Constriction-minimizing microfluidic devices

Abstract

Disclosed herein is a microfluidic 3D cell culture device that supports the housing and swelling of cell culture scaffolding substances without causing damage to the cells and / or cell aggregates cultured therein, optionally wherein the device has an open-top design. The device is configured to comprise networks allowing for perfusion of fluid throughout, and the configuration of the networks is tunable to the biological question being addressed. The open-top design allows for access to the cells, scaffolding substances and fluid, such that the system is amenable to easy experimental manipulation.

Description

[0001] CONSTRICTION-MINIMIZING MICROFLUIDIC DEVICES

[0002] GOVERNMENT SUPPORT

[0003] This invention was made with government support under NS 115064 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] CROSS REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 582,636, filed September 14, 2023, entitled “CONSTRICTIONMINIMIZING MICROFLUIDIC DEVICES,” the entire disclosure of which is hereby incorporated by reference in its entirety.

[0006] BACKGROUND OF INVENTION

[0007] The utility and fidelity of laboratory models in mimicking human physiology and pathology critically dictate the extent of mechanistic understanding of disease and the effectiveness of therapeutics discovered and developed to ameliorate disease states. The recent advent of microphysiological in vitro systems provides great promise for accelerating discovery and development of disease treatments.

[0008] These systems typically involve cells cultured in a microfluidic device or similar device platform, often in combination with a 3D matrix or scaffolding substance to provide 3D tissuemimetic architecture. Simultaneously, new engineered and synthetic biomaterials provide new capabilities in mimicking human pathogenesis and creating biomimetic tissue mimics. However, there has been limited success in combining these engineered and synthetic materials with microfluidic device platforms due to their characteristic swelling properties. In confined regions of microfluidic devices, hydrogel swelling introduces mechanical stress, which is deleterious for cell culture and often results in cell death. Furthermore, these systems typically facilitate fluid flow via a closed channel system, thus limiting access to the fluids, scaffolds, and cells.

[0009] SUMMARY OF INVENTION

[0010] The present disclosure is based, at least in part, on the development of a constrictionminimizing microfluidic cell culture device that, in some aspects, supports 3D co-culturing of cells in a scaffold such that the scaffold may swell within the device without causing stress or damage to the cultured cells. The microfluidic devices described herein provide, in some aspects, a culture system wherein fluid may be perfused throughout the device, the channels of which provide easy access to the fluid, scaffold and cells due to their open-top design.

[0011] Furthermore, in some aspects, the configuration of the channels within the device may be designed to address the biological question being interrogated, based on the arrangement, size, shape and order of channels and chambers.

[0012] Thus, in some embodiments, provided herein are minimally restrictive microfluidic 3D cell culture devices (also referred to interchangeably herein as GelChip) comprising a body (also referred to interchangeably herein as base) (1), the body comprising: (i) at least one inlet chamber (4) comprised of a bottom surface, side walls and an open top; (ii) at least one outlet chamber (5) comprised of a bottom surface, side walls and an open top; and (iii) at least one channel (6); wherein the dimensions of the at least one channel support the housing and swelling of a scaffolding substance. In some embodiments, the minimally restrictive microfluidic 3D cell culture device further comprises: (iv) at least one cell culture chamber (3) comprised of a bottom surface, side walls and an open top, wherein the dimensions of the at least one cell culture chamber support the housing and swelling of a scaffolding substance. In some embodiments, the minimally restrictive microfluidic 3D cell culture device comprises a first channel of the at least one channel extending between the at least one inlet chamber and the at least one cell culture chamber, and a second channel of the at least one channel extending between the at least one outlet chamber and the at least one cell culture chamber.

[0013] In some embodiments, the at least one channel has a height of at least 0.5 mm. In some embodiments, the at least one channel has a height of about 0.6 mm. In some embodiments, the at least one channel has a height between 0.50 mm and 0.55 mm, between 0.55 mm and 0.60 mm, between 0.60 mm and 0.65 mm, between 0.65 and 0.70 mm, between 0.70 mm and 0.75 mm, between 0.75 mm and 0.80 mm, between 0.80 mm and 0.85 mm, between 0.85 mm and 0.90 mm, between 0.90 mm and 0.95 mm, between 0.95 mm and 1.00 mm, between 1.00 mm and 1.05 mm, between 1.05 mm and 1.10 mm, between 1.10 mm and 1.15 mm, between 1.15 mm and 1.20 mm, between 1.20 mm and 1.25 mm, between 1.25 mm and 1.30 mm, between 1.30 mm and 1.35 mm, between 1.35 mm and 1.40 mm, between 1.40 mm and 1.45 mm, or between 1.45 mm and 1.50 mm. In some embodiments, the at least one channel has a length of at least 1.5 mm. In some embodiments, the at least one channel has a length of about 2 mm. In some embodiments, the at least one channel has a length between 1.5 mm and 1.6 mm, between

[0014] I.6 mm and 1.7 mm, between 1.7 mm and 1.8 mm, between 1.8 mm and 1.9 mm, or between 1.9 mm and 2.0 mm. In some embodiments, the at least one channel has a width of at least 0.15 mm. In some embodiments, the at least one channel has a width of about 0.85 mm. In some embodiments, the at least one channel has a width between 0.15 mm and 0.20 mm, between 0.20 mm and 0.25 mm, between 0.25 mm and 0.30 mm, between 0.30 mm and 0.35 mm, between 0.35 mm and 0.40 mm, between 0.40 mm and 0.45 mm, between 0.45 mm and 0.50 mm, between 0.50 mm and 0.55 mm, between 0.55 mm and 0.60 mm, between 0.60 mm and 0.65 mm, between 0.65 mm and 0.70 mm, between 0.70 mm and 0.75 mm, between 0.75 mm and 0.80 mm, between 0.80 mm and 0.85 mm, between 0.85 mm and 0.90 mm, between 0.90 mm and 0.95 mm, between 0.95 mm and 1.00 mm, between 1.00 mm and 1.05 mm, between 1.05 mm and 1.10 mm, between 1.10 mm and 1.15 mm, or between 1.15 mm and 1.20 mm.

[0015] In some embodiments, the at least one inlet chamber and the at least one outlet chamber have a width of at least 0.5 mm. In some embodiments, the at least one inlet chamber and the at least one outlet chamber have a length of at least 0.5 mm.

[0016] In some embodiments, the minimally restrictive microfluidic 3D cell culture device comprises at least two cell culture chambers. In some embodiments, the minimally restrictive microfluidic 3D cell culture device further comprises at least one propagation channel extending between the at least two cell culture chambers. In some embodiments, the at least one propagation channel has a height of no more than 0.5 mm. In some embodiments, the at least one propagation channel has a width between 0.1 mm and 6 mm. In some embodiments, the at least one propagation channel has a length between 0.5 mm and 6 mm.

[0017] In some embodiments, the minimally restrictive microfluidic 3D cell culture device is comprised of synthetic polymers, biological polymers, and / or glass. In some embodiments, the device is fabricated from a material chosen from the list comprising polydimethylsiloxane (PDMS), polycarbonate (PC), polystyrene (PS), polyamide (PA), polyethylene glycol (PEG), thermoplastic elastomers (TPE), polyaryletherketones (PAEK), polytetrafluoroethylene (PTFE), cyclic olefin copolymer (COC), glass, or other translucent material. In some embodiments, the biological polymers are chosen from dextran, PEG, gelatin, fibrin, Matrigel, Collagen (types I,

[0018] II, and III), Laminin, Fibronectin, Fibrinogen, Tropoelastin, Elastin, Hyaluronic Acid (Hyaluronan), Chondroitin Sulfate, Alginate, Chitosan, Polyacrylamide (PAAm), Polylactic acid (PLA), Polylactic-co-glycolic acid (PLGA), Polycaprolactone (PCL), Poly(vinyl alcohol) (PVA), poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), Poly(N- isopropylacrylamide) (PNIPAm), Poly(acrylic acid) (PAAc), PNIPAAm-PAAc, and / or variants thereof.

[0019] In some embodiments, the minimally restrictive microfluidic 3D cell culture device is formed through 3D printing, injection molding, hot embossing, micromilling, micromachining, or etching. In some embodiments, the method of 3D printing is chosen from the list comprising Stereolithography (SLA), Selective Laser Sintering (SLS), Eused Deposition Modeling (EDM), Digital Light Processing (DLP), Multi Jet Fusion (MJF), PolyJet, Direct Metal Laser Sintering (DMLS), and / or Electron Beam Melting (EBM).

[0020] In some embodiments, the minimally restrictive microfluidic 3D cell culture device comprises a microfluidic network comprising at least two configurations of the at least one cell culture chamber, the at least one inlet chambers, the at least one outlet chambers, the at least one channel, and the at least one propagation channel. In some embodiments, the minimally restrictive microfluidic 3D cell culture device supports perfusion of fluid between the microfluidic network of at least two configurations.

[0021] In some embodiments, the scaffolding substance is a gel or gel-precursor selected from the list comprising Hydrogel, Fibrin, Collagen (types I, II, and III) Laminin, Fibronectin, Fibrinogen, Tropoelastin, Elastin, Hyaluronic Acid (Hyaluronan), Matrigel, Chondroitin Sulfate, Alginate, Chitosan, Dextran, and / or variants thereof. In some embodiments, the hydrogel is comprised of Polyethylene glycol (PEG), Polyacrylamide (PAAm), Polylactic acid (PLA), Polylactic-co-glycolic acid (PLGA), Polycaprolactone (PCL), Poly(vinyl alcohol) (PVA), poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), Poly(N- isopropylacrylamide) (PNIPAm), Poly(acrylic acid) (PAAc), PNIPAAm-PAAc, and / or variants thereof.

[0022] In some aspects, provided herein are methods of 3D culturing cells in the minimally restrictive microfluidic 3D cell culture device as described above, comprising: (a) introducing one or more cell types into the at least one input chamber, the at least one output chamber, and / or the at least one channel; (b) contacting the one or more cell types with culture media; and (c) incubating the minimally restrictive microfluidic 3D cell culture device containing the one or more cell types in an environment supporting cell growth. In some embodiments, the method of 3D culturing comprises introducing the culture media through the at least one input chamber and removing the culture media through the at least one output chamber, thereby perfusing the culture media through the at least two chambers and the at least one channel. In some embodiments, the one or more cell types is human.

[0023] In some aspects, provided herein are perfusable microfluidic cell culture networks, comprising at least two cell culture chambers, at least one inlet chamber, at least one outlet chamber, at least two channels, and at least one propagation channel, wherein: the at least two channels comprise a first channel extending between the at least one inlet chamber and a first cell culture chamber, and a second channel extending between the at least one outlet chamber and a second cell culture chamber; wherein the dimensions of the at least one cell culture chamber, the at least one propagation channel, and / or the at least two channels support the housing and swelling of a synthetic hydrogel scaffolding substance with minimal cell compression.

[0024] In some embodiments, the at least two channels have a height of at least 0.5 mm. In some embodiments, the at least two channels and / or the at least one propagation channels have a height of about 0.6 mm.

[0025] In some embodiments, the perfusable microfluidic cell culture network comprises at least two inlet chambers, at least two outlet chambers, and at least four channels, wherein the at least four channels comprise a first set of channels extending between the at least two inlet chambers and at least two cell culture chambers, and a second set of channels extending between the at least two outlet chambers and at least two cell culture chambers. In some embodiments, the at least two cell culture chambers, at least two inlet chambers, and at least two outlet chambers are comprised of a bottom surface, side walls and an open top. In some embodiments, the perfusable microfluidic cell culture network supports the perfusion of fluid between the at least one inlet chamber and the at least one outlet chamber.

