Cell culture method

Abstract

A method for culturing cells utilizing one or more interlocking porous hydrogel blocks (IPHBs) as a scaffold for cell growth and expansion is provided. The IPHBs include a continuous polymeric matrix material and a network of microporous channels and / or chambers extending through the continuous polymeric matrix material. The method includes seeding one or more target cells into a first IPHB, providing a first culture medium to the one or more target cells and allowing the one or more target cells to expand in the network of microporous channels and / or chambers, expanding the original scaffold by interlocking a second IPHB with the first IPHB, and allowing the one or more target cells to expand from the first IPHB into the second IPHB and providing the first culture medium or the second culture medium to the one or more target cells in the second IPHB.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 296,267, filed January 4, 2022, which is expressly incorporated by reference in its entirety.

[0002] Inventive embodiments of the present disclosure generally relate to cell culture propagation methods that utilize one or more interlocking porous hydrogel blocks (IPHBs) that can be interdigitated with one another to provide continuous 3D growth of various cells and / or tissues. The IPHBs provide increased flexibility to implement a wide variety of unique cell culture propagation methods (e.g., sequential propagation of cells, simultaneous multiple cell propagation, etc.). [Background technology]

[0003] Cells (e.g., human, animal, plant, bacterial, fungal) are traditionally cultured on plastic substrates such as Petri dishes, undivided petri dishes, well plates, flasks, and scaffolds. However, each of these cell culture vessels is individual and finite, or has limited surface area for cell growth expansion. For example, these in vitro expansion culture systems involve the use of rigid 2D plastic culture vessels to expand cells as monolayers and require multiple protease-dependent subculture (passage) events to achieve sufficiently large cell numbers. Evidence suggests that traditional 2D culture systems are not ideal for stem cell expansion, and may result in loss of multipotency, reduced viability, induce senescence, and reduce secretion of regenerative factors. Moreover, these types of vessels do not mimic the internal environment (e.g., the human body, where different cell types arise) in which the cells of interest grow naturally. Although these cell culture vessels allow for easy propagation of cells, once the cells outgrow the original cell culture vessel, they must be dissociated using a toxin against the cells and transferred to an additional cell culture vessel. As referenced above, this process is known as subculture, also known as passaging. The process of expanding cells is time consuming, error prone, laborious, and expensive. When primary cells derived directly from tissue are subcultured multiple times, these primary cells eventually lose their natural characteristics (phenotype) and function. At some point, the primary cells will stop dividing and no longer function in the same way as when they were first extracted from the primary tissue. This cessation of function is known as senescence, and may limit the life span of the expansion of cells from the original sample of primary cells of interest. Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there remains a need in the art for cell culture methods that provide one or more of the following: mimic the internal environment; eliminate the need for subculturing cells; allow for continuous cell growth while optionally harvesting simultaneously grown cells; and allow for simultaneous multiple cell culture growth where interfaces between different cells of interest can be created for further use and / or analysis. [Means for solving the problem]

[0005] One or more embodiments of the present invention may address one or more of the problems discussed above. Certain embodiments according to the present invention provide a method of culturing cells. The method may include the steps of: (i) providing an initial scaffold comprising a first interlocking porous hydrogel block (IPHB), wherein the first IPHB comprises a three-dimensional (3D) macrostructure defined by a continuous polymeric matrix material and a network of microporous channels and / or chambers extending into the continuous polymeric matrix material, wherein the 3D macrostructure comprises a top surface, a bottom surface, and a thickness defined by at least one lateral edge extending from the top surface to the bottom surface, wherein the structure of the 3D macrostructure includes at least one interlocking male component and at least one interlocking female component; (ii) seeding one or more target cells onto the first IPHB. (iii) providing a first culture medium to the one or more target cells and allowing the one or more target cells to grow and expand in the network of microporous channels and / or chambers; (iv) expanding the initial scaffold by mating a second IPHB to the first IPHB, wherein at least one mating male component or at least one mating female component of the first IPHB is joined to a corresponding mating male component or corresponding mating female component of the second IPHB; and (v) allowing the one or more target cells to grow and expand from the first IPHB into the second IPHB and providing the first culture medium or the second culture medium to the one or more target cells in the second IPHB. [Brief description of the drawings]

