcutaneous models, with or without cells. EXAMPLES

[0159] The following examples are illustrative of the beneficial effects that can be achieved with the present technology and 3D printed hydrogels of the present technology and should not be construed as limiting.

[0160] material 4-arm PEG maleimide (PEG-Mal, molecular weight 5, 10, and 20 kDa, JenKem), PEG bisthiol (molecular weight 1 kDa, Sigma Aldrich), gelatin (from bovine, fish, or porcine sources, Sigma Aldrich), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, Sigma Aldrich), N-hydroxysulfosuccinimide (Sulfo-NHS, Sigma Aldrich), sodium hydroxide (NaOH), CRGDS, CIKVAV, CYIGSR, GCRDGPQGIWGQDRCG, GCRDPLGLDRCG, and GCIPVSLRSGCG (>90%, Genscript), phosphate buffered saline without calcium chloride (PBS, Gibco), Dulbecco's modified Eagle's medium (DMEM, Gibco), calcium-free DMEM (Gibco), sodium pyruvate (Gibco), L-glutamine (Gibco), trypsin (Sigma Aldrich), 0.22 μm syringe filters (polyethersulfone membrane, Merck Millipore) T75 and T150 flasks (Corning), 15 mL centrifuge tubes (Corning), and ethanol (Sigma Aldrich) were used as received.

[0161] Example 1 - Preparation of PEG-Mal polymer bioink Example 1a - Preparation of polymer bioink in PBS Three types of PEG-Mal polymer bioinks were prepared in PBS buffer.

[0162] PEG-Mal polymer bioink PEG-Mal polymer bioink was prepared by mixing PEG-Mal (7.5 wt%) in PBS buffer at room temperature. After complete dissolution, the solution was sterile filtered through a 0.22 μm syringe filter.

[0163] PEG-RGD polymer bioink RGD-containing PEG-Mal (PEG-RGD) polymer bioink was prepared by mixing CRGDS with PEG-Mal (at 10 wt%) in PBS buffer for 30 min at room temperature to obtain a total RGD concentration of 5 mM. Completion of the reaction was confirmed by Ellman's assay when no free thiols were recorded. The reaction product contained PEG-RGD at 10 wt% and 5 mM RGD concentration and was dissolved in PBS. After complete dissolution, NaOH at equimolar concentration to the RGD concentration was added to the solution to raise the pH of the solution to 7.4. The solution was sterilized by aseptic filtration through a 0.22 μm syringe filter.

[0164] PEG-tripeptide polymer bioink PEG-Mal containing RGD, IKVAV and YIGSR (PEG-tripeptide) polymer bioink was prepared by mixing CRGDS, CIKVAV and CYIGSR (at the desired molar concentration) with PEG-Mal (at 15 wt%) in PBS buffer. The solution was allowed to dissolve at room temperature for 30 min to obtain 15 wt% PEG-tripeptide and 0.67 mM RGD, YIGSR, and IKVAV. After complete dissolution, the pH of the solution was raised to 7.4 by dropwise addition of 1 M NaOH. The resulting solution was then sterilized by aseptically filtering through a 0.22 μm syringe filter.

[0165] Figure 2 shows the analysis of the modification of 4-arm PEG-Mal with CRGDS. In the UV analysis, a clear peak was observed at 412 nm at the beginning of the reaction (Figure 2(a)). This is the TNB produced when the disulfide bond of Ellman's reagent is cleaved and ionized by the thiol moiety (CRGDS in this case). 2- This represents the presence of the (2-nitro-5-thiobenzoate) ion. In contrast, such a peak was not present in the 4-arm PEG-Mal solution without CRGDS, suggesting that the 4-arm PEG-Mal had not reacted with Ellman's reagent after the reaction was allowed to proceed for 30 min, whereas CRGDS reacted completely after 30 min, respectively. Quantification of CRGDS binding was 1This was confirmed by comparing the amide and maleimide proton peaks using 1 H NMR (Figure 2(b)).

[0166] Example 1b - Preparation of polymer bioink in cell culture medium An RGD-containing PEG-Mal polymer bioink in cell culture medium was prepared according to the procedure in Example 1a, using the appropriate cell culture solution for the cells to be printed instead of the PBS solution. The resulting polymer bioink was aseptically sterilized by filtration (0.22 μm filter).

[0167] The polymer bioink prepared according to Example 1 was stored at -20°C until required for further use. The polymer bioink can be stored at -20°C for at least 6 months without affecting its functionality.

[0168] Example 2 - Preparation of PEG bisthiol and GCIPVSLRSGCG activator Example 2a - Preparation of activator in PBS Activators were prepared by mixing dithiothreitol (DTT), PEG bisthiols, GCIPVSLRSGCG, and GCRDPLGLDRCG, or various combinations thereof, in PBS at a total thiol concentration equimolar to the maleimide concentration of the complementary polymer bioink. The resulting solution was sterilized by aseptic filtration through a 0.22 μm syringe filter.

[0169] Example 2b - Preparation of activator in cell culture medium The activators were prepared by mixing dithiothreitol (DTT), PEG bisthiol, GCIPVSLRSGCG, and GCRDPLGLDRCG, at a total thiol concentration equimolar to the maleimide concentration of the complementary bioink, in a cell culture solution appropriate for the cells to be printed. The resulting solution was sterilized by aseptic filtration (0.22 μm filter).