[0026] In some embodiments, the device includes a scaffolding substance. In some embodiments the scaffolding substance is a gel or gel-precursor selected from the list comprising Hydrogel, Fibrin, Collagen (types I, II, and III) Laminin, Fibronectin, Fibrinogen, Tropoelastin, Elastin, Hyaluronic Acid (Hyaluronan), Matrigel, Chondroitin Sulfate, Alginate, Chitosan, Dextran, and / or variants thereof. In some embodiments, the hydrogel is comprised of Polyethylene glycol (PEG), Polyacrylamide (PAAm), Polylactic acid (PLA), Polylactic-co- glycolic acid (PLGA), Polycaprolactone (PCL), Poly(vinyl alcohol) (PVA), poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), Poly(N-isopropylacrylamide) (PNIPAm), Poly(acrylic acid) (PAAc), PNIPAAm-PAAc, and / or variants thereof. In some embodiments, provided herein are methods of culturing cells and / or cell aggregates on a device, comprising introducing one or more cell types in a scaffolding substance to a device, the device comprising: (i) at least one inlet chamber comprised of a bottom surface, side walls and an open top; (ii) at least one outlet chamber comprised of a bottom surface, side walls and an open top; and (iii) at least two channels, wherein the dimensions of the at least one channel supports the housing and swelling of the scaffolding substance. In some embodiments, the device further comprises: (iv) at least one cell culture chamber comprised of a bottom surface, side walls and an open top, wherein the dimensions of the at least one cell culture chamber support the housing and swelling of a scaffolding substance. In some embodiments, the device comprises a first channel of the at least one channel extending between the at least one inlet chamber and the at least one cell culture chamber, and a second channel of the at least one channel extending between the at least one outlet chamber and the at least one cell culture chamber. In some embodiments, the device is the device described above.

[0027] In some embodiments, the cultured cells and / or cell aggregates comprise more than one cell type. In some embodiments, the cultured cells and / or cell aggregates comprise mammalian cells. In some embodiments, the mammalian cells are selected from the list comprising endothelial cells, neuronal cells, connective tissue cells, fibroblasts, epithelial cells, epidermal or dermal cells, chondrocytes, osteocytes (e.g., osteoblasts), blood cells (e.g., red or white blood cells), plasma cells, adipocytes, myocytes, hepatocytes, cholangiocytes, stellate cells, Kupffer cells, glial cells, endocrine cells (e.g., cells of the islet), reticular cells, cells of sensory organs, mesenchymal cells, immune cells, and / or stem cells (e.g., embryonic or induced pluripotent stem cells).

[0028] In some embodiments, the method of culturing cells and / or cell aggregates further comprises vascularizing the cells and / or cell aggregates in the scaffolding substance. In some embodiments, the method of culturing cells and / or cell aggregates further comprises inducing a flow of a fluid through the microfluidic network. In some embodiments, the fluid comprises one or more active agents. In some embodiments, the one or more active agent comprises small molecules, drugs, nucleic acids, proteins, peptides, lipids, carbohydrates, growth factors, bacteria, viruses, variants thereof and / or any other type of biological or synthetic molecules.

[0029] In some embodiments, provided herein is an in vitro tissue mimic comprising a 3- dimensional (3D) matrix comprising a perfusable vascular network in a synthetic hydrogel, wherein the hydrogel comprises a dextran-based polysaccharide macromer, a cell-degradable crosslinker and a tissue-specific extracellular matrix protein and further comprising at least one neural specific cell type dispersed in the hydrogel. In some embodiments, the tissue- specific extracellular matrix protein is versican or a versican-like peptide.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIGs. 1A-1C depict a schematic of an example structure of an embodiment of the minimally restrictive microfluidic 3D cell culture device described herein. FIG. 1A shows the components of the device, comprising a base 1 and at least one core microfluidic network 2, wherein the core microfluidic network comprises at least one cell culture chamber 3, at least one inlet chamber 4, at least one outlet chamber 5, and at least two channels 6. FIG. IB shows an example configuration of an embodiment of the device of the present application wherein two core microfluidic networks are connected via a propagation channel 7. FIG. 1C shows an embodiment of the device of the present application wherein multiple bases comprising microfluidic networks are incorporated on a single device.

[0032] FIGs. 2A-2H depict examples of microfluidic network configurations compatible for use in the present device. FIG. 2A shows a 3-dimensional rendering of a microfluidic network comprising two core microfluidic networks (wherein each core microfluidic network comprises at least one cell culture chamber 3, at least one inlet chamber 4, at least one outlet chamber 5, and at least two channels 6) connected via a propagation channel 7, and different vectorial directions in which the components of the device may be altered (e.g., length, width or height). FIGs. 2B-2H provide non-limiting examples of shape combinations and configurations of microfluidic networks compatible for use in the device of the present application, wherein the microfluidic networks comprise at least one inlet chamber 4, at least one outlet chamber 5, and at least two channels 6. In some embodiments, the configuration comprises a propagation channel 7 (FIG. 2H).

[0033] FIGs. 3A-3E show an example of an embodiment of the device provided herein formed through 3D printing. FIG. 3A shows an example of a 3D printed mold used to form an embodiment of the device of the present invention. FIG. 3B shows an example of a device formed by casting polymers off of molds such as the mold shown in FIG. 3A. FIG. 3C demonstrates the functionality of 3D culturing cells in the device of the present invention. An immunofluorescent microscopy image is shown of endothelial cells grown in the device provided herein (scale bar = 30 pm). FIG. 3D shows an immunofluorescent image of a 3D culture of neurons cultured in a propagation channel of the present device. FIG. 3E shows an example of a device formed through stereolithography (SLA) 3D printing molds using resin and casting devices with polydimethylsiloxane (PDMS), wherein the device comprises the microfluidic network design of FIG. IB.

[0034] FIGs. 4A-4F show experimental setups used to screen parameter spaces for the constriction-minimizing microfluidic 3D cell culture devices described herein. FIG. 4A demonstrates the inability of complex 3D co-cultures to grow and form physiological structures in conventional devices. FIG. 4B depicts the results of an experiment to test the ability of channels of various heights to support the formation of endothelial networks. Immunofluorescent microscopy images (left) demonstrate the improved formation of vascular networks with increasing channel height (quantified right). FIG. 4C depicts the dimensions used in an experiment to test varying lengths and heights of channels for culturing perfusable neurovascular units. FIGs. 4D-4E depict example configurations of the “GelChip” microfluidic device, which comprise channels with a height of at least 600 pm. In some embodiments, the channels of the GelChips have tapered channels, with a minimal width of about 150 pm and a maximal width of about 850 pm, and a height of about 600 pm. FIG. 4F shows a GelChip microfluidic device comprising tapered channels, wherein the minimal and maximal widths and lengths are altered as labeled. Scale bars, 0.2 mm. In some embodiments, a device provided herein comprises multiple core microfluidic networks on a single base, allowing for multiplexed experiments.

[0035] FIGs. 5A-5B demonstrate the functionality of the device provided herein for culturing vascularized 3D cell cultures. FIG. 5A shows a comparison of the formation of miBRAIN 3D endothelial cell networks cultured in a conventional microfluidic device in an engineered hydrogel (left) versus in a constriction-minimizing GelChip microfluidic device (right). FIG. 5B shows a fluorescence microscopy image demonstrating the successful growth of neurons of a miBRAIN when cultured in the device provided herein.

[0036] FIG. 6 demonstrates the functionality of the device provided herein for perfusing vascularized 3D cell cultures and for assessing 3D cell migration. FIG. 6 shows the characterization of barrier function of these vascular networks via quantification of their permeability, which shows size-dependence. FIGs. 7A-7C provide examples of microfluidic network configurations useful for achieving different arrangements of cells and fluids for culturing and creating a wide variety of cell environments and tissues.

[0037] FIGs. 8A-8C provide examples of microfluidic network configurations useful for achieving different arrangements of cells and fluids for culturing and creating a wide variety of cell environments and tissues.

[0038] FIGs. 9A-9B provide examples of microfluidic network configurations useful for achieving different arrangements of cells and fluids for culturing and creating a wide variety of cell environments and tissues.

[0039] FIG. 10 provides examples of microfluidic network configurations useful for achieving different arrangements of cells and fluids for culturing and creating a wide variety of cell environments and tissues.

[0040] FIG. 11 shows an experimental setup used to screen length and width parameter spaces for the constriction-minimizing microfluidic 3D cell culture devices described herein.

[0041] FIG. 12 depicts an example of a constriction-minimizing microfluidic 3D cell culture devices (i.e. also referred to as a GelChip Plate), which, in some embodiments, comprises at least one channel with a height of at least 0.6 mm, a width of at least 0.85 mm, and a length of at least 03 mm. The example GelChip Plate comprises a multitude of perfusion systems, comprising multiple inlet chambers, outlet chambers, channels, and propagation channels.

[0042] DETAILED DESCRIPTION OF INVENTION

[0043] 3D culture systems provide promising methods through which complex cell-cell interactions, and even disease states, can be mimicked in vitro without the risk and cost associated with in vivo models. These 3D culture methods can even model crosstalk between cells of different types, allowing for the simulation of multiple organ systems on a plate. In order to provide these in vitro cell networks with growth support that is normally provided in vivo through extracellular matrix proteins and fibers, scaffolding substances have been designed to mimic this support and maintain cell survival and growth in a 3D environment. These scaffolding substances, such as hydrogel or fibrin, are widely applied and have the useful characteristic of being perfusable. Through perfusion, the 3D cultured cells may be contacted with fluid in order to introduce flow and agents (e.g., active agents) to the 3D cultured cells and / or cell aggregates. However, when perfused, these same scaffolding substances swell in volume. Current perfusable 3D cell culture devices have such restrictive parameters that perfusion of the scaffolding substance with fluid often causes pressure-induced strain on the cells and / or cell aggregates cultured in them, leading to cell injury and even death. Thus, there is a need for 3D cell culture devices with parameters amenable to the swelling of scaffolding substances in order to maintain cell health and survival.

[0044] Some embodiments of the invention described herein relate generally to 3D cell culture devices designed to support cell culture scaffolding substances such that the scaffolding substances may expand during fluid perfusion. While the description sets forth various embodiment specific details, it will be appreciated that the description is illustrative only and should not be construed in any way as limiting the invention. Furthermore, various applications of the invention and modifications thereto, which may occur to those who are skilled in the art, are also encompassed by the general concepts described herein.

[0045] Definitions

[0046] As described herein, “minimally restrictive” refers to device parameters (e.g., dimensions of the device components) that allow for at least partial swelling of a scaffolding substance embedded within the device without imparting additional strain on the substance. Swelling of a scaffolding substance refers to the change in volume of the substance as it absorbs a compatible solvent, such as water for hydrogels. Swelling is possible because the solid component of the scaffolding substance (e.g., a polymer network) is both elastic and hydrophilic, enabling enough water to be absorbed to increase the volume, but creating elastic tension within the solid component that prevents total dissolution. In some aspects, swelling is characterized by swelling ratios, which describe how much a hydrogel swells from one state to another. A swelling ratio can be calculated by dividing the scaffolding substance’s swollen volume by its dry volume. Additional methods to measure swelling include polymer volume fractions, reference ratio, and polymer mass fractions. In some aspects, the minimally restrictive device described herein supports swelling of the scaffolding substance up to 100 times its original volume without imparting additional strain. In some aspects, the device described herein supports swelling of the scaffolding substance about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, 15 times, about 20 times, about 25 times, about 30 times, about 50 times, about 75 times, about 95 times, about 100 times, or about 200 times its original volume without additional strain. In some aspects, the strain of the scaffolding substance is measured through pressure of the scaffolding substance against the walls of the device (e.g., in units of Pascals). In other aspects, the strain of the scaffolding substance is measured by the survival, cytotoxicity, proliferation, and / or viability of the cells cultured within the substance following perfusion of the substance. Cell survival, cytotoxicity, proliferation, and / or viability can be measured through any method known in the art (see, e.g., Riss et al., “Cell Viability Assays.” 2013 May 1 [Updated 2016 Jul 1]. In: Markossian et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences, 2004-.; Adan et al., “Cell Proliferation and Cytotoxicity Assays.” Curr Pharm Biotechnol. 2016; 17(14): 1213- 1221 ; Riss et al., “Cytotoxicity Assays: In Vitro Methods to Measure Dead Cells.” 2019 May 1. In: Markossian, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004).

[0047] A “minimally restrictive microfluidic 3D cell culture device” as used herein is a device having multiple channels and chambers sized and configured to minimally restrict a variety of cells and tissues while providing adequate fluid flow.