[0006] [Figure 1] FIG. 1 is a visual illustration of a method according to certain embodiments of the present invention. [Diagram 2] FIG. 2 illustrates two separate IPHBs according to certain embodiments of the present invention. [Diagram 3] FIG. 3 illustrates three interlocking IPHBs according to certain embodiments of the present invention. [Figure 4] FIG. 4 depicts top and side views of an IPHB in accordance with certain embodiments of the present invention, illustrating the continuous polymer matrix material and the network of microporous channels and / or chambers extending through the continuous polymer matrix material exposed at the surface of the IPHB. [Diagram 5] FIG. 5 illustrates an IPHB that includes a mating male component on the top surface and a mating female component that protrudes into the bottom surface, according to certain embodiments of the present invention. [Figure 6] FIG. 6 illustrates another IPHB that includes mating male components on the top surface and mating female components protruding into the bottom surface, according to certain embodiments of the present invention. [Figure 7] FIG. 7 illustrates cell migration from a first IPHB to a second IPHB when interdigitated together, according to certain embodiments of the present invention. [Figure 8A] ~ [Figure 8F] 8A-8F illustrate cell growth patterns within microchannels according to certain embodiments of the present invention. [Figure 9] FIG. 9 is a schematic diagram illustrating the nomenclature and conceptual cell migration of multiple IPHBs according to certain embodiments of the present invention. [Figure 10] FIG. 10 is a schematic diagram illustrating additional options for adding additional IPHBs of various numbers and arrangements to the IPHB of FIG. [Figure 11]Figure 11 is a schematic diagram for the invasion assay setup of a series of meshed empty IPHBs. Four seeded IPHBs are meshed as side chains to a series of meshed empty IPHBs. [Figure 12] 12 is a schematic diagram of a dual invasion assay setup of a series of interdigitated empty IPHBs. A first set of four IPHBs seeded with a first cell type are interdigitated as side chains to the series of interdigitated empty IPHBs, and a second set of four IPHBs seeded with a second cell type capable of producing or secreting a therapeutic agent are interdigitated as side chains on the opposite side of the series of interdigitated empty IPHBs. [Figure 13] Figure 13 illustrates a multiplex invasion assay configuration in which eight IPHBs are attached as side chains to a series of interdigitated empty IPHBs, where each attached IFPB is seeded with a different cell type, each of which can produce or secrete a different therapeutic agent. [Figure 14] Figure 14 illustrates the migration assay configuration, in which a series of seeded IPHBs have eight side chains of unseeded or empty IPHBs attached to them. [Figure 15] Figure 15 illustrates a dual migration assay configuration in which a series of seeded IPHBs includes a first group of IPHBs seeded with a first cell type and a second group of IPHBs seeded with a second cell type, in which the eight side chains of unseeded or empty IPHBs are attached to them. [Figure 16] Figure 16 illustrates a multiplex migration assay configuration in which a series of seeded IPHBs, each IPHB seeded with a different cell type, and eight side chains of unseeded or empty IPHBs are attached to them. [Figure 17] Figure 17 illustrates an 8-chamber omnitray loaded with multiple IPHBs and spacers, where each chamber is loaded with a group of four interdigitated IPHBs seeded with a different cell type (e.g., each chamber has its own unique cell type within it). [Figure 18]Figure 18 illustrates an 8-chamber omnitray loaded with multiple IPHBs and spacers, in which the IPHBs are configured as a unidirectional paracrine signaling assay. [Figure 19] Figure 19 illustrates an 8-chamber omnitray loaded with multiple IPHBs and spacers, in which the IPHBs are configured as a two-way paracrine signaling assay. [Figure 20] Figure 20 illustrates an 8-chamber omnitray loaded with multiple IPHBs and spacers, in which the IPHBs are configured as a paracrine signaling assay. [Figure 21] Figure 21 illustrates an 8-chamber omnitray loaded with multiple IPHBs and spacers, in which the IPHBs are configured as a 4-way paracrine signaling assay. [Figure 22A] Figure 22A illustrates the macrostructure of the hydrogel system, where (i) the left image is a single photographic image of a 3D hydrogel system of approximately 1 cm3, (ii) the middle image is a photographic image of four hydrogels connected to each other on a 6-well plate, and (iii) the right image is a photographic image of four connected hydrogels, with annotations illustrating migration of cells from the initially seeded hydrogel into / towards the additional attached hydrogel without cells. [Figure 22B]FIG. 22B illustrates the microstructure of the hydrogel system, where (i) the left image is a confocal microscopy image of a cross section of the hydrogel's internal structure illustrating adipose-derived mesenchymal stem / stromal cells (ASCs) migrating and proliferating within the porous architecture, where (a) small white arrows point to the polymeric struts of the hydrogel, (b) blue=Hoechst, (c) green=phalloidin, and (d) red=Mitotracker. (ii) The middle image is a fluorescent image of ASCs lining individual pores within the hydrogel system's microstructure, where (a) blue=Hoechst, (b) green=wheat germ agglutinin, and (c) red=Mitotracker. and (iii) the right image is a confocal microscopy z-stack image of ASCs migrating from the originally seeded hydrogel (hydrogel #1) into the newly attached hydrogel (hydrogel #2), in which the large white arrow indicates the direction of ASC migration and the dotted white line indicates the junction of the two