[0170] The activator prepared according to Example 2 was stored at -20°C until required for further use. The activator can be stored at -20°C for at least 6 months without affecting its functionality.

[0171] Example 3 - Cell culture SK-N-BE(2) (human neuroblastoma), Kras G12D and p53R 172H (pancreatic ductal adenocarcinoma), MCF7 (human breast cancer), and U87vIII (human glioblastoma) cells were maintained separately in 10% fetal calf serum (FCS) / DMEM at 37°C / 5% CO2. Human non-small cell lung cancer H460 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium and 10% FCS at 37°C / 5% CO2. Cell lines were routinely screened and free of mycoplasma contamination.

[0172] Example 4 - Trypan blue dye exclusion test SK-N-BE(2), Kras G12D and p53R 172H , H460, and U87vIII cells were harvested from T75 flasks using trypsin / PBS and pelleted using centrifugation. Cells were resuspended in polymer bioink containing PEG-Mal and PEG-RGD at 10 wt % and 5 mM RGD concentration to a cell concentration of 2 × 10 6 cells / mL. Samples were divided into two groups: printed and non-printed controls. Printed samples were passed through a 3D bioprinter and kept in sterile conditions at room temperature for 30 min. Dead cells were identified using 0.4% trypan blue staining (Invitrogen). Cell counts were obtained by counting the number of live and dead cells in each sample. Cell viability was determined by dividing the total number of live cells by the total number of cells (live + dead).

[0173] Example 5 – Preparation of cell-polymer bioink Cultured cells of interest at a particular culture density were harvested according to the procedure in Example 4. To construct the cell-polymer bioink, the harvested cells were resuspended in the sterile polymer bioink prepared according to Example 1(a) or 1(b) at the appropriate cell concentration, up to 2 × 10 6 Obtain 1 mL of sterile polymer bioink with cells / mL.

[0174] Example 6 - Preparation of cell activator The cultured cells of interest at a specific culture density were harvested according to the procedure of Example 4. To construct the cell activator, the harvested cells were resuspended at an appropriate cell concentration in the sterile activator prepared according to Example 2(a) or 2(b).

[0175] Example 7 -3D cell viability assay Live / dead staining was used to monitor the viability of hydrogel-encapsulated cells. Calcein AM (1 μL mL) in DMEM was used. -1 ) and ethidium homodimer-I (4 μL mL -1 ) were used to stain live and dead cells, respectively. Hydrogels with encapsulated cells were 3D bioprinted using the cell-polymer bioink prepared according to Example 5. After the required incubation period, the bioprinted hydrogels were incubated with the live / dead stain for at least 2 hours at 37° C. / 5% CO2 before confocal fluorescence microscopy analysis.

[0176] Example 8 - Rheological Analysis The mechanical properties were studied on hydrogels prepared from 4-arm PEG-Mal hydrogels with 5 mM RGD (10 wt %) (prepared according to Example 1(a)) and crosslinked with PEG bis-thiol and the MMP2-sensitive bis-cysteine ​​peptide GCIPVSLRSGCG (mixed at 50:50 thiol molar concentration) (prepared according to Example 2(a)).

[0177] Hydrogel rheology was measured using an Anton Paar Modular Compact Rheometer (MCR) 302 equipped with a concentric cylindrical spindle and cup system. Viscosity measurements were performed from 1 to 1000 s -1The shear rate values ​​were performed at 100-200 Hz. The rheological properties of the hydrogels were measured using the same machine equipped with a parallel plate geometry (25 mm diameter) system and a solvent trap to minimize evaporation of the liquid. The rheological measurements were performed on a preheated plate and maintained at a constant temperature of 37 °C. Amplitude strain sweep experiments (γ: 1 100%) were performed to determine the linear viscoelastic region (LVR) at a fixed frequency of 10 Hz. Having established the LVR for each sample, dynamic frequency sweeps were performed at fixed strains in the linear viscoelastic region of the hydrogels (ω: 0.1–10 Hz). Time sweep measurements were typically performed at a fixed frequency (10 Hz) and strain amplitude (within the LVR).

[0178] As shown in Figure 3, frequency (Figure 3(a)) and strain (Figure 3(b)) sweep studies confirmed that the mechanical properties of the gels were independent of these two parameters.

[0179] Subsequent stiffness determinations were performed at constant frequency and strain values ​​within the viscoelastic region of the hydrogel (Figure 3(c)). Measurements were performed within 1 min of casting the hydrogel. Figure 3(c) shows that the storage modulus (G') values ​​were always higher than the loss modulus (G'') values ​​throughout the measurement period. The constant storage modulus value of approximately 1.1 kPa indicates that the gelation process was complete within 1 min. Viscosity studies of both the polymer bioink and the active agent showed viscosity values ​​below 10 cP, making both the polymer bioink and the active agent highly suitable for the drop-on-demand bioprinting process (Figure 3(d)).