[0048] As used herein, a “scaffolding substance” refers to any substance capable of maintaining living cells in a spatial relation in multiple dimensions, preferably 3 dimensions. Scaffolding substances may be liquids, gels or solids. Non-limiting examples of scaffolding substances for use in the microfluidic device described herein include water, gels or gel precursors, micropore scaffolds, microfiber scaffolds, membranes and hollow fibers. In some aspects, the gel or gel precursor is comprised of agar, hydrogel, fibrin, collagen (types I, II, and III), laminin, fibronectin, fibrinogen, tropoelastin, elastin, hyaluronic acid (hyaluronan), matrigel, chondroitin sulfate, alginate, chitosan, dextran, and / or variants thereof. In some aspects, the hydrogel is comprised of Polyethylene glycol (PEG), Polyacrylamide (PAAm), Polylactic acid (PLA), Polylactic-co-glycolic acid (PLGA), Polycaprolactone (PCL), Poly(vinyl alcohol) (PVA), poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), Poly(N- isopropylacrylamide) (PNIPAm), Poly(acrylic acid) (PAAc), PNIPAAm-PAAc, and / or variants thereof. The scaffolding substances are capable of swelling when used in the devices disclosed herein.

[0049] As used herein, the term “fluid” refers to any substance that continually flows under an applied shear stress. Fluids as used herein may include liquids, gases and plasmas. The device described herein is “perfusable” in that it is capable of maintaining fluid flow throughout the chambers and channels of the device.

[0050] The minimally restrictive microfluidic 3D cell culture device supports perfusion of fluid between the microfluidic network of at least two configurations. “At least two configurations” refers to at least two distinct arrangements of microfluidic networks of chambers and channels. For instance a number of examples of different chambers and channels is shown in Figures 7-10. The “microfluidic network” is a set of channels and chambers which are arranged together such that a composition such as a fluid can be passed or exchanged or in contact through the channels and chambers.

[0051] As used herein, the term “active agent” refers to any molecule (e.g., protein, nucleic acid, sugar, small molecule, lipid, chemical, drug, cell, virus, antibody, etc.) capable of activity and / or inducing activity in a cell.

[0052] As used herein, the term “about” refers to a value within ±10% variability from the reference value, unless otherwise specified.

[0053] The terms “a” and “an” mean “one or more of.” For example, “a device” is understood to represent one or more such devices. As such, the terms “a” and “an,” “one or more of a (or an),” and “at least one of a (or an)” are used interchangeably herein.

[0054] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0055] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0056] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0057] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein, it is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. For any conflict in definitions between various sources or references, the definition provided herein shall control.

[0058] 3D cell culture

[0059] 3D cell culture is an artificial environment in which biological cells are permitted to grow and / or interact with their surroundings in all three dimensions. Unlike in 2D environments, 3D cell culture permits cells in vitro to grow in all directions, more similarly modeling an in vivo environment. Within living tissues, cells exist in 3D microenvironments that comprise intricate cell-cell and cell-matrix interactions and complex transport dynamics. Standard 2D (i.e., monolayer) cell cultures are inadequate representations of this environment, often causing unreliability in interpreting in vivo physiology, such as measuring drug efficacy or toxicity. Thus, 3D cultures present superior models of in vivo tissues, as they more similarly resemble the architecture and interactions that cells experience in vivo. Importantly, the third dimension in 3D cultures provides cells with the ability to generate networks and to migrate in all directions, which are important quantifiable parameters in many disease states. In addition, studies have shown that gene expression of cells cultured in 3D more closely resembles gene expression of in vivo cells compared to cells in 2D culture. These findings are likely supported by the fact that cells 3D culture survive much longer than 2D cultured cells, allowing for studies to be carried out over longer periods of time (e.g., weeks to months) without disruption.

[0060] Furthermore, 3D cell culture presents the ability to create co-culture systems that can be vascularized in all dimensions. This is an important feature, as in vivo tissues must be vascularized for molecules (e.g., nutrients, oxygen, drugs, etc.) to be transported to them. By vascularizing 3D cell cultures, this creates a model that can be used to study the transport of such molecules between cell types under a variety of conditions. In some embodiments of the present invention, the 3D cultured cells and / or cell aggregates are vascularized within the scaffolding substance in the device. Vascularization of 3D cultures can be carried out by any method known in the art.

[0061] Microfluidic 3D cell culture

[0062] Microfluidic cell culture techniques, often referred to as “organs-on-chip”, allow for precise control of fluids and particles down to the nanoliter scale. Microfluidic cell culture is performed on microfluidic devices (also referred to as “microfluidic chips”), which contain micro-channels through which fluid can flow. These micro-channels can be connected together in a configuration to allow for the desired features and functionalities of the microfluidic device being used, such as mixing, pumping, sorting, or controlling the biochemical environment. An attractive characteristic of microfluidic devices is the requirement for less volume of samples, chemicals, and reagents compared to conventionally sized cell culture systems. These techniques facilitate simultaneous manipulation and analysis of cultured cells from single cells to intact tissues, leading to considerable advances in the fields of quantitative and systems biology. Because microfluidic cell cultures enable modeling of tissue-tissue-interfaces, they can provide in-depth findings about physiological barriers. Furthermore, these techniques offer clear approaches for investigating signaling between different organs. Integration of microfluidic cell culture techniques with 3D scaffolding systems makes it possible to adapt culture conditions both biochemically and biomechanically, such as creating dynamic 3D structures and providing a microenvironment that allows for formation of artificial tissues from cultured cells. Microfluidic cell culture devices allow precise control of cell numbers and cell density in a given area or volume and can provide placement of cells in complex geometries. Combining these techniques provides high level of precision, throughput capabilities and relevant modeling of in vivo physiologies and disease states.

[0063] In order to control and confine fluids at the micro-scale, microfluidic chips with microchannels are conventionally fabricated into devices in which the micro-channels have heights of approximately 100-250 pm. An individual cell is on the order of 20-30 pm. Thus, the height of conventional micro-channels are less than ten times the size of individual cells. The small scale of these micro-channels, along with the characteristic swelling properties of hydrogels in which 3D cell cultures are traditionally cultured, often lead to cellular stress that can be cytotoxic and has been shown to be prohibitive to the growth and formation of 3D cell cultures. Therefore, provided herein are microfluidic devices that support 3D cell culture at larger length scales, that that they minimize constrictive forces in 3D cell culture, enable successful, interconnected, perfusable 3D cell networks.

[0064] The microfluidic device according to the invention may be configured as one or multiple arranged systems. As shown in FIG. 1A, the device comprises a body 1 in which there is a configuration of at least one microfluidic system 2. The core unit of the microfluidic system comprise at least one cell culture chamber 3, at least one inlet chamber 4, at least one outlet chamber 5, and at least two channels 6 that extend between the cell culture chamber and the inlet and outlet chambers. The inlet and outlet chambers may be located anywhere relative to the cell culture chamber, and relative to one another within the body. For example, the location of the inlet chamber 4 and the outlet chamber 5 may be switched in FIG. 1A.

[0065] Inlet and outlet chambers, independently of one another have a bottom surface and side walls, which together form a chamber. Each of the inlet and outlet chambers have at least one connection to a channel such that a substance such as a fluid or gel may flow in or out of the chambers. In some embodiments these chambers and / or channels do not have a top surface. The lack of at least one surface, such as a top surface in the channels and / or chambers allows a scaffolding substance present with the channels or chambers to swell, thus avoiding overly restrictive conditions which can alter cell properties. The dimensions of the channels or chambers which support swelling of a scaffolding substance, thus can be achieved by designing the outer surfaces of the channels or chambers such that one side (i.e., the top) is missing or by providing larger dimensions than are necessary to otherwise house the scaffolding substance. The excess space provided for the scaffolding substance enables control over the external forces that are exerted on cells and tissues used in the device.

[0066] As shown in FIG. IB, the body 1 may comprise a microfluidic system 2 in which there is more than one core unit (i.e., configuration of cell culture chambers 3, inlet chambers 4, outlet chambers 5 and channels 6) connected via one or more propagation channels 7. The device may consist of multiple bodies 1 comprising microfluidic systems, as shown in FIG. 1C. The multiplicity of bodies on the device may have the same or different microfluidic device designs.

[0067] As used herein, a “cell culture chamber” or a “culture chamber” refers to a space within the device designated for growing cells or cell aggregates in a scaffolding substance (see, e.g., FIG. 1A, 3). In some aspects, the cell culture chamber is spatially connected to inlet and outlet chambers such that fluid can be introduced via the inlet chamber (see, e.g., FIG 1A, 4), flow through the cell culture chamber, and removed via the outlet chamber (see, e.g., FIG 1A, 5). In some embodiments, the device has one cell culture chamber, as shown in FIG. 1A. In some embodiments, the device has more than one cell culture chamber, as shown in FIG. IB. In other embodiments, the device has 3, 4, 5, 6, 7, 8, 9, 10 or more cell culture chambers. The number of cell culture chambers need not be the same in separate microfluidic networks of a single device. The size, shape and number of the cell culture chambers can vary within a single device or microfluidic network.

[0068] As used herein, an “inlet chamber” is a space in the device through which fluid is introduced into the microfluidic system. In some embodiments, the device has one inlet chamber, as shown in FIG. 1A. In some embodiments, the device has more than one inlet chamber, as shown in FIG. IB. In other embodiments, the device has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more inlet chambers. As used herein, an “outlet chamber” is a space in the device through which fluid is removed from the microfluidic system. In some embodiments, the device has one outlet chamber, as shown in FIG. 1A. In some embodiments, the device has more than one outlet chamber, as shown in FIG. IB. In other embodiments, the device has 3, 4, 5, 6, 7, 8, 9, 10 or more outlet chambers. The number of inlet and outlet chambers need not be the same in a single device or single microfluidic network. The size, shape and number of the inlet and outlet chambers can vary within a single device or microfluidic network.

[0069] In some aspects, the inlet and outlet chambers are physically connected, either directly or indirectly, to a cell culture chamber through channels (see, e.g., FIG 1A, 6). As used herein, a “channel” is a space through which fluid can flow between the inlet chamber and the outlet chamber. In some embodiments, a channel supports fluid flow between an inlet chamber or an outlet chamber and a cell culture chamber. In some embodiments, the device comprises two channels, as shown in FIG. 1A. In some embodiments, the device comprises more than two channels, as shown in FIG. IB. In other embodiments, the device comprises 4, 5, 6, 7, 8, 9, 10 or more channels. The number of channels need not be the same in separate microfluidic networks of a single device. The size, shape and number of channels can vary within a single device or microfluidic network.

[0070] In some aspects, the cell culture chamber is physically connected to a second cell culture chamber through a propagation channel (see, e.g., FIG. IB, 7). As used herein, a “propagation channel” is a space within the device that spatially connects two cell culture chambers, either directly or indirectly. In some embodiments, a propagation channel supports the ability of the 3D cultured cells and / or cell aggregates to migrate from one cell culture chamber to another. In a preferred embodiment, the propagation channel supports migration of 3D cultured cells through the scaffolding substance housed in the propagation channel(s). In some embodiments, the device comprises one propagation channel (7), as shown in FIG. IB. In some embodiments, the device comprises more than one propagation channel, as shown in FIGs. 7A-8C. In other embodiments, the device comprises 3, 4, 5, 6, 7, 8, 9, 10 or more propagation channels. The number of propagation channels need not be the same in separate microfluidic networks of a single device. The size, shape and number of propagation channels can vary within a single device or microfluidic network.

[0071] The dimensions (e.g., height, width, length, diameter and / or shape) of the channels, cell culture chambers, inlet chambers, outlet chambers, and propagation channels may be altered to tailor the device to specific experimental setups (e.g., differences in cell types, cell numbers, perfusion rates, fluid types and / or scaffold types). As an example, FIG. 2A depicts example vectorial directions in which height, length, and width of the channels and chambers may be altered. Additionally, FIGs. 2B-2H show non-limiting examples of shape combinations that may be used for cell culture chambers 3, inlet chambers 4, outlet chambers 5, channels 6 and propagation channels 7. The shapes and sizes of the components of the device may be altered in any way such that the device supports (1) survival and / or growth of cells in a scaffolding substance, and (2) the flow of fluid throughout the device. The dimensions for each device component suitable for a particular scaffolding substance or cell type may be systematically determined through the method taught in Example 1.

[0072] In some embodiments, the channels and / or propagation channel(s) of the devices disclosed herein have a height of at least 0.05 mm, at least 0.1 mm, at least 0.15 mm, at least 0.2 mm, at least 0.25 mm, at least 0.3 mm, at least 0.35 mm, at least 0.4 mm, at least 0.45 mm, at least 0.50 mm, at least 0.55 mm, at least 0.60 mm, at least 0.65 mm, at least 0.70 mm, at least

[0073] 0.75 mm, at least 0.80 mm, at least 0.85 mm, at least 0.90 mm, at least 0.95 mm, at least 1.00 mm, at least 1.05 mm, at least 1.10 mm, at least 1.15 mm, at least 1.20 mm, at least 1.25 mm, at least 1.30 mm, at least 1.35 mm, at least 1.40 mm, at least 1.45 mm, at least 1.50 mm, or more.