attached hydrogels. [Figure 22C] FIG. 22C illustrates three images depicting 3D z-stack images within a single pore channel, highlighting the 3D network formed by ASCs (middle image) and cell processes protruding from the cells (right image), while the image on the left is a low-magnification image of the entire stained hydrogel. Here, all images were acquired by Nikon Corporation with their AXR confocal imaging system. In these, blue=Hoechst, green=phalloidin, and pink=Mitotracker. [Figure 23A] ~ [Figure 23B]Figures 23A-23B illustrate the results of ASCs seeded at passage 1 (P1) in the 3D hydrogel system or in conventional 2D culture, where ASCs were serially passaged and assessed in 2D culture at P2, P6, and P10, and ASCs in the 3D hydrogel system were compared to their respective 2D counterparts at the corresponding passage time points. Figure 23A illustrates that representative images of 2D (leftmost column) and 3D (rightmost column) cultured ASCs stained for either CD73 (left), CD90 (center), or CD105 (right) at each respective time point (P2, P6, and P10) are shown. CD marker staining is shown in green in the images. Samples were counterstained with Hoechst (blue) and phalloidin (not shown). Figure 23B illustrates quantification of imaging data and the percentage of total positive cells, shown by box plots for each marker, where each individual point represents the quantification of a single image of ASCs in 2D (black circles) or 3D (cyan diamonds) culture. A 20x objective was used. Samples were analyzed in quadruplicate (n=4). Scale bar=100 μm. Error bars are standard error of the mean, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 24A] ~ [Figure 24N]Figures 24A-24N illustrate the reduced induction of senescence in 3D hydrogels over time compared to conventional 2D culture systems, where ASCs were seeded into 3D hydrogel systems or conventional 2D cultures at passage 1 (P1), and ASCs were serially passaged in 2D cultures and evaluated at P2, P6, and P10. ASCs in the 3D hydrogel systems were compared to their respective 2D counterparts depending on the passage time point. To provide sufficient surface area for continued cell growth, three additional hydrogels were added to each individual hydrogel at the 2-week mark and left for the remainder of the culture period. FIG. 24A illustrates that representative images of 2D (upper row) and 3D (lower row) cultured ASCs stained for β-galactosidase (green) at each respective time point (P2, P6, and P10) are shown. Samples were counterstained with Hoechst (blue) and phalloidin (not shown). FIG. 24B illustrates quantification of imaging data and the total percentage of positive cells, shown by box plots for each marker. Each individual point indicates quantification of a single image of ASCs in 2D (black circles) or 3D (cyan diamonds). A 20× objective was used. Samples were analyzed in quadruplicate (n=4). Scale bar=100 μm. Error bars are standard error of the mean, ***p<0.001, ****p<0.0001. [Figure 25A] ~ [Figure 25D]Figures 25A-25D illustrate the retention of wound healing capacity in adipose-derived mesenchymal stem / stromal cell conditioned medium (ASC-CM) from 2D and 3D from each respective time point, which was used to treat keratinocytes, which were then assessed for changes in their migration, metabolic, and proliferation activities. Figures 25A and 25B illustrate migration activity assessed by scratch assay, where Figure 25A illustrates the whole well image scans acquired, and a representative image of the wound image is provided. Here, the solid white line indicates the original wound border, and the solid black line outlines the remaining wound site. Figure 25B illustrates that the average wound area was determined after 22 hours, and was performed in triplicate. Figure 25C illustrates metabolic activity, which was quantified by PrestoBlue and displayed as the average relative fluorescence units. Figure 25D illustrates proliferation activity, which was quantified by PicoGreen and the average cell number was determined. 2D ASC-CM treated keratinocytes are shown in black bars. 3D ASC-CM treated keratinocytes are shown in cyan bars. Scale bar = 500 μm. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars are standard error of the mean. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the present invention are shown. Indeed, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0008] The disclosed invention generally relates to a cell culture method utilizing a platform whereby cells of interest can be continuously grown and expanded on a substrate (e.g., a 3D scaffold) without the need for subculture. According to certain embodiments of the present invention, the method of culturing cells of interest utilizes one or more interlocking porous hydrogel blocks (IPHBs), which can be interlocked with each other to provide continuous 3D growth of various cells and / or tissues. In this regard, the IPHBs provide a substrate that is modular, connecting or linking together in a manner similar to the fitting of puzzle pieces. Cells growing on one IPHB can migrate and grow and expand on the second IPHB when the second IPHB is linked or connected to the original IPHB. Additional IPHBs (e.g., scaffold substrates) can be connected or linked to the original IPHB or to IPHBs linked or connected to the original IPHB. Advantageously, cells can thus continue to grow and expand as long as the additional IPHBs are linked or connected together. Thus, a system of interconnected IPHBs may define a collective 3D network of microporous channels and / or chambers that extends in a continuous manner throughout the entirety of the interconnected IPHBs (e.g., each IPHB has its own unique 3D network of microporous channels and / or chambers, which may be the same as or different from any of the others).