[0180] Mechanical properties were also studied for hydrogels prepared from a 4-arm PEG-Mal blank polymer bioink (prepared according to Example 1(a)) crosslinked with PEG bisthiol (prepared according to Example 2(a)), and a hydrogel prepared from a PEG tripeptide crosslinked with an MMP-sensitive activator ((GCIPVSLRSGCG) prepared according to Example 2(a)). Stiffness determinations were performed at constant frequency and strain values ​​within the viscoelastic region for PEG-Mal hydrogels (Figure 4(a)) and PEG tripeptide hydrogels (Figure 4(b)). Measurements were performed within 1 min of casting the hydrogels. Figures 4(a) and 4(b) show that the storage modulus (G') values ​​were always higher than the loss modulus (G'') values ​​throughout the measurement period. This observation confirmed the presence of a hydrogel when the bioink and activator were mixed. Quantitatively, the stiffness value of the hydrogel formed from the PEG-Mal blank polymer bioink and the PEG bis-activator was approximately 0.2 kPa, and the stiffness value of the PEG tripeptide / MMP-sensitive hydrogel was approximately 1.6 kPa.

[0181] Example 9 -3D Bioprinting Platform The components of the 3D bioprinter used in this example include a three-axis motion control stage, a drop-on-demand droplet dispensing system, and a pressure regulator to control the pressure in the fluid reservoir. The droplet dispensing system and three-axis stage can be housed in a sterile chamber such as a laminar flow cabinet. The printing platform includes adapters for printing on various types of substrates, such as microwell plates and petri dishes. The printing platform can be heated to 37°C to support cell growth.

[0182] A three-axis motion control stage (MX80S, Parker) can precisely position the droplet dispensing systems with a resolution of 10 μm in all three (X, Y and Z) dimensions. The 10 droplet dispensing systems consist of a solenoid valve (VHS series solenoid valve, The Lee Company) with a jewelled orifice dispensing nozzle (MINSTAC Nozzle, The Lee Company) controlled by a microcontroller (Arduino Mega 2560, Arduino). The inner diameter of the jewelled orifice nozzle can be from 127 to 254 μm, depending on the viscosity of the fluid and the desired droplet volume. Each droplet dispensing system is attached to a static pressure reservoir through which the polymer bioink and activator solution are dispensed via flexible tubing. The back pressure of the fluid reservoir and the opening time of the solenoid valve can also be used to adjust the required droplet volume. Typically, the back pressure is set at a pressure of 1 to 300 kPa, the opening time of the solenoid valve is 0.3 ms or more, and the droplet volume is 1 to 500 nL. The flexible tubing connecting the polymer bioink and the activator reservoir was kept as short as possible to minimize the time taken for priming the system and purging at the end of the printing routine.

[0183] Example 10 -Control Software The 3D bioprinter was controlled via custom software developed to print the biological assays. The software includes a graphical user interface (GUI) and is written in the Python programming language. Through the GUI, end users can select different assay printing routines and modify assay parameters such as droplet spacing and droplet volume. Users can also manually control the spatial position of the droplet dispensing system and create custom patterns of droplets. Additional features of the software include routines for priming and purging the droplet dispensing system.

[0184] Example 11 -3D Bioprinting Sterile polymer bioink and activator were heated to room temperature prior to printing. Cells of interest were mixed with the polymer bioink and / or activator at the desired cell concentration. All solutions were then loaded into the sample tray of the bioprinter. Bioprinting was performed using a Rastrum™ modular 3D bioprinter (Inventia Life Science (ILS)). Structures were designed using Rastrum™ software (ILS). Two different bioprint samples were printed; a cylindrical structure with a diameter of approximately 6.35 cm and a thickness of 400 μm and a hemispherical structure with a diameter of 100 μm, on top of an inert gel layer with a diameter of 6.35 cm and a thickness of 100 μm. Both polymer bioink and activator solutions were printed with a printing pressure of 20-100 kPa. Prior to the bioprinting process, the Rastrum™ bioprinter performed an automated sterilization and priming step.

[0185] The generation of 3D objects was achieved by the deposition of a polymer bioink droplet, followed immediately after by the deposition of an activator droplet on top of it, at a position determined by the software. All printing was performed in a 96-well plate (Corning). Once complete, cell culture medium (150 μL) associated with the cells was added and the plate was moved to an incubator.

[0186] FIG. 1 is a representative schematic of the 3D bioprinting process. The polymer bioink used contained 4-arm PEG maleimide (PEG-Mal), CRGDS, and the desired cancer cells (prepared according to Examples 1(a) and 5). The activator contained a mixture of PEG bis-thiol and bis-thiol MMP-sensitive peptide GCIPVSLRSGCG (prepared according to Example 2(a)). Both solutions were loaded into an ILS 3D bioprinter system according to Example 9, which deposited the polymer bioink and activator combination in a spatially controlled manner. Mixing both solutions resulted in immediate gelation, generating cells three-dimensionally encapsulated within the hydrogel in a 96-well plate.

[0187] Example 12 - Pancreatic ductal adenocarcinoma (Kras G12D and p53R 172H ) 3D Bioprinting Multiple samples of three-dimensionally encapsulated cancer cells within thiol-maleimide cross-linked PEG hydrogels were bioprinted using an ILS 3D bioprinter. Specifically, simultaneous delivery of cancer cells, polymer bioink, and activating agents was performed using the bioprinter to achieve rapid creation of in situ cell encapsulation within the bioprinted hydrogels.