[0074] In some embodiments, the channels and / or propagation channel(s) have a height between 0.05 and 0.1 mm, between 0.1 and 0.15 mm, between 0.15 and 0.2 mm, between 0.2 and 0.25 mm, between 0.25 and 0.3 mm, between 0.3 and 0.35 mm, between 0.35 and 0.4 mm, between 0.4 and 0.45 mm, between 0.45 and 0.5 mm, between 0.50 mm and 0.55 mm, between 0.55 mm and 0.60 mm, between 0.60 mm and 0.65 mm, between 0.65 mm and 0.70 mm, between 0.70 mm and 0.75 mm, between 0.75 mm and 0.80 mm, between 0.80 mm and 0.85 mm, between 0.85 mm and 0.90 mm, between 0.90 mm and 0.95 mm, between 0.95 mm and 1.0 mm, between 1.0 mm and 1.05 mm, between 1.05 mm and 1.10 mm, between 1.10 mm and 1.15 mm, between 1.15 mm and 1.20 mm, between 1.20 mm and 1.25 mm, between 1.25 mm and 1.30 mm, between 1.30 mm and 1.35 mm, between 1.35 mm and 1.40 mm, between 1.40 mm and 1.45 mm, between 1.45 mm and 1.50 mm, between 0.1 mm and 0.2 mm, between 0.1 mm and 0.3 mm, between 0.1 mm and 0.4 mm, between 0.1 mm and 0.5 mm, between 0.1 mm and 0.6 mm, between 0.1 mm and 0.7 mm, between 0.1 mm and 0.8 mm, between 0.1 mm and 0.9 mm, between 0.1 mm and 1 mm, between 0.1 mm and 1.1 mm, between 0.1 and 1.2 mm, between 0.1 and 1.3 mm, between 0.1 and 1.4 mm, between 0.1 and 1.5 mm, between 0.2 mm and 0.3 mm, between 0.3 mm and 0.4 mm, between 0.4 mm and 0.5 mm, between 0.5 mm and 0.6 mm, between 0.6 mm and 0.7 mm, between 0.8 mm and 0.9 mm, between 0.9 mm and 1 mm, between 1 mm and 1.1 mm, between 1.1 mm and 1.2 mm, between 1.2 mm and 1.3 mm, between 1.3 mm and 1.4 mm, between 1.4 mm and 1.5 mm, between 0.3 mm and 0.5 mm, between 0.4 mm and 0.6 mm, between 0.5 mm and 0.7 mm, between 0.6 mm and 0.8 mm, between 0.7 mm and 0.9 mm, between 0.8 mm and 1 mm, between 0.9 mm and 1.1 mm, between 1 mm and 1.2 mm, between 1.1 mm and 1.3 mm, between 1.2 mm and 1.4 mm, between 1.3 mm and 1.5 mm, between 0.5 and 1 mm, between 1 mm and 1.5 mm, or between 0.05 and 1.5 mm. In some embodiments, the channels and / or propagation channel(s) have a height of about 0.60 mm. In some embodiments, the channels and / or propagation channel(s) have a height of 0.6 mm.

[0075] In some embodiments, the channels and / or propagation channel(s) of the devices disclosed herein have tapered ends (e.g., wherein the width of the channel entrance is less than the width at the center of the channel).

[0076] In some embodiments, the channels and / or propagation channel(s) of the devices disclosed herein have a width of at least 0.15 mm, at least 0.20 mm, at least 0.25 mm, at least 0.30 mm, at least 0.35 mm, at least 0.40 mm, at least 0.45 mm, at least 0.50 mm, at least 0.55 mm, at least 0.60 mm, at least 0.65 mm, at least 0.70 mm, at least 0.75 mm, at least 0.80 mm, at least 0.85 mm, at least 0.90 mm, at least 0.95 mm, at least 1.0 mm, at least 1.05 mm, at least 1.1 mm, at least 1.15 mm, at least 1.20 mm, or more. In some embodiments, the channels and / or propagation channel(s) have a width between 0.15 mm and 0.20 mm, between 0.20 mm and 0.25 mm, between 0.25 mm and 0.30 mm, between 0.30 mm and 0.35 mm, between 0.35 mm and 0.40 mm, between 0.40 mm and 0.45 mm, between 0.45 mm and 0.50 mm, between 0.50 mm and 0.55 mm, between 0.55 mm and 0.60 mm, between 0.60 mm and 0.65 mm, between 0.65 mm and 0.70 mm, between 0.70 mm and 0.75 mm, between 0.75 mm and 0.80 mm, between 0.80 mm and 0.85 mm, between 0.85 mm and 0.90 mm, between 0.90 mm and 0.95 mm, between 1.00 mm and 1.05 mm, between 1.05 mm and 1.10 mm, between 1.10 mm and 1.15 mm, between 1.15 mm and 1.20 mm, between 0.1 mm and 0.2 mm, between 0.2 mm and 0.3 mm, between 0.3 mm and 0.4 mm, between 0.4 mm and 0.5 mm, between 0.5 mm and 0.6 mm, between 0.6 mm and 0.7 mm, between 0.7 mm and 0.8 mm, between 0.8 mm and 0.9 mm, between 0.9 mm and 1 mm, between 1 mm and 1.1 mm, between 1.1 mm and 1.2 mm, between 0.1 mm and 0.3 mm, between 0.1 mm and 0.4 mm, between 0.1 mm and 0.5 mm, between 0.1 mm and 0.6 mm, between 0.1 mm and 0.7 mm, between 0.1 mm and 0.8 mm, between 0.1 mm and 0.9 mm, between 0.1 mm and 1 mm, between 0.1 mm and 1.1 mm, between 0.1 mm and 1.2 mm, between 0.2 mm and 0.4 mm, between 0.3 mm and 0.5 mm, between 0.4 mm and 0.6 mm, between 0.5 mm and 0.7 mm, between 0.6 mm and 0.8 mm, between 0.7 mm and 0.9 mm, between 0.8 mm and 1 mm, between 0.9 mm and 1.1 mm, between 1 mm and 1.2 mm, between

[0077] 1.1 mm and 1.3 mm, between 0.1 mm and 1.2 mm, between 0.5 mm and 1 mm, or between 0.8 mm and 0.9 mm. In some embodiments, the channels and / or propagation channel(s) have a width of about 0.85 mm. In some embodiments, the channels and / or propagation channel(s) have a width of 0.85 mm.

[0078] In some embodiments, the channels and / or propagation channel(s) of the devices disclosed herein have a length of at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 0.6 mm, at least 0.7 mm, at least 0.8 mm, at least 0.9 mm, at least 1 mm, at least 1.2 mm, at least 1.4 mm, at least 1.6 mm, at least 1.8 mm, at least 2 mm, at least 2.2 mm, at least 2.4 mm, at least 2.6 mm, at least 2.8 mm, at least 3 mm, at least 3.2 mm, at least 3.4 mm, at least 3.6 mm, at least 3.8 mm, at least 4 mm, at least 4.2 mm, at least 4.4 mm, at least 4.6 mm, at least 4.8 mm, at least 5 mm, at least 5.2 mm, at least 5.4 mm, at least 5.6 mm, at least 5.8 mm, at least 6 mm, or more. In some embodiments, the channels and / or propagation channel(s) of the devices disclosed herein have a length between 0.1 mm and 0.2 mm, between 0.2 mm and 0.3 mm, between 0.3 mm and 0.4 mm, between 0.4 mm and 0.5 mm, between 0.5 mm and 0.6 mm, between 0.6 mm and 0.7 mm, between 0.7 mm and 0.8 mm, between 0.8 mm and 0.9 mm, between 0.9 mm and 1 mm, between 1 mm and 1.1 mm, between 1.1 mm and 1.2 mm, between

[0079] 1.2 mm and 1.3 mm, between 1.3 mm and 1.4 mm, between 1.4 mm and 1.5 mm, between 1.5 mm and 2 mm, between 2 mm and 2.5 mm, between 2.5 mm and 3 mm, between 3 mm and 3.5 mm, between 3.5 mm and 4 mm, between 4 mm and 4.5 mm, between 4.5 mm and 5 mm, between 5 mm and 5.5 mm, between 5.5 mm and 6 mm, between 0.1 mm and 0.5 mm, between 0.2 mm and 0.5 mm, or between 0.1 mm and 6 mm. In some embodiments, the channels and / or propagation channel(s) of the devices disclosed herein have a length of about 0.3 mm. In some embodiments, the channels and / or propagation channel(s) of the devices disclosed herein have a length of 0.3 mm.

[0080] In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) of the devices disclosed herein have a height of at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 1.0 mm, at least 1.5 mm, at least 2.0 mm, at least 2.5 mm, at least 3.0 mm, at least 3.5 mm, at least 4.0 mm, at least 4.5 mm, at least 5.0 mm, at least 5.5 mm, at least 6.0 mm, at least 6.5 mm, at least 7 mm, or more. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) have a height between 0.1 mm and 0.2 mm, between 0. 2 mm and 0.3 mm, between 0.3 mm and 0.4 mm, between 0.4 mm and 0.5 mm, between 0.5 mm and 0.6 mm, between 0.6 mm and 0.7 mm, between 0.7 mm and 0.8 mm, between 0.8 mm and 0.9 mm, between 0.9 mm and 1 mm, between 1 mm and 1.1 mm, between 1.1 mm and 1.2 mm, between 1.2 mm and 1.3 mm, between 1.3 mm and 1.4 mm, between 1.4 mm and 1.5 mm, between 1.5 mm and 2 mm, between 2 mm and 2.5 mm, between 2.5 mm and 3 mm, between 3 mm and 3.5 mm, between

[0081] 3.5 mm and 4 mm, between 4 mm and 4.5 mm, between 4.5 mm and 5 mm, between 5 mm and

[0082] 5.5 mm, between 5.5 mm and 6 mm, between 6 mm and 6.5 mm, between 6.5 mm and 7 mm, between 0.5 mm and 0.7 mm, between 0.1 mm and 1 mm, between 0.5 mm and 1.0 mm, between 1.0 mm and 1.5 mm, between 1.5 mm and 2.0 mm, between 2.0 mm and 2.5 mm, between 2.5 mm and 3.0 mm, between 3.0 mm and 3.5 mm, between 3.5 mm and 4.0 mm, between 4.0 mm and 4.5 mm, between 4.5 mm and 5.0 mm, or between 5.5 mm and 6.0 mm. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) have a height of about 0.6 mm. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) have a height of 0.6 mm.

[0083] In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) of the devices disclosed herein have a width of at least at least 0.5 mm, at least 1.0 mm, at least 1.5 mm, at least 2.0 mm, at least 2.5 mm, at least 3.0 mm, at least 3.5 mm, at least 4.0 mm, at least 4.5 mm, at least 5.0 mm, at least 5.5 mm, at least 6.0 mm, or more. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) have a width between 0.5 mm and 1.0 mm, between 1.0 mm and 1.5 mm, between 1.5 mm and 2.0 mm, between 2.0 mm and 2.5 mm, between 2.5 mm and 3.0 mm, between 3.0 mm and 3.5 mm, between 3.5 mm and 4.0 mm, between 4.0 mm and 4.5 mm, between 4.5 mm and 5.0 mm, or between 5.5 mm and 6.0 mm. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) have a width of about 0.5 mm. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) have a width of 0.5 mm. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) of the devices disclosed herein have a length of at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 1.0 mm, at least 1.5 mm, at least 2.0 mm, at least 2.5 mm, at least 3.0 mm, at least 3.5 mm, at least 4.0 mm, at least 4.5 mm, at least 5.0 mm, at least 5.5 mm, at least 6.0 mm, or more. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) have a length between 0.1 and 0.2 mm, between 0.2 and 0.3 mm, between 0.3 and 0.4 mm, between 0.4 and 0.5 mm, between 0.5 and 0.6 mm, between 0.6 and 0.7 mm, between 0.7 and 0.8 mm, between 0.8 and 0.9 mm, between 0.9 and 1 mm, between 1 mm and 1.1 mm, between 1.1 mm and 1.2 mm, between 1.2 mm and 1.3 mm, between 1.3 mm and 1.4 mm, between 1.4 mm and 1.5 mm, between 1.5 mm and 2 mm, between 2 mm and 2.5 mm, between 2.5 mm and 3 mm, between 3 mm and 3.5 mm, between

[0084] 3.5 mm and 4 mm, between 4 mm and 4.5 mm, between 4.5 mm and 5 mm, between 5 mm and

[0085] 5.5 mm, between 5.5 mm and 6 mm, between 0.2 mm and 0.4 mm, between 0.1 mm and 1 mm, between 0.5 mm and 1.0 mm, between 1.0 mm and 1.5 mm, between 1.5 mm and 2.0 mm, between 2.0 mm and 2.5 mm, between 2.5 mm and 3.0 mm, between 3.0 mm and 3.5 mm, between 3.5 mm and 4.0 mm, between 4.0 mm and 4.5 mm, between 4.5 mm and 5.0 mm, or between 5.5 mm and 6.0 mm. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) have a length of about 0.5 mm. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) have a length of 0.5 mm. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) have a length of about 0.3 mm, In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) have a length of 0.3 mm.