[0009] According to certain embodiments, the cell culture propagation method lacks any subculturing step. Such methods that avoid subculturing preserve the cellular characteristics for more than twice as long as when the cells are grown in a vessel that requires subculturing. The modular IPHB (e.g., substrate) can be two-dimensional (2D) or 3D, although a 3D configuration may be more desirable. As discussed above and in more detail below, the IPHB may contain void spaces, channels, or may be permeable to gas or liquid substrates. According to certain embodiments of the invention, the continuous polymeric material of the IPHB may be a hydrogel material that includes or consists mostly or exclusively of synthetic polymers, biologically derived polymers, and / or tissue-derived components, such extracellular matrices. Depending on the use of hydrogel materials, the IPHB may be individually tailored to have characteristics that mimic the environment of virtually any environment, for example, within the human body, allowing the cells to grow in a more natural state and maintain the cellular characteristics for a longer period of time than when grown in a cell culture vessel that requires subculturing. Moreover, different IPHBs can be combined (e.g., interdigitated) together to allow different cell types to intermix and form tissues or produce multicellular organoids. IPHBs, according to certain embodiments of the invention, can be fixed in situ to preserve the cells for analysis, or the matrix can be degraded to release the cells for analysis.

[0010] According to certain embodiments of the present invention, the method of culturing cells may allow the end user to continuously culture virtually any cell type without stopping and without sub-culturing. This advantageously allows for a continuity of experimentation and continuity of cell production not possible with conventional platforms. Moreover, the method of culturing cells may include: Minimize human interaction. Since the need for subculture is eliminated, this reduces human error, risk of contamination, and other inconsistencies. Furthermore, methods utilizing IPHB according to certain embodiments of the present invention can be compatible with current cell culture trays and plates, meaning that these methods can be used with automated systems that can move standard cell culture trays and plates between incubators and liquid handlers to dispense and aspirate cell culture media. With fewer manual steps and subcultures, the methods disclosed herein can also dramatically reduce consumption of consumables, including media and plasticware. Methods utilizing IPHB according to certain embodiments of the present invention also allow for uniform nutrient and gas exchange within the IPHB (e.g., hydrogel matrix) based on the microchannel architecture built into the IPHB. These methods utilizing IPHBs allow for customization to facilitate the growth of specific cell types, for example, by constructing a hydrogel material that forms a continuous polymer network and / or a network of microporous channels and / or chambers that extend into the continuous polymer matrix material of the IPHB. Additionally, methods according to certain embodiments of the invention allow for the culture of multiple different cell types in combination, allowing the cells to secrete and form extracellular matrices that can be harvested as raw materials for therapeutic or diagnostic purposes, and facilitating the secretion of bioproducts from the cells into the luminal space of the microchannels for collection.