[0188] cell culture Pancreatic ductal adenocarcinoma Kras cells at 80% confluency in a T150 flask G12D and p53R 172H The flask was washed with 3 mL of PBS. After aspirating excess PBS, 3 mL of trypsin was added and the flask was incubated at 37 °C to dissociate the cells from the flask surface for 5 min. Then, 7 mL of tissue culture medium prepared by mixing DMEM and FCS at 10 v / v% was added, and the dissociated cells were transferred to a 15 mL tube. The cell dispersion was centrifuged at 400 g for 3 min. The supernatant was discarded and the cell pellet was resuspended in 5 mL of medium. Cell counting was then performed by mixing equal volumes of cell suspension and trypan blue stain to determine the cell concentration.

[0189] substrate The assembled 96-well plates supplied by Corning were used as received. The sterile packaging was opened in a sterile environment to maintain the sterility of the contents.

[0190] Polymer Bioink To prepare the polymer bioink, PEG-Mal (0.150 g) was dissolved in 1.5 mL of PBS to obtain a 10 wt% polymer bioink. For the incorporation of PEG-RGD, CRGDS (1.34 mg) was added to the solution and stirred for 30 min to obtain a 10 wt% PEG-Mal / PEG-RGD polymer bioink with 5 mM RGD. To this solution, 2.5 μL of 1 M NaOH was added to raise the pH of the solution to 7.4. The solution was then filtered through a 0.22 μm syringe filter in a sterile environment.

[0191] 2×10 6 Collected Kras cells / mL dispersed in 1 mL of DMEM G12D and p53R 172H The pellet was then redispersed in polymer bioink (1 mL) to obtain a cell pellet of 500,000 cells. 6 Kras cells / mL G12D and p53R 172H A cell-polymer bioink solution in which cells were dispersed was obtained.

[0192] Activator Two different activators were prepared to generate both non-MMP and MMP-sensitive hydrogels. The two activators were prepared from either PEG bisthiol (0.01975 g) alone or a combination of PEG bisthiol (0.0096 g) and GCIPVSLRSGCG (0.0111 g), each dissolved in 1 mL PBS to yield an activator with a total thiol concentration equimolar to the maleimide concentration of the polymer bioink. Each solution was then filtered through a 0.22 μm syringe filter in a sterile environment.

[0193] Cell printing conditions The cellular polymer bioink and activator were loaded into associated cartridges that were connected to the bioprinter. Both the cellular polymer bioink and activator were connected to a 0.007” nozzle and operated at 10 Psi.

[0194] The printer was first cleaned by wiping it down with ethanol (70 v / v% in water) and air drying. The fluid lines, valves, and nozzles were also sterilized by purging with ethanol, water, and tissue culture medium, in that order. Next, the cellular polymer bioink and activator were loaded into their respective vials. The printhead was then primed before starting the printing routine.

[0195] The 3D structure of the assay was designed using ILS custom-made software. The 3D assay consisted of three layers of circular gels with a total diameter of 6.35 mm. Bioprinting of the 3D assay was performed by repeatedly depositing droplets of polymer bioink and activator pairs at positions determined via the software.

[0196] cell concentration 2×10 6 A cell-polymer bioink at a concentration of cells / mL was used in this experiment.

[0197] Cell viability More than 95% cell viability was obtained.

[0198] Example 13 – 3D bioprinting of human breast cancer cells (MCF7) cell culture MCF7 cells at 80% confluency in a T75 flask were washed with 3 mL of PBS. After aspirating excess PBS, 1 mL of trypsin was added and the flask was incubated at 37°C to dissociate the cells from the flask surface for 3 min. Then, 7 mL of tissue culture medium prepared by mixing DMEM and FCS at 10 v / v% was added, and the dissociated cells were transferred to a 15 mL tube. The cell dispersion was centrifuged at 400 g for 3 min. The supernatant was discarded and the cell pellet was resuspended in 5 mL of medium. Cell counting was then performed by mixing equal volumes of cell suspension and trypan blue stain to determine the cell concentration.

[0199] substrate The assembled 96-well plates supplied by Corning were used as received. The sterile packaging was opened in a sterile environment to maintain the sterility of the contents.

[0200] Polymer Bioink To prepare the polymer bioink, PEG-Mal (0.1125 g) was dissolved in 1.5 mL of PBS to obtain a 7.5 wt% polymer bioink. The PEG tripeptide was prepared by mixing CRGDS (0.54 mg), CIKVAV (0.64 mg), CYIGSR (0.70 mg), and PEG-Mal (0.225 g) in 1.5 mL of PBS for 30 min to obtain 15 wt% PEG-tripeptide and 0.67 mM RGD, YIGSR, and IKVAV. The pH of the solution was raised to 7.4 by dropwise addition of 1 M NaOH. Both solutions were sterile filtered through a 0.22 μm syringe filter.

[0201] Activator Two different activators were prepared to generate both non-MMP and MMP-sensitive hydrogels. The two activators were prepared from either PEG bisthiol (0.0015 g), or GCRDPLGLDRCG (0.019 g), each dissolved in 1 mL PBS to yield an activator with a total thiol concentration equimolar to the maleimide concentration of the blank bioink and PEG tripeptide, respectively. The MMP-sensitive activator solution was then added dropwise with 1 M NaOH to raise the pH to 7.4. Each solution was then filtered through a 0.22 μm syringe filter in a sterile environment.

[0202] The collected MCF7 cells dispersed in 1 mL of DMEM were centrifuged to obtain a cell pellet of 10.000,000 cells. The resulting pellet was then resuspended in activator (1 mL) to obtain a cell pellet of 1 × 10 MCF7 cells. 7 A cell-MMP-activator solution was obtained, dispersed in cells / mL.