[0086] The channels, propagation channels, cell culture chambers, inlet chambers, and outlet chambers of the present invention need not be limited by shape. Non-limiting examples of such shapes include said channels being cylindrical or cuboidal. In some embodiments, the channels and / or propagation channel(s) of the devices disclosed herein are cylindrical. In some embodiments, the channels and / or propagation channel(s) are cuboidal. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) of the devices disclosed herein are cylindrical. In some embodiments, the cell culture chamber(s), inlet chamber(s), and / or outlet chamber(s) are cuboidal.

[0087] The device described herein may be formed through any method known in the art, including, but not limited to, injection molding, etching, hot embossing, extrusion molding, compression molding, blow molding, rotational molding, vacuum casting, thermoforming, micromilling, micromachining, and vacuum forming. In some embodiments, the microfluidic device according to the invention is formed through 3D printing. Non-limiting examples of 3D printing methods for use in the present application include Stereolithography (SLA), Selective Laser Sintering (SLS), Fused Deposition Modeling (FDM), Digital Light Processing (DLP), Multi Jet Fusion (MJF), PolyJet, Direct Metal Laser Sintering (DMLS), and / or Electron Beam Melting (EBM). In some aspects, the material used for formulating a microfluidic device according to the present invention is chosen from the list comprising polyethylene terephthalate (PET), high- and low-density polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), polydimethylsiloxane (PDMS), polycarbonate (PC), polystyrene (PS), polyamide (PA), polyethylene glycol (PEG), thermoplastic elastomers (TPE), poly aryletherketones (PAEK), polytetrafluoroethylene (PTFE), cyclic olefin copolymer (COC), glass, or any other translucent material. An example of a minimally restrictive microfluidic 3D cell culture device formed through SLA 3D printing molds using resin and casting devices with PDMS is shown in FIGs. 3A, 3B and 3E.

[0088] In some embodiments, the device described herein is comprised of synthetic polymers, biological polymers, and / or glass. Non-limiting examples of biological polymers include dextran, gelatin, fibrin, Matrigel, Collagen (types I, II, and III), Laminin, Fibronectin, Fibrinogen, Tropoelastin, Elastin, Hyaluronic Acid (Hyaluronan), Chondroitin Sulfate, Alginate, Chitosan, Polyacrylamice (PAAm), Polylactic acid (PLA), Polylactic-co-glycolic acid (PLGA), Polycaprolactone (PCL), Poly(vinyl alcohol) (PVA), poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), Poly(N-isopropylacrylamide) (PNIPAm), Poly(acrylic acid) (PAAc), PNIPAAm-PAAc, or variants thereof.

[0089] In some embodiments, the microfluidic device according to the invention has an opentop design, such that the chambers of the device are open to the air. This open-top design provides easy accessibility to the cells and / or cell aggregates cultured in the device, along with the scaffolding substance and any fluid being perfused. Through this open-top design, agents (e.g., active agents, e.g., drugs, cells, dyes, etc.) can easily be administered into a specific chamber. In some embodiments, an agent is administered into a specific cell culture chamber, as opposed to introducing the agent to the entire system through an inlet chamber. In some embodiments, at least one of the chambers (e.g., cell culture chambers, inlet chambers and / or outlet chambers) of the device described herein has an open top. In other embodiments, all the chambers (e.g., cell culture chambers, inlet chambers and outlet chambers) of the device described herein have open tops.

[0090] One important aspect of 3D cell culture in the microfluidic device described herein is the ability to spatially control the introduction of fluids and agents to distinct regions of the cells and / or cell aggregates. For example, FIGs. 7A-10 provide possible microfluidic network configurations through which fluid is introduced to a particular area of the device, potentially without contacting other areas. Methods such as this can provide measurements of cell and / or cell aggregate migration and chemotropism, for example. In some embodiments, the propagation channels place the cell culture chambers in direct contact.

[0091] Aspects of the disclosure relate to methods of culturing human 3D in vitro models of tissues for developing or identifying treatment strategies for diseases or disorders associated with those tissues. For instance, a 3D model of liver tissue can be used to study liver disease or other properties of liver. A 3D model of brain tissue can be used to study neurodegenerative diseases such as Alzheimer’s disease (AD). The 3D models of tissue can be cultured using the devices disclosed herein. Importantly, the devices disclosed herein provide 3D cell cultures to avoid pressure-induced cell stress, which leads to sub-optimal growth and prevents cells from forming physiological structures. In some embodiments, the microfluidic devices disclosed herein include cell culture media and components conditioned for optimal tissue development that is suitable for determining genetic and molecular internetworks of tissue specific diseases.

[0092] Without models that can accurately mimic pathogenesis of complex diseases such as Alzheimer’s disease in humans, drug screenings for personalized treatments are limited. The devices disclosed herein are particularly useful for culturing human 3D in vitro tissue models, such as the brain, which recapitulates numerous molecular and physiological features of the intact tissue, such as brain tissue. Thus, improved microfluidic 3D cell culture devices capable of supporting the growth and formation of these 3D in vitro tissue models while also allowing perfusion are disclosed herein.

[0093] Aspects of the disclosure provide a device for culturing tissue engineering that harnesses the innate ability of mature cells to self-assemble into complex 3D tissues, making perfusable tissues. For example, the blood-brain barrier can be formed through the interaction of at least three cell types including brain endothelial cells (BECs), astrocytes, and pericytes. Capillary formation can be initiated by culturing endothelial cells in malleable ECM.

[0094] Brain tissues can comprise a cerebrovascular network comprised of human brain endothelial cell (BEC) vessel comprised of a large, interconnected network of human pluripotent-derived positive endothelial cells and human pluripotent-derived pericytes proximal to the BEC vessel on an apical surface. In some embodiments, human pluripotent-derived astrocytes may be dispersed throughout the 3D matrix and human myelinated cells may be dispersed throughout the 3D matrix and immune cells. In some embodiments, the cells may be derived from human hematopoietic progenitor cells (HPCs). It should be appreciated that the cells can be derived from any cells or stem cells capable of assisting with the preparation and production of cells for the tissue culture described herein.

[0095] In some embodiments, the perfusable tissue in the device includes a culture media that supports the diverse cell-types present. In some embodiments, the culture media comprises hESFM, AGS, B27 and one or more supplements selected from the group consisting of NT3, T3, IGF, PDGFB, cAMP, AA, BDNF, GDNF, mCSF, IL34, and VegF-A.

[0096] In some embodiments, the scaffold material comprises a synthetic hydrogel, a cell- degradable crosslinker and a tissue specific extracellular matrix (ECM) protein. In some embodiments the synthetic hydrogel comprises a dextran-based polysaccharide macromer. In some embodiments the ECM protein comprises at least one brain-mimicking ECM. Various ECM proteins can be used, for instance, RGD peptides, collagens, fibronectins, laminins, aggrecans, hyaluronic acids, heparan sulphates, thrombospondin, tenascin R, tenascin C, reelin, versicans or versican like peptides, which include but are not limited to neurocan and brevican, and other chondroitin sulphates, and peptides that mimic key signaling regions of these proteins and proteoglycans. Versican is a chondroitin sulfate proteoglycan that is found in the ECM of many human tissues.

[0097] EXAMPLES

[0098] In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the methods, devices, and systems provided herein and are not to be construed in any way as limiting their scope.

[0099] Example 1: Screening parameter space for constriction- minimizing microfluidic “GelChip” and “GelChip Plate” 3D cell culture devices enabling 3D vascular network formation

[0100] Engineered hydrogels swell and in confined environments like microfluidic devices. This swelling leads to deleterious forces on cells cultured in 3D in the hydrogels, inducing cellular stress that can be cytotoxic and has been shown to be prohibitive to 3D cultures, including perfusable 3D endothelial network formation (FIG. 4A, left). While conventional microfluidic devices support 3D endothelial cell network formation in fibrin and protein-based materials (FIG. 4A, middle), networks are inhibited from forming in engineered hydrogels even with simple endothelial cell and fibroblast co-cultures, and fully connected networked cannot be attained nor perfused. Moreover, fibrin and protein-based gels cannot support complex cell cocultures, such as miBrain, which contains 6 different cell types. Attempts to grow miBrain cultures in conventional gels (e.g., hydrogel) showed a failure to form lumenized 3D endothelial cell networks in culture (FIG. 4A, right).

[0101] An individual cell is on the order of 20-30 pm and conventional microfluidic chambers for cell culture are fabricated at heights of 100-250 pm, less than ten times the scale of a single cell. Therefore, devices were designed and fabricated to determine if devices at larger length scales could be successfully constructed for 3D cell culture, that they would minimize constrictive forces in 3D cell culture, and that they would enable successful interconnected, perfusable 3D endothelial cell networks.

[0102] A first screening experiment was conducted to identify device dimensions capable of supporting 3D cell culture and growth of perfusable vascular networks. Endothelial Cells and Fibroblasts were seeded in a dextran-based engineered hydrogel in the cell culture chambers (3) of a constriction-minimizing microfluidic 3D cell culture device with the network configuration shown in FIG. 2D. The channels of all microfluidic networks of the device were 3 mm long and 0.85 mm wide, and the height of the channels was varied between 0.05 mm and 0.6 mm. Cells were cultured with media introduced via the inlet chamber and removed via the outlet chamber for 1 week, then assessed for vessel network formation. Devices with a height of 0.6 mm and width of 0.85 mm were found to support maximal growth of endothelial networks (FIG. 4B).

[0103] Additional experiments were designed and carried out to determine if further altering the channel lengths, widths, or heights would alter vessel network formation. Exemplary experimental designs of such experiments are shown in FIGs. 4C and 11. Devices comprising channels with a height of 0.6 mm, a length of 0.3 mm, and a width of 0.85 mm demonstrated high levels of vessel network formation. Indeed, co-culture of endothelial cells and fibroblasts in a device with these dimensions successfully showed the formation of vascular networks which were perfusable within the cell scaffolds of the device.

[0104] Given these promising results, devices comprising channels having at least 0.6 mm height, 0.3 mm length, and 0.85 mm width were named “GelChips” and used for further characterization. FIGs. 4D-4E depict examples of GelChip configurations. In some embodiments, the channels connecting the culture chamber and the inlet and / or outlet chambers have tapered ends (e.g., wherein the channel has an entrance width of 0.15 mm with a full width of 0.85 mm, as shown in FIG. 4D, middle). In some embodiments, the device of the present invention comprises multiple core microfluidic networks on a single base (e.g., for use in high- throughput experiments), as shown in FIG. 4E. In some embodiments, the channels of the GelChip device have varying lengths and / or maximum and minimum widths. FIG. 4F shows an example of a GelChip device with: (1) a tapered channel having a minimum width of 0.15 mm (e.g., at the intersection between the channel and the inlet chamber) with an overall length of 2 mm (left); (2) a tapered channel having a minimum width of 0.15 mm and an overall length of 1.5 mm (middle); and (3) a tapered channel having a minimum width of 0.3 mm and an overall length of 1.5 mm (right).

[0105] The GelChip device was further modified to produce a scaled-up device to support higher throughput experiments, hereinafter called a “GelChip Plate.” In some embodiments, the GelChip Plate comprises a multitude of inlet chambers, outlet chambers, and channels. In some embodiments, a GelChip Plate comprises two or more core microfluidic networks, each of which comprises at least one inlet chamber, at least one outlet chamber, at least two channels, at least two culture chambers, and at least one propagation channel. An example of a GelChip Plate configuration is shown in FIG. 12. The example GelChip Plate shown in FIG. 12 comprises 10 core microfluidic networks, each microfluidic network comprising one inlet chamber, one outlet chamber, two major channel systems, eight culture chambers, and four propagation channels. A GelChip Plate may comprise any microfluidic network design that supports perfusion, and may comprise any number of microfluidic networks on a single GelChip Plate.

[0106] Example 2: Validating novel GelChips for complex 3D cell culture and vessel network formation within miBrain

[0107] A third experiment was designed and carried out to determine if GelChips of 600 pm height and 850 pm width could support the network formation of iPSC-derived endothelial cells and of multiple iPS-derived cell types co-cultured with endothelial cells and fibroblasts. Multicellular integrated brains (miBRAINs) (for example described in US patent application 2022 / 0127581) comprised of all six CNS cell types (endothelial cells, pericytes, astrocytes, neurons, oligodendrocytes, and microglia), all of which were derived from iPSCs, were cultured with fibroblasts in the device provided herein or in conventional commercially-available microfluidic devices (AIM Biotech). The co-culture was then assessed for vessel network formation. Methods of generating and culturing in vitro brain (miBRAIN) are described in U.S. Patent Application 17 / 458,838, the contents of which are incorporated herein. The novel GelChip demonstrated the ability to form fully interconnected miBrain vessel networks while commercially available devices did not support miBrain endothelial cell network formation (FIG. 5A).

[0108] To fully characterize the miBRAIN cultured in GelChip, and to ensure that the novel devices provided for robust phenotypes across miBRAIN cells, each cell type was assessed. In addition to the endothelial cells that formed robust interconnected vascular networks, neurons extended interconnected processes throughout the culture (FIG. 5B).

[0109] Example 3. Perfusable 3D brain-chip platform for evaluating delivery to the brain

[0110] To test if GelChips enabled perfusion through the lumens of 3D vessel networks, fluorescent nanoparticles were used to assess vessel perfusion. Endothelial cells and fibroblasts were cultured for 1 week in GelChips. Additional endothelial cells were added to the inlet and outlet chambers to form an outer barrier to the gel and to connect with the 3D vessel networks. Nanoparticles were introduced into one inlet chamber, and nanoparticle motion was tracked on a confocal imaging system over time. Nanoparticles entered the lumenized vessels and moved throughout the vessel network, validating the perfusability of the vessels. Fluorescent dextran tracers of 70 kDa and 500 kDa were used to quantify the permeability of the barrier (FIG. 6).

[0111] To test if the novel GelChips could enable perfusable 3D vessels for iPSC-derived endothelial cells and within complex cell co-culture, miBRAINs with iPSC-derived cell types of endothelial cells, pericytes, astrocytes, neurons, oligodendrocytes, and microglia along with fibroblasts were cultured in GelChips for 2 weeks. A fluorescent dextran tracer was perfused through one inlet chamber of the GelChip and tracked on a confocal microscope through the lumenized 3D vessels of the miBRAIN. The tracer entered the lumenized vessels and progressed through the networks, validating the perfusability of the vessels in the miBRAINs cultured in GelChips.

[0112] Example 4: Propagation- GelChip to probe phenotype progression and microglia migration

[0113] Given the motility of microglia through the miBrain and the interest in the field to understand how microglia could potentially spread pathological effectors between disparate areas of brain tissue, Propagation-GelChips were designed. miBRAINs were cultured in the culture chambers and the propagation channels connecting the culture chambers of the Propagation-GelChips for 2 weeks. Microglia were added to one chamber, and CCL2 was added to the opposite chamber to provide a chemoattractant to drive migration across the device. Within 3 days, migration of microglia was detected through the propagation chamber in the direction of CCL2. Varying the concentration of CCL2 from 0 to 200 ng / mL demonstrated a dose-effect on microglia migration, with 200 ng / mL leading to the highest number of migrating microglia.

[0114] To harness Propagation-GelChips to model microglia response to injury, miBRAINs were cultured in Propagation-GelChips and subjected to laser ablation. miBRAINs were cultured for 2 weeks with fluorescent neurons in one culture chamber, with and fluorescently- labeled microglia were incorporated in another culture chamber in the same microfluidic network. High intensity laser power was used to ablate neurons in one chamber, and microglia response to the ablation was monitored longitudinally. 4 days post-ablation, migration of microglia was detected and a population of microglia was detected at the site of the injury, demonstrating the 3D longitudinal cell-cell communication and migration supported by the culture devices described herein.

[0115] Example 5: Modulation of spatial configurations and designs of microfluidic networks

[0116] The microfluidic networks of the device described herein may be arranged in any way in order to probe the biological question being addressed. The network configurations described herein are offered to illustrate example configurations and are not to be construed in any way as limiting their scope.

[0117] FIG. 7A provides an example of an “Axis-GelChip” or “2Region-GelChip” network design that can be used, for example, to study interactions between two tissues or two tissue regions (e.g., cells and / or cell aggregates). The number of tissues or tissue regions (e.g., cells and / or cell aggregates) can be increased by building further configurations off this design. In some embodiments, the 2Region-GelChip configuration is used, for example, to study the interaction between two brain regions connected by neuronal projections and / or migrating immune cells. In some embodiments the 2Region-GelChip configuration is used, for example, to study the interaction between an engineered gut and an engineered brain model connected by a vagus nerve mimic.

[0118] FIG. 7B provides an example of a “Peripheral-GelChip” network design to study interactions between two tissues or two tissue regions (e.g., cells and / or cell aggregates). The number of tissues or tissue regions (e.g., cells and / or cell aggregates) can be increased by building further configurations off of this design. In some embodiments, the peripheral chip configuration is used, for example, to study the interaction between two tissues connected only by the bloodstream, such as a mini-liver that secretes factors that are passed via the bloodstream through the vessels infiltrating a mini-brain tissue, which can be studied for passage across the blood-brain barrier to examine the effect on brain tissue. In other embodiments, the peripheral chip configuration is used to study whether direct contact between the tissues is needed, or if the peripheral interactions are sufficient to induce the observed biological effect.

[0119] FIG. 7C provides an example of a “DualNetwork-GelChip” network design, which can be used, for example, to co-culture two tubular cellular networks connected to independent side ports, wherein different factors (e.g., active agents) can be introduced into each network. In some embodiments, the dual-network chip configuration is used, for example, to co-culture lymphatic vessels and endothelial networks for the construction of a mini-brain model with both blood and lymphatic flow mimics, and for modeling glymphatic clearance or interactions between glymphatics and cerebral blood flow.

[0120] FIG. 8A provides examples of “2Region-GelChip” configurations and example variations thereof, demonstrating the tunability in the length of the propagation channel(s) of the present invention. In some embodiments, the 2Region-GelChip configurations are used, for example, to study whether cholinergic neuron length contributes to neuronal susceptibility to tau pathology. This can be performed by studying the transmission of signaling cues that trigger immune cell responses to neurovascular injury.

[0121] FIG. 8B provides examples of “3Region-GelChip” configurations and example variations thereof, demonstrating the tunability in propagation channel lengths and vectorial placement in relation to the cell culture chambers and inlet and outlet chambers of the present device. In some embodiments, the 3Region-GelChip configurations are used, for example, to study three different tissues or three tissue regions (e.g., cells and / or cell aggregates), such as between brain regions, or more specifically, between hippocampal-thalamic-cortical circuits required for memory consolidation.

[0122] FIG. 8C provides examples of “4Region-GelChip” configurations and example variations thereof, demonstrating the tunability in propagation channel lengths and vectorial placement in relation to cell culture chambers and inlet and outlet chambers of the present device. In some embodiments, the 4Region-GelChip configurations are used to study a hippocampal circuit such as one connecting the hippocampus, cortex, thalamus and hypothalamus, which is responsible for consolidation of new declarative memories, or an amygdala circuit such as one connecting the amygdala, cortex, thalamus and hypothalamus, which is responsible for emotional memories and behaviors.

[0123] FIG. 9A provides examples of “4RegionCircuit-GelChip” configurations and example variations thereof, demonstrating the tunability in configurations, dimensions and contacts of the propagation and channels of the present device. In some embodiments, the 4RegionCircuit- GelChip configurations enable, for example, dissection of various interactions such as circuit interactions in brain-mimics (e.g., decision trees). In some embodiments, the 4RegionCircuit- GelChip supports, for example, the study of neuronal activity changes in one region with additional inlets projecting into it. In another embodiment, the 4RegionCircuit-GelChip supports, for example, the study of decision trees wherein various factors (e.g., active agents) can be placed in connecting regions, and the effect on cells in an adjacent region is examined. In another embodiment, the 4RegionCircuit-GelChip supports, for example, a study wherein immune cells are placed in one cell culture chamber, and their preference for various factors (e.g., active agents) in a mini-brain tissue mimic are studied.

[0124] FIG. 9B provides examples of “ProjectionLogic-GelChip” configurations and example variations thereof, further demonstrating the tunability in configurations, dimensions and contacts of the propagation channels of the present device. In some embodiments, the projection logic chip configuration is an adaptation of the 3Region-GelChip configuration, which allows, for example, the parsing of effects of various interactions between tissues and tissue regions (e.g., cells and / or cell aggregates). In some embodiments, the projection logic chip assesses the effect of various inlets and combinations of inlets (e.g., decision trees), wherein an “inlet” may be, for example, neuronal projections from one or more different neuronal subtypes or genetic backgrounds. In some embodiments, the projection logic chip assesses the effect on neuronal activity of additional neuronal inlets.

[0125] FIG. 10 provides examples of “NeuralCircuit-GelChip” and “NetworkTree-GelChip” configurations. In some embodiments, the networks are arranged to mimic, for example, various brain regions connected via neuronal projections, or wherein various chambers are used to create decision scenarios.

[0126] The following are exemplary embodiments of the claimed invention: 1. A minimally restrictive microfluidic 3D cell culture device comprising a body, the body comprising:

[0127] (i) at least one inlet chamber comprised of a bottom surface, side walls and an open top;

[0128] (ii) at least one outlet chamber comprised of a bottom surface, side walls and an open top; and

[0129] (iii) at least one channel; wherein the dimensions of the at least one channel support the housing and swelling of a scaffolding substance.

[0130] 2. The minimally restrictive microfluidic 3D cell culture device of embodiment 1, wherein the body further comprises: (iv) at least one cell culture chamber comprised of a bottom surface, side walls and an open top, wherein the dimensions of the at least one cell culture chamber support the housing and swelling of a scaffolding substance.

[0131] 3. The minimally restrictive microfluidic 3D cell culture device of embodiment 2, wherein the at least one channel comprises a first channel extending between the at least one inlet chamber and the at least one cell culture chamber, and a second channel extending between the at least one outlet chamber and the at least one cell culture chamber.

[0132] 4. The minimally restrictive microfluidic 3D cell culture device of any one of embodiments 1-3, wherein the at least one channel has a height of at least 0.05 mm.

[0133] 5. The minimally restrictive microfluidic 3D cell culture device of embodiment 4, wherein the at least one channel has a height of about 0.6 mm.

[0134] 6. The minimally restrictive microfluidic 3D cell culture device of embodiment 4, wherein the at least one channel has a height between 0.05 mm and 0.1 mm, between 0.1 and 0.15 mm, between 0.15 and 0.2 mm, between 0.2 and 0.25 mm, between 0.25 and 0.3 mm, between 0.3 and 0.35 mm, between 0.35 and 0.4 mm, between 0.4 and 0.45 mm, between 0.45 and 0.5 mm, between 0.5 mm and 0.55 mm, between 0.55 mm and 0.6 mm, between 0.6 mm and 0.65 mm, between 0.65 and 0.7 mm, between 0.7 mm and 0.75 mm, between 0.75 mm and 0.8 mm, between 0.8 mm and 0.85 mm, between 0.85 mm and 0.9 mm, between 0.90 mm and 0.95 mm, or between 0.95 mm and 1.00 mm. 7. The minimally restrictive microfluidic 3D cell culture device of any one of embodiments 1-6, wherein the at least one channel has a length of at least 0.1 mm.

[0135] 8. The minimally restrictive microfluidic 3D cell culture device of embodiment 7, wherein the at least one channel has a length of about 0.3 mm.