[0203] Cell printing conditions The polymer bioink, PEG bis-thiol activator, and MMP-sensitive cell activator were loaded into associated cartridges connected to the bioprinter. The polymer bioink, PEG bis-thiol activator, and MMP-sensitive cell activator were each connected to a 0.007” nozzle and operated at 25–30 kPa.

[0204] The printer was first cleaned by wiping with ethanol (70 v / v% in water) and air drying. The fluid lines, valves, and nozzles were also sterilized by ethanol, then a sterile water purge, in that order. Next, the polymer bioink, PEG bisthiol activator, and MMP-sensitive cell activator were loaded into their respective vials. The printhead was then primed before starting the printing routine.

[0205] The 3D structure of the assay was designed using ILS custom-made software. The 3D assay consisted of a 6.35 cm diameter, 100 μm thick lower gel layer and a 1 mm diameter, 200 μm thick dome-shaped gel containing the cells.

[0206] cell concentration 1×10 7 Cell activators at a concentration of cells / mL were used in this experiment.

[0207] Cell viability More than 95% cell viability was obtained.

[0208] Example 14 - Cell sequence comparison The effect of hydrogel microenvironment on pancreatic cancer cell arrangement was tested using the polymer bioink and activator of the present technology. Pancreatic cancer cells were chosen because their biological properties are highly dependent on the cell culture environment. Two different cell arrangements were tested by bioprinting hydrogel samples with a diameter of 6 mm and a height of 0.4 mm into 96-well plates. First, cells were 3D bioprinted on top of the hydrogel, and then cells were printed inside the hydrogel (i.e., encapsulated). In both cell arrangements tested, live cells were observed. Furthermore, cell-gel interactions were visible, as represented by the ability of the cells to form protrusions into the hydrogel. Overall, biocompatible and biorelevant 3D assays were successfully bioprinted with the PEG-based polymer bioink.

[0209] Pancreatic cancer cells seeded on hydrogel The cell-polymer bioink was developed by injecting collected pancreatic ductal adenocarcinoma cells (Kras G12D and p53R 172H ) and a 4-arm PEG-Mal (10 kDa) with 5 mM RGD polymer bioink for 3D bioprinting. Pancreatic cancer cells were dispersed in the cell-polymer bioink at a concentration appropriate for seeding an estimated 5,000 cells / well.

[0210] Acellular hydrogels were first 3D bioprinted onto 96-well plates from 4-arm PEG-Mal (10 kDa) with 5 mM RGD polymer bioink and PEG bis-thiol and MMP2 (GCIPVSLRSGCG) activator (at a thiol concentration ratio of 50:50). A cellular polymer bioink was then printed on top of the hydrogel with PEG bis-thiol and MMP2 activator (at a thiol concentration ratio of 50:50) and seeded with an estimated 5,000 cells / well.

[0211] Microscopic images were taken of pancreatic cancer cells seeded on hydrogels. After 3 days of incubation, morphological differences were observed for the same cells when seeded on tissue culture plastic. Seeding on hydrogels promoted both cell-gel and cell-cell interactions, resulting in the formation of extended cell networks with extensive cell processes in all three dimensions. After 3 days of incubation, cultured cells were confirmed to be viable by live / dead staining assay. Cell viability was confirmed to be >98%, with small individual dead cells clearly visible within larger live cell clusters.

[0212] Pancreatic cancer cells were also seeded on various substrates for comparison. Microscopic images showed that the pancreatic cancer cells seeded on the tissue culture well plate were highly individual. In contrast, the pancreatic cancer cells formed an extended network between multiple cells when seeded on the hydrogel. While seeded on the hydrogel, the cells were able to invade the hydrogel and form a 3D cellular network. Scan the gel in the z-axis with a microscope to confirm the presence of a 3D cellular network within the hydrogel.

[0213] Pancreatic cancer cells encapsulated in biologically inert hydrogels Pancreatic ductal adenocarcinoma cells (Kras G12D and p53R 172H ) into the polymer bioink without PEG-RGD at 2 × 10 6Dispersed at 1000 cells / mL, the cell-polymer bioink and cell-polymer bioink were printed with activators without MMP2-sensitive peptide. In situ encapsulation was achieved by direct 3D bioprinting of the cell bioink with PEG bisthiol activator on 96-well plates.

[0214] Confocal images showed pancreatic cancer cells encapsulated within the hydrogel. The cell morphology was confirmed over the incubation period. Due to the absence of RGD and MMP2 crosslinkers, the cells had a rounded morphology within the gel. This morphology indicated that the cells were unable to interact with the hydrogel to generate focal adhesions and were unable to migrate within the gel. Live / dead assays were performed on the encapsulated cells after a 3-day incubation period. Fluorescence images showed live cells (>95% viability) after 3 days of incubation.

[0215] Pancreatic cancer cells encapsulated in biologically active hydrogels Pancreatic ductal adenocarcinoma cells (Kras G12D and p53R 172H ) to 2 x 10 6 Cells were dispersed in a cell-polymer bioink at 1000 x 1000 cells / mL and printed together with an MMP2-sensitive peptide activator (at a 50:50 thiol concentration ratio). In situ encapsulation was achieved by direct 3D bioprinting of the cell-polymer bioink with PEG bis-thiol and MMP-2 activator onto 96-well plates. The MMP-2 activator was used to allow cell infiltration.