[0136] 9. The minimally restrictive microfluidic 3D cell culture device of embodiment 7, wherein the at least one channel has a length between 0.1 mm and 0.2 mm, between 0.2 mm and 0.3 mm, between 0.2 mm and 0.4 mm, between 0.3 mm and 0.4 mm, between 0.4 mm and 0.5 mm, between 0.5 mm and 0.6 mm, between 0.6 mm and 0.7 mm, between 0.7 mm and 0.8 mm, between 0.8 mm and 0.9 mm, between 0.9 mm and 1.0 mm, between 1.0 mm and 1.1 mm, between 1.1 mm and 1.2 mm, between 1.2 mm and 1.3 mm, or between 1.3 mm and 1.4 mm.

[0137] 10. The minimally restrictive microfluidic 3D cell culture device of any one of embodiments 1-9, wherein the at least one channel has a width of at least 0.5 mm.

[0138] 11. The minimally restrictive microfluidic 3D cell culture device of embodiment 10, wherein the at least one channel has a width of about 0.85 mm.

[0139] 12. The minimally restrictive microfluidic 3D cell culture device of embodiment 10, wherein the at least one channel has a width between 0.5 mm and 0.55 mm, between 0.55 mm and 0.6 mm, between 0.6 mm and 0.65 mm, between 0.65 mm and 0.7 mm, between 0.7 mm and 0.75 mm, between 0.75 mm and 0.8 mm, between 0.8 mm and 0.85 mm, between 0.8 mm and 0.9 mm, between 0.85 mm and 0.9 mm, between 0.9 mm and 0.95 mm, between 0.95 mm and 1.0 mm, between 1.0 mm and 1.1 mm, between 1.1 mm and 1.2 mm, between 1.2 mm and 1.3 mm, or between 1.3 mm and 1.4 mm.

[0140] 13. The minimally restrictive microfluidic 3D cell culture device of any one of embodiments 1-12, wherein the at least one inlet chamber and / or the at least one outlet chamber has a width of at least 0.5 mm.

[0141] 14. The minimally restrictive microfluidic 3D cell culture device of any one of embodiments 1-13, wherein the at least one inlet chamber and / or the at least one outlet chamber has a length of at least 0.5 mm. 15. The minimally restrictive microfluidic 3D cell culture device of any one of embodiments 1-14, wherein the device comprises at least two cell culture chambers.

[0142] 16. The minimally restrictive microfluidic 3D cell culture device of embodiment 15, further comprising at least one propagation channel extending between the at least two cell culture chambers.

[0143] 17. The minimally restrictive microfluidic 3D cell culture device of embodiment 16, wherein the at least one propagation channel has a height between 0.05 mm and 1 mm, optionally wherein the at least one propagation channel has a height of about 0.6 mm.

[0144] 18. The minimally restrictive microfluidic 3D cell culture device of embodiments 16 or 17, wherein the at least one propagation channel has a width between 0.1 mm and 6 mm, optionally wherein the at least one propagation channel has a width of about 0.85 mm.

[0145] 19. The minimally restrictive microfluidic 3D cell culture device of any one of embodiments 16-18, wherein the at least one propagation channel has a length between 0.1 mm and 6 mm, optionally wherein the at least one propagation channel has a length of about 0.3 mm.

[0146] 20. The minimally restrictive microfluidic 3D cell culture device of any one of embodiments 1-19, wherein the device is comprised of synthetic polymers, biological polymers, and / or glass.

[0147] 21. The minimally restrictive microfluidic 3D cell culture device of embodiment 20, wherein the device is fabricated from a material chosen from the list comprising polydimethylsiloxane (PDMS), polycarbonate (PC), polystyrene (PS), polyamide (PA), polyethylene glycol (PEG), thermoplastic elastomers (TPE), polyaryletherketones (PAEK), polytetrafluoroethylene (PTFE), cyclic olefin copolymer (COC), glass, or other translucent material.

[0148] 22. The minimally restrictive microfluidic 3D cell culture device of embodiment 20, wherein the biological polymers are chosen from dextran, PEG, gelatin, fibrin, Matrigel, Collagen (types I, II, and III), Laminin, Fibronectin, Fibrinogen, Tropoelastin, Elastin, Hyaluronic Acid (Hyaluronan), Chondroitin Sulfate, Alginate, Chitosan, Polyacrylamide (PAAm), Polylactic acid (PLA), Polylactic-co-glycolic acid (PLGA), Polycaprolactone (PCL), Poly(vinyl alcohol) (PVA), poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), Poly(N- isopropylacrylamide) (PNIPAm), Poly(acrylic acid) (PAAc), PNIPAAm-PAAc, and / or variants thereof.

[0149] 23. The minimally restrictive microfluidic 3D cell culture device of any one of embodiments 1-22, wherein the device is formed through 3D printing, injection molding, hot embossing, micromilling, micromachining, or etching.

[0150] 24. The minimally restrictive microfluidic 3D cell culture device of embodiment 23, wherein the method of 3D printing is chosen from the list comprising Stereolithography (SLA), Selective Laser Sintering (SLS), Fused Deposition Modeling (FDM), Digital Light Processing (DLP), Multi Jet Fusion (MJF), PolyJet, Direct Metal Laser Sintering (DMLS), and Electron Beam Melting (EBM).

[0151] 25. The minimally restrictive microfluidic 3D cell culture device of any one of embodiments 16-24, comprising a microfluidic network comprising at least two configurations of the at least one cell culture chamber, the at least one inlet chamber, the at least one outlet chamber, the at least one channel, and the at least one propagation channel.

[0152] 26. The minimally restrictive microfluidic 3D cell culture device of embodiment 25, wherein the device supports perfusion of fluid between the microfluidic network of at least two configurations.

[0153] 27. The minimally restrictive microfluidic 3D cell culture device of any one of embodiments 1-26, wherein the scaffolding substance is a gel or gel-precursor selected from the list comprising Hydrogel, Fibrin, Collagen (types I, II, and III) Laminin, Fibronectin, Fibrinogen, Tropoelastin, Elastin, Hyaluronic Acid (Hyaluronan), Matrigel, Chondroitin Sulfate, Alginate, Chitosan, Dextran, and / or variants thereof.

[0154] 28. The minimally restrictive microfluidic 3D cell culture device of embodiment 27, wherein the hydrogel is comprised of Polyethylene glycol (PEG), Polyacrylamide (PAAm), Polylactic acid (PLA), Polylactic-co-glycolic acid (PLGA), Polycaprolactone (PCL), Poly (vinyl alcohol) (PVA), poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), Poly(N- isopropylacrylamide) (PNIPAm), Poly(acrylic acid) (PAAc), PNIPAAm-PAAc, and / or variants thereof. 29. A method of 3D culturing cells in the minimally restrictive microfluidic 3D cell culture device of any one of embodiments 1-28, comprising:

[0155] (a) introducing one or more cell types into the at least one input chamber and / or the at least one output chamber;

[0156] (b) contacting the one or more cell types with culture media; and

[0157] (c) incubating the minimally restrictive microfluidic 3D cell culture device containing the one or more cell types in an environment supporting cell growth.

[0158] 30. The method of embodiment 29 further comprising introducing the culture media through the at least one input chamber and removing the culture media through the at least one output chamber, thereby perfusing the culture media through the at least two channels and the at least one channel.

[0159] 31. The method of embodiments 29 or 30, wherein the one or more cell types is human.

[0160] 32. A perfusable microfluidic cell culture network, comprising at least two cell culture chambers, at least one inlet chamber, at least one outlet chamber, at least two channels, and at least one propagation channel, wherein: the at least two channels comprise a first channel extending between the at least one inlet chamber and a first cell culture chamber, and a second channel extending between the at least one outlet chamber and a second cell culture chamber; wherein the at least one propagation channel extends between the first cell culture chamber and the second cell culture chamber; and wherein the dimensions of the at least one cell culture chamber, the at least one propagation channel, and / or the at least two channels support the housing and swelling of a synthetic hydrogel scaffolding substance with minimal cell compression.

[0161] 33. The perfusable microfluidic cell culture network of embodiment 32, wherein the at least two channels have a height of at least 0.5 mm.

[0162] 34. The perfusable microfluidic cell culture network of embodiment 33, wherein the at least two channels and / or the at least one propagation channel have a height of about 0.6 mm. 35. The perfusable microfluidic cell culture network of any one of embodiments 32-34, wherein the perfusable microfluidic cell culture network comprises at least two inlet chambers, at least two outlet chambers, and at least four channels, wherein the at least four channels comprise a first set of channels extending between the at least two inlet chambers and the at least two cell culture chambers, and a second set of channels extending between the at least two outlet chambers and at least two cell culture chambers.

[0163] 36. The perfusable microfluidic cell culture network of embodiment 35, wherein the at least two cell culture chambers, the at least two inlet chambers, and the at least two outlet chambers are comprised of a bottom surface, side walls and an open top.

[0164] 37. The perfusable microfluidic cell culture network of any one of embodiments 32-36, wherein the perfusable microfluidic cell culture network supports the perfusion of fluid between the at least one inlet chamber and the at least one outlet chamber.

[0165] 38. The perfusable microfluidic cell culture network of any one of embodiments 32-37, wherein the scaffolding substance is a gel or gel-precursor selected from the list comprising Hydrogel, Fibrin, Collagen (types I, II, and III) Laminin, Fibronectin, Fibrinogen, Tropoelastin, Elastin, Hyaluronic Acid (Hyaluronan), Matrigel, Chondroitin Sulfate, Alginate, Chitosan, Dextran, and / or variants thereof.

[0166] 39. The perfusable microfluidic cell culture network of embodiment 38, wherein the hydrogel is comprised of Polyethylene glycol (PEG), Polyacrylamide (PAAm), Polylactic acid (PLA), Polylactic-co-glycolic acid (PLGA), Polycaprolactone (PCL), Poly(vinyl alcohol) (PVA), poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), Poly(N- isopropylacrylamide) (PNIPAm), Poly(acrylic acid) (PAAc), PNIPAAm-PAAc, and / or variants thereof

[0167] 40. A method of culturing cells and / or cell aggregates on a device, comprising introducing one or more cell types in a scaffolding substance to the device, the device comprising:

[0168] (i) at least one inlet chamber comprised of a bottom surface, side walls and an open top;

[0169] (ii) at least one outlet chamber comprised of a bottom surface, side walls and an open top; and (iii) at least two channels, wherein the dimensions of the at least one channel supports the housing and swelling of the scaffolding substance.

[0170] 41. The method of embodiment 40, wherein the device further comprises: (iv) at least one cell culture chamber comprised of a bottom surface, side walls and an open top, wherein the dimensions of the at least one cell culture chamber support the housing and swelling of a scaffolding substance.

[0171] 42. The method of embodiment 41, wherein the at least one channel comprises a first channel extending between the at least one inlet chamber and the at least one cell culture chamber, and a second channel extending between the at least one outlet chamber and the at least one cell culture chamber.

[0172] 43. The method of any one of embodiments 40-42, wherein the device is the minimally restrictive microfluidic 3D cell culture device of any one of embodiments 1-28.

[0173] 44. The method of any one of embodiments 40-43, wherein the cultured cells and / or cell aggregates comprise more than one cell type.

[0174] 45. The method of any one of embodiments 40-44, wherein the cultured cells and / or cell aggregates comprise mammalian cells.

[0175] 46. The method of embodiment 45, wherein the mammalian cells are selected from the list comprising endothelial cells, neuronal cells, connective tissue cells, fibroblasts, epithelial cells, epidermal or dermal cells, chondrocytes, osteocytes (e.g., osteoblasts), blood cells (e.g., red or white blood cells), plasma cells, adipocytes, myocytes, hepatocytes, cholangiocytes, stellate cells, Kupffer cells, glial cells, endocrine cells (e.g., cells of the islet), reticular cells, cells of sensory organs, mesenchymal cells, immune cells, and / or stem cells (e.g., embryonic or induced pluripotent stem cells).

[0176] 47. The method of any one of embodiments 40-46, further comprising vascularizing the cells and / or cell aggregates in the scaffolding substance. 48. The method of any one of embodiments 40-47, further comprising inducing a flow of a fluid through the microfluidic network.

[0177] 49. The method of embodiment 48, wherein the fluid comprises one or more active agents.

[0178] 50. The method of embodiment 49, wherein the one or more active agent comprises small molecules, drugs, nucleic acids, proteins, peptides, lipids, carbohydrates, growth factors, bacteria, viruses, variants thereof and / or any other type of biological or synthetic molecules.

[0179] 51. An in vitro tissue mimic comprising a 3-dimensional (3D) matrix comprising a perfusable vascular network in a synthetic hydrogel, wherein the hydrogel comprises a dextran- based polysaccharide macromer, a cell-degradable crosslinker and a tissue specific extracellular matrix protein and further comprising at least one neural specific cell type dispersed in the hydrogel.

[0180] 52. The 3D matrix of embodiment 51, wherein the tissue specific extracellular matrix protein is versican or a versican-like peptide.

Claims

CLAIMSWhat is claimed is:

1. A minimally restrictive microfluidic 3D cell culture device comprising a body, the body comprising:(i) at least one inlet chamber comprised of a bottom surface, side walls and an open top;(ii) at least one outlet chamber comprised of a bottom surface, side walls and an open top; and(iii) at least one channel; wherein the dimensions of the at least one channel support the housing and swelling of a scaffolding substance.

2. The minimally restrictive microfluidic 3D cell culture device of claim 1, wherein the body further comprises: (iv) at least one cell culture chamber comprised of a bottom surface, side walls and an open top, wherein the dimensions of the at least one cell culture chamber support the housing and swelling of the scaffolding substance and optionally has a height of at least 0.05 mm.

3. The minimally restrictive microfluidic 3D cell culture device of claim 2, wherein the at least one channel comprises a first channel extending between the at least one inlet chamber and the at least one cell culture chamber, and a second channel extending between the at least one outlet chamber and the at least one cell culture chamber.

4. The minimally restrictive microfluidic 3D cell culture device of any one of claims 1-3, wherein the at least one channel has(i) a height of about 0.6 mm, between 0.05 mm and 0.1 mm, between 0.1 and 0.15 mm, between 0.15 and 0.2 mm, between 0.2 and 0.25 mm, between 0.25 and 0.3 mm, between 0.3 and 0.35 mm, between 0.35 and 0.4 mm, between 0.4 and 0.45 mm, between 0.45 and 0.5 mm, between 0.5 mm and 0.55 mm, between 0.55 mm and 0.6 mm, between 0.6 mm and 0.65 mm, between 0.65 and 0.7 mm, between 0.7 mm and 0.75 mm, between 0.75 mm and 0.8 mm, between 0.8 mm and 0.85 mm, between 0.85 mm and 0.9 mm, between 0.90 mm and 0.95 mm, or between 0.95 mm and 1.00 m;(ii) a length of at least 0.1 mm, about 0.3 mm, between 0.1 mm and 0.2 mm, between 0.2 mm and 0.3 mm, between 0.2 mm and 0.4 mm, between 0.3 mm and 0.4 mm, between 0.4 mm and 0.5 mm, between 0.5 mm and 0.6 mm, between 0.6 mm and 0.7 mm, between 0.7 mm and 0.8 mm, between 0.8 mm and 0.9 mm, between 0.9 mm and 1.0 mm, between 1.0 mm and 1.1 mm, between 1.1 mm and 1.2 mm, between 1.2 mm and 1.3 mm, or between 1.3 mm and 1.4 mm; and / or(iii) a width of at least 0.5 mm, about 0.85 mm, between 0.5 mm and 0.55 mm, between 0.55 mm and 0.6 mm, between 0.6 mm and 0.65 mm, between 0.65 mm and 0.7 mm, between 0.7 mm and 0.75 mm, between 0.75 mm and 0.8 mm, between 0.8 mm and 0.85 mm, between 0.8 mm and 0.9 mm, between 0.85 mm and 0.9 mm, between 0.9 mm and 0.95 mm, between 0.95 mm and 1.0 mm, between 1.0 mm and 1.1 mm, between 1.1 mm and 1.2 mm, between 1.2 mm and 1.3 mm, or between 1.3 mm and 1.4 mm.

5. The minimally restrictive microfluidic 3D cell culture device of any one of claims 1-4, wherein the at least one inlet chamber and / or the at least one outlet chamber has a width of at least 0.5 mm, at least 0.5 mm and / or a height between 0.05 mm and 1 mm.

6. The minimally restrictive microfluidic 3D cell culture device of any one of claims 1-5, wherein the device comprises at least two cell culture chambers and further comprises at least one propagation channel extending between the at least two cell culture chambers, wherein the at least one propagation channel has a width between 0.1 mm and 6 mm, a length between 0.1 mm and 6 mm, and optionally wherein the at least one propagation channel has a height of about 0.6 mm a width of about 0.85 mm and a length of about 0.3 mm.

7. The minimally restrictive microfluidic 3D cell culture device of any one of claims 1-6, wherein the device is comprised of synthetic polymers, biological polymers, and / or glass, and optionally wherein the device is fabricated from a material chosen from the list comprising polydimethylsiloxane (PDMS), polycarbonate (PC), polystyrene (PS), polyamide (PA), polyethylene glycol (PEG), thermoplastic elastomers (TPE), poly aryletherketones (PAEK), polytetrafluoroethylene (PTFE), cyclic olefin copolymer (COC), glass, or other translucent material, wherein the biological polymers are chosen from dextran, PEG, gelatin, fibrin, Matrigel, Collagen (types I, II, and III), Laminin, Fibronectin, Fibrinogen, Tropoelastin, Elastin, Hyaluronic Acid (Hyaluronan), Chondroitin Sulfate, Alginate, Chitosan, Polyacrylamide(PAAm), Polylactic acid (PLA), Polylactic-co-glycolic acid (PLGA), Polycaprolactone (PCL), Poly (vinyl alcohol) (PVA), poly (ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), Poly(N-isopropylacrylamide) (PNIPAm), Poly(acrylic acid) (PAAc), PNIPAAm- PAAc, and / or variants thereof.

8. The minimally restrictive microfluidic 3D cell culture device of any one of claims 1-7, wherein the device is formed through 3D printing, injection molding, hot embossing, micromilling, micromachining, or etching, optionally wherein the method of 3D printing is chosen from the list comprising Stereolithography (SLA), Selective Laser Sintering (SLS), Eused Deposition Modeling (EDM), Digital Light Processing (DLP), Multi Jet Fusion (MJF), PolyJet, Direct Metal Laser Sintering (DMLS), and Electron Beam Melting (EBM).

9. The minimally restrictive microfluidic 3D cell culture device of any one of claims 6-8, comprising a microfluidic network comprising at least two configurations of the at least one cell culture chamber, the at least one inlet chamber, the at least one outlet chamber, the at least one channel, and the at least one propagation channel, optionally wherein the device supports perfusion of fluid between the microfluidic network of at least two configurations.

10. The minimally restrictive microfluidic 3D cell culture device of any one of claims 1-9, wherein the scaffolding substance is a gel or gel-precursor selected from the list comprising Hydrogel, Fibrin, Collagen (types I, II, and III) Laminin, Fibronectin, Fibrinogen, Tropoelastin, Elastin, Hyaluronic Acid (Hyaluronan), Matrigel, Chondroitin Sulfate, Alginate, Chitosan, Dextran, and / or variants thereof, optionally wherein the hydrogel is comprised of Polyethylene glycol (PEG), Polyacrylamide (PAAm), Polylactic acid (PLA), Polylactic-co-glycolic acid (PLGA), Polycaprolactone (PCL), Poly(vinyl alcohol) (PVA), poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), Poly(N-isopropylacrylamide) (PNIPAm), Poly(acrylic acid) (PAAc), PNIPAAm-PAAc, and / or variants thereof.

11. A method of 3D culturing cells in the minimally restrictive microfluidic 3D cell culture device of any one of claims 1-10, comprising:(a) introducing one or more cell types into the at least one input chamber and / or the at least one output chamber;(b) contacting the one or more cell types with culture media; and(c) incubating the minimally restrictive microfluidic 3D cell culture device containing the one or more cell types in an environment supporting cell growth.

12. The method of claim 11, further comprising introducing the culture media through the at least one input chamber and removing the culture media through the at least one output chamber, thereby perfusing the culture media through the at least two channels and the at least one channel.

13. A perfusable microfluidic cell culture network, comprising at least two cell culture chambers, at least one inlet chamber, at least one outlet chamber, at least two channels, and at least one propagation channel, wherein: the at least two channels comprise a first channel extending between the at least one inlet chamber and a first cell culture chamber, and a second channel extending between the at least one outlet chamber and a second cell culture chamber; wherein the at least one propagation channel extends between the first cell culture chamber and the second cell culture chamber; and wherein the dimensions of the at least one cell culture chamber, the at least one propagation channel, and / or the at least two channels support the housing and swelling of a synthetic hydrogel scaffolding substance with minimal cell compression such that the at least two channels have a height of at least 0.5 mm and optionally the at least two channels and / or the at least one propagation channel have a height of about 0.6 mm.

14. The perfusable microfluidic cell culture network of claim 13, wherein the perfusable microfluidic cell culture network comprises at least two inlet chambers, at least two outlet chambers, and at least four channels, wherein the at least four channels comprise a first set of channels extending between the at least two inlet chambers and the at least two cell culture chambers, and a second set of channels extending between the at least two outlet chambers and at least two cell culture chambers, optionally wherein the at least two cell culture chambers, the at least two inlet chambers, and the at least two outlet chambers are comprised of a bottom surface, side walls and an open top.

15. The perfusable microfluidic cell culture network of any one of claims 13-14, wherein the perfusable microfluidic cell culture network supports the perfusion of fluid between the at least one inlet chamber and the at least one outlet chamber.

16. The perfusable microfluidic cell culture network of any one of claims 13-15, wherein the scaffolding substance is a gel or gel-precursor selected from the list comprising Hydrogel, Fibrin, Collagen (types I, II, and III) Laminin, Fibronectin, Fibrinogen, Tropoelastin, Elastin, Hyaluronic Acid (Hyaluronan), Matrigel, Chondroitin Sulfate, Alginate, Chitosan, Dextran, and / or variants thereof and wherein the hydrogel is comprised of Polyethylene glycol (PEG), Polyacrylamide (PAAm), Polylactic acid (PLA), Polylactic-co-glycolic acid (PLGA), Polycaprolactone (PCL), Poly(vinyl alcohol) (PVA), poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), Poly(N-isopropylacrylamide) (PNIPAm), Poly(acrylic acid) (PAAc), PNIPAAm-PAAc, and / or variants thereof17. A method of culturing cells and / or cell aggregates on a device, comprising introducing one or more cell types in a scaffolding substance to the device, the device comprising:(i) at least one inlet chamber comprised of a bottom surface, side walls and an open top;(ii) at least one outlet chamber comprised of a bottom surface, side walls and an open top; and(iii) at least two channels, wherein the dimensions of the at least one channel supports the housing and swelling of the scaffolding substance.

18. The method of claim 17, wherein the device further comprises: (iv) at least one cell culture chamber comprised of a bottom surface, side walls and an open top, wherein the dimensions of the at least one cell culture chamber support the housing and swelling of a scaffolding substance.

19. The method of claim 18, wherein the at least one channel comprises a first channel extending between the at least one inlet chamber and the at least one cell culture chamber, and a second channel extending between the at least one outlet chamber and the at least one cell culture chamber.

20. The method of any one of claims 17-19, wherein the device is the minimally restrictive microfluidic 3D cell culture device of any one of claims 1-10.

21. The method of any one of claims 17-20, wherein the cultured cells and / or cell aggregates comprise more than one cell type and optionally comprise mammalian cells.

22. The method of any one of claims 17-20, wherein the method further comprises:(i) vascularizing the cells and / or cell aggregates in the scaffolding substance; and / or (ii) inducing a flow of a fluid through the microfluidic network, optionally, wherein the fluid comprises one or more active agents selected from small molecules, drugs, nucleic acids, proteins, peptides, lipids, carbohydrates, growth factors, bacteria, viruses, variants thereof and / or any other type of biological and synthetic molecules.

23. An in vitro tissue mimic comprising a 3-dimensional (3D) matrix comprising a perfusable vascular network in a synthetic hydrogel, wherein the hydrogel comprises a dextran- based polysaccharide macromer, a cell-degradable crosslinker and a tissue specific extracellular matrix protein and further comprising at least one neural specific cell type dispersed in the hydrogel.

24. The 3D matrix of claim 23, wherein the tissue specific extracellular matrix protein is versican or a versican-like peptide.

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