[0216] Confocal images showed pancreatic cancer cells encapsulated within the hydrogel. Morphological changes of the cells were confirmed over the incubation period. Inside the gel, cell-cell interactions were represented by the formation of cell clusters, while cell-gel interactions and cell invasion were confirmed by the presence of cell membrane protrusions. Live / dead assays were performed on the encapsulated cells after 3-day and 6-day incubation periods. Bright field and fluorescent images showed live cells (>98% viability) after 3 days of incubation, confirming the presence of cell-cell interactions and cell-gel interactions. Cell invasion was more prominent at 6 days of incubation, indicating strong cell-gel interactions. At the same time, cell viability above 98% was maintained after 6 days of culture within the gel.

[0217] MCF7 breast cancer cells seeded in hydrogels Cell activity was confirmed via observation of cell morphology under a bright-field microscope after 3 days of incubation. After the incubation period, aggregation of single MCF7 cells was observed, forming multiple spheroids in predefined gel regions, indicating that individual MCF7 cells could migrate relative to each other within the hydrogel.

[0218] Example 15 - Modification of gelatin from cold water fish skin with maleimide groups Gelatin (1 g) from fish skin was dissolved in 20 mL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer at 40 °C. In a separate container, 6-maleimidohexanoic acid (MHA; 0.211 g), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, at 1:20 EDC:MHA molar ratio), and N-hydroxysuccinimide (NHS, at 1:2 NHS:EDC molar ratio) were dissolved in 1 mL of MES buffer and stirred for 20 min to activate the carboxylic acid (COOH) groups. The solution of activated COOH groups was then transferred to the gelatin solution, and the reaction was allowed to proceed for 24 h. The resulting solution was purified by dialysis against 10 mM hydrochloric acid (HCl) and 1 wt% sodium chloride (NaCl) for 2 days, followed by dialysis against 10 mM HCl for 1 day. The purified gelatin solution was then freeze-dried to obtain maleimide-modified fish gelatin.

[0219] Example 16 - Modification of gelatin from pigskin with maleimide groups Gelatin from porcine skin (gel strength 300, type A, 1 g) was dissolved in 20 mL of MES buffer at 40 °C. In a separate container, MHA (0.139 g), EDC (at 1:20 EDC:MHA molar ratio) and N-hydroxysuccinimide (NHS, at 1:2 NHS:EDC molar ratio) were dissolved in 1 mL MES buffer and stirred for 20 min to activate the COOH groups. The solution of activated COOH groups was then transferred to the gelatin solution and the reaction was allowed to proceed for 24 h. The resulting solution was purified by dialysis against 10 mM HCl and 1 wt% NaCl for 2 days, followed by dialysis against 10 mM HCl for 1 day. The purified gelatin solution was then freeze-dried to obtain maleimide-modified porcine gelatin.

[0220] Example 17 - Gelation of maleimide-modified gelatin The maleimide-modified gelatins from both pig and cold water fish skins, synthesized in Examples 15 and 16, were dissolved in 20 wt% PBS to obtain polymer bioinks. The pH of each solution was neutralized to pH 7.4 with 1 M NaOH. To prepare the activator, PEG bisthiol with SH molar concentration equal to the maleimide molar concentration was dissolved in PBS. Gelation was achieved by mixing equal amounts of each polymer bioink and the activator.

[0221] Example 18 – 3D bioprinting of collagen-containing PEG hydrogels substrate Cell culture dishes supplied by Thermofisher were etched with a target grid that mimicked the dimensions of a 96-well plate.

[0222] Polymer Bioink To prepare the polymer bioink, PEG-Mal (0.15 g) was dissolved in 1.5 mL of PBS to obtain a 10 wt% polymer bioink. The solution was sterile filtered through a 0.22 μm syringe filter.

[0223] Activator A collagen-containing activator was prepared by mixing GCRDPLGLDRCG (0.01 g) into 0.5 mL of PBS. Then, 0.5 mL of type I bovine collagen (3.1 mg) was mixed into the activator solution to obtain a collagen-containing MMP activator. The pH of the solution was then adjusted to pH 7.4 by adding 1 M NaOH dropwise. The solution was then sterile filtered through a 0.22 μm syringe filter.

[0224] Printing conditions The polymer bioink and collagen-containing MMP activator were loaded into appropriate cartridges that were connected to the bioprinter. Both the polymer bioink and activator were connected to a 0.007” nozzle and operated at 25-30 kPa.

[0225] The 3D structure of the assay was designed using ILS custom-made software. The 3D rectangular prism structure consisted of a lower gel layer with a length and width of approximately 4.5 cm and a thickness of 150 μm.

[0226] Validating the structure The resulting hydrogel structures were imaged using a USB digital microscope.

[0227] Example 19 - Fibroblast culture in collagen-containing PEG hydrogels Collagen-containing polymer bioink was prepared by mixing 10 wt% PEG-Mal and type I bovine collagen in PBS in a 1:1 v / v ratio. The pH of the solution was raised to 7.4 by dropwise addition of 1 M NaOH solution. PEG bis-thiol activator with equimolar thiol to maleimide concentration was prepared in PBS. Harvested human lung fibroblast (MRC-5) cells were pelleted and resuspended in collagen-containing polymer bioink. PEG hydrogels were prepared by first transferring the polymer bioink into a well plate, followed by transferring an equal amount of cell-containing activator into the same well. Then, 100 μL of DMEM + 10 v / v% FBS was added to each well, and the samples were incubated for 6 days, with bright field images taken at days 0, 3, and 6 to monitor cell activity.

[0228] The biocompatibility of the gels for fibroblast culture was assessed by examining the change in fibroblast morphology from round to spindle-like morphology.

[0229] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. The invention as broadly described should be understood to include all such variations and modifications without departing from the spirit or scope of the invention. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0230] The present disclosure also includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of any two or more of such steps, features, compositions, and compounds.

[0231] References 1. Caliari, SR; Burdick, JA, A practical guide to hydrogels for cell culture. Nature Methods 13, 405 (2016). 2. Murphy, S. V.; Atala, A., 3D bioprinting of tissues and organs. Nature Biotechnology 32, 773 (2014). 3. Donderwinkel, I.; van Hest, J. C. M.; Cameron, N. R., Bio-inks for 3D bioprinting: recent advances and future prospects. Polymer Chemistry 8, 4451-4471 (2017). 4. Jungst, T.; Smolan, W.; Schacht, K.; Scheibel, T.; Groll, J., Strategies and Molecular Design Criteria for 3D Printable Hydrogels. Chemical Reviews 116, 1496-1539 (2016). 5. Lowe, S. B.; Tan, V. T. G.; Soeriyadi, A. H.; Davis, T. P.; Gooding, J. J., Synthesis and High-Throughput Processing of Polymeric Hydrogels for 3D Cell Culture. Bioconjugate Chemistry 25, 1581-1601 (2014). 6. Gao, G.; Hubbell, K.; Schilling, A. F.; Dai, G.; Cui, X., Bioprinting Cartilage Tissue from Mesenchymal Stem Cells and PEG Hydrogel. In 3D Cell Culture: Methods and Protocols, Koledova, Z., Ed. Springer New York: New York, NY, 2017; pp 391-398. 7. Gao, G.; Yonezawa, T.; Hubbell, K.; Dai, G.; Cui, X., Inkjet-bioprinted acrylated peptides and PEG hydrogel with human mesenchymal stem cells promote robust bone and cartilage formation with minimal printhead clogging. Biotechnology Journal 10, 1568-1577 (2015).

Claims

1. 3D printed hydrogels formed from maleimide-containing polymers crosslinked using a bis-thiol-containing crosslinker having at least two thiol functional groups.

2. Maleimide-containing polymers include maleimide-containing polysaccharides, including polymers containing fructose, sucrose, or glucose monomers; poly(ethylene glycol) (PEG) maleimide, poly(hydroxyethyl methacrylate) (PHEMA) maleimide, poly(ε-caprolactone) (PCL) maleimide, poly(vinyl alcohol) (PVA) maleimide, poly(vinylpyrrolidone) (PVP) maleimide, poly(N-isopropylacrylamide) (NIPAAM) maleimide, poly(propylene fumarate) (PPF) maleimide, poly(ethyleneimine) (PEI) maleimide, poly(3-methacrylamidopropyl)trimethylammonium (PMAETMA) maleimide, poly( L 2. The 3D printed hydrogel of claim 1, wherein the hydrogel is selected from the group consisting of synthetic polymers including poly(acrylic acid) (PAA) maleimide, poly(styrenesulfonate) (PSS) maleimide, poly(acrylic acid-stat-dimethylaminoethyl methacrylamide) (P(AA-stat-DMAEMA)) maleimide, and poly(arginine methacrylate) maleimide, or derivatives thereof; maleimide-containing biopolymers including gelatin maleimide, cellulose maleimide, hyaluronic acid maleimide, and alginate maleimide; maleimide-containing nucleobase polymers comprising adenine, thiamine, guanine, and / or cytosine repeating units; and any combination thereof.

3. 3. The 3D printed hydrogel of claim 2, wherein the maleimide-containing polymer comprises PEG-maleimide.

4. 3. The 3D printed hydrogel of claim 2, wherein the maleimide-containing polymer comprises gelatin maleimide.

5. The 3D printed hydrogel of any one of claims 1 to 4, wherein the bis-thiol containing crosslinker is selected from the group consisting of synthetic polymers, biopolymers, small molecules, bioactive molecules, and any combination thereof.

6. 6. The 3D printed hydrogel of claim 5, wherein the synthetic polymer is selected from the group consisting of PEG-bis-thiol, poly(N-isopropylacrylamide) bisthiol (NIPAAM bisthiol), poly(acrylic acid) bisthiol, poly(methacrylic acid) bisthiol, poly(styrenesulfonate) bisthiol, poly(amide) bisthiol, and any combination thereof.

7. 7. The 3D printed hydrogel of claim 6, wherein the bis-thiol containing crosslinker is PEG bis-thiol.

8. 6. The 3D printed hydrogel of claim 5, wherein the biopolymer is selected from the group consisting of thiol-gelatin, thiol-cellulose, thiol-chitosan, thiol-hyaluronic acid, and any combination thereof.

9. 6. The 3D printed hydrogel of claim 5, wherein the small molecule is dithiothreitol (DTT).

10. The 3D printed hydrogel of claim 5, wherein the bioactive molecule is selected from the group consisting of short peptides having at least two cysteine ​​amino acid groups, inactive short peptides, enzyme-sensitive short peptides, matrix metalloproteinase (MMP)-sensitive peptides, and any combination thereof.

11. The 3D printed hydrogel of claim 10, wherein the bioactive molecule is an MMP-sensitive bis-cysteine ​​peptide.

12. 12. The 3D printed hydrogel of claim 11, wherein the MMP sensitive peptide is selected from the group consisting of GCIPVSLRSGCG, GCRDGPQGIWGQDRCG, GCRDPLGLDRCG, GCRDEAPLKQDRCG, and any combination thereof.

13. 2. The 3D printed hydrogel of claim 1, wherein the maleimide-containing polymer is a PEG-maleimide and the bis-thiol-containing crosslinker is selected from the group consisting of PEG-bis-thiol, an MMP-sensitive peptide, and combinations thereof.

14. 2. The 3D printed hydrogel of claim 1, wherein the maleimide-containing polymer is a PEG maleimide substituted with at least one bioactive molecule selected from RGD, YIGSR, and IKVAV, and the bis-thiol-containing crosslinker is selected from a PEG bis-thiol, an MMP-sensitive peptide, and combinations thereof.

15. The 3D printed hydrogel of claim 13 or 14, wherein the MMP sensitive peptide is GCIPVSLRSGCG, GCRDPLGLDRCG, or a combination thereof.

16. 2. The 3D printed hydrogel of claim 1, wherein the maleimide-containing polymer is gelatin maleimide and the bis-thiol-containing crosslinker is PEG bis-thiol.

17. 17. The 3D printed hydrogel of any one of claims 1 to 16, wherein the molar ratio of maleimide-containing polymer to bisthiol-containing crosslinker ranges from 10:1 to 1:

10.

18. 18. The 3D printed hydrogel of any one of claims 1 to 17, comprising cells.

19. 20. The 3D printed hydrogel of claim 18, wherein the cells are suspended within a portion of the hydrogel, or the cells are suspended substantially uniformly throughout the hydrogel.

20. The concentration of printed cells is 1 × 10 5 ~5×10 8 20. The 3D printed hydrogel of claim 18 or 19, in the range of cells / mL.

21. 21. The 3D printed hydrogel of any one of claims 18 to 20, wherein the cells are selected from the group consisting of hepatic cells, gastrointestinal cells, pancreatic cells, renal cells, lung cells, tracheal cells, vascular cells, skeletal muscle cells, cardiac cells, skin cells, smooth muscle cells, connective tissue cells, corneal cells, urogenital cells, breast cells, germ cells, endothelial cells, epithelial cells, fibroblasts, neural cells, Schwann cells, adipocytes, bone cells, bone marrow cells, chondrocytes, pericytes, mesothelial cells, endocrine tissue derived cells, stromal cells, stem cells, progenitor cells, lymphatic cells, blood cells, endoderm derived cells, ectoderm derived cells, and mesoderm derived cells, and any combination thereof.

22. 22. The 3D printed hydrogel of any one of claims 1 to 21, further comprising a bioactive molecule.

23. 23. The 3D printed hydrogel of claim 22, wherein the bioactive molecule is a free bioactive molecule or is conjugated to a maleimide-containing polymer, a bis-thiol-containing crosslinker, or both a maleimide-containing polymer and a bis-thiol-containing crosslinker.

24. 24. The 3D printed hydrogel of claim 22 or 23, wherein the bioactive molecule is selected from the group consisting of peptides, MMP sensitive peptides, proteins, polysaccharides, drugs, therapeutic agents, antibodies, small molecule inhibitors, kinase inhibitors, phosphatase inhibitors, antigens, pathogens, platelets, growth factors, cytokines, amino acids, nutrients, conditioned media, antibiotics, antivirals, RNA, and any combination thereof.

25. 25. The 3D printed hydrogel of claim 24, wherein the bioactive molecule is collagen.

26. The 3D printed hydrogel of any one of claims 1 to 24, further comprising a cell culture medium.

27. providing a polymer bioink comprising a maleimide-containing polymer; providing an activating agent comprising a bis-thiol-containing crosslinker having at least two thiol functional groups; and Printing the polymer bioink and activator to form a 3D printed hydrogel A method for preparing a 3D printed hydrogel comprising:

28. 28. The method of claim 27, further comprising providing cells to form a 3D printed hydrogel containing cells.

29. 29. The method of claim 28, wherein the cells are present in the polymer bio-ink, in the activator, in both the polymer bio-ink and the activator, or in a separate medium prior to 3D printing.

30. 30. The method of any one of claims 27-29, wherein the 3D printed hydrogel is formed within 30 minutes, or within 10 minutes, or within 1 minute, or within 30 seconds, or within 10 seconds, or within 1 second of printing the polymer bioink and activator.

31. 31. The method of any one of claims 27 to 30, wherein the polymer bio-ink, the activator, or both are adjusted to about pH 7.4 prior to printing.

32. 32. The method of claim 31 , wherein the pH is adjusted using NaOH.

33. A cellular assay comprising a 3D printed hydrogel comprising the cells of any one of claims 18 to 21.

34. Use of a 3D printed hydrogel comprising cells according to any one of claims 18 to 21 for a cellular assay.

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