[0011] According to certain embodiments of the present invention, the IPHB allows for perfusion of liquid medium and / or gas within the microchannels, allowing cells to organize according to the microarchitectural design in order to form specific tissue structures. Advantageously, the cell culture growth method according to certain embodiments of the present invention overcomes the problem of spheroid necrosis by allowing the spheroids to dissolve within the microchannels and form cylinders and other higher order structures. In this regard, the method facilitates the creation of a native microenvironment that promotes cell proliferation, migration, survival, and function. Moreover, the flexible and robust nature of the method utilizing the IPHB-based system may enable custom growth of cell cultures.

[0012] According to certain embodiments of the present invention, the cell culture propagation method may be applied to the production of biologics, such as exosomes, extracellular vesicles, growth factors, monoclonal antibodies, peptides, proteins, viral particles, oligonucleotides, and organelles. In this regard, cells producing or secreting a biologic of interest may be cultured as disclosed herein, and the produced or secreted biologic may be isolated for further processing, use, and / or analysis. Additional applications to which the cell culture propagation method may be applied include, for example, tissue formation, organoid formation, filtration, regenerative medicine, autograft generation, meat production, cell culture, plant culture, protein production, physiological modeling, infectious disease, wound healing, cell reprogramming, disease modeling, cancer research, bioreactors, mass production of cells, and microenvironment formation.

[0013] As noted above, the disclosed invention generally relates to a method for culturing cells. The method may, according to certain embodiments of the present invention, include the steps of: (i) providing an initial scaffold comprising a first interlocking porous hydrogel block (IPHB), wherein the first IPHB comprises a three-dimensional (3D) macrostructure defined by a continuous polymeric matrix material and a network of microporous channels and / or chambers extending through the continuous polymeric matrix material, wherein the 3D macrostructure comprises a top surface, a bottom surface, and a thickness defined by at least one lateral edge extending from the top surface to the bottom surface, wherein the structure of the 3D macrostructure includes at least one mating male component and at least one mating female component; (ii) attaching one or more target cells to the first IPHB; (iii) providing a first culture medium to the one or more target cells and allowing the one or more target cells to grow and expand into the network of microporous channels and / or chambers; (iv) expanding the initial scaffold by mating a second IPHB to the first IPHB, wherein at least one mating male component or at least one mating female component of the first IPHB is joined to a corresponding mating male component or corresponding mating female component of the second IPHB; and (v) allowing the one or more target cells to grow and expand from the first IPHB into the second IPHB and providing the first culture medium or the second culture medium to the one or more target cells in the second IPHB.

[0014] 1, for example, illustrates one exemplary method 200 of culturing cells, including the optional step 210 of coating a first IPHB 1 (e.g., a primary or seeding block) with a compatibilizer, which may promote or facilitate adhesion of target cells to the polymeric matrix material (e.g., walls defining a network of microporous channels and / or chambers extending into the continuous polymeric matrix material) within the first IPHB. As shown in FIG. 1, the compatibilizer coating step may be performed simply by applying a flowable or liquid composition including the compatibilizer to the top surface of the first IPHB and allowing the composition to diffuse and / or flow into and through the network of microporous channels and / or chambers extending into the continuous polymeric matrix material. According to certain embodiments of the present invention, a gas, or a combination of a liquid composition and a gas, may be delivered through the first IPHB for the purpose of altering the texture of the network of microporous channels and / or chambers to facilitate cell adhesion, cell migration, cell proliferation, or other cell behaviors and functions. In this regard, the first IPHB may be immersed or soaked in a composition having a compatibilizer to ensure a desired coating of the network of microporous channels and / or chambers with the compatibilizer. FIG. 1 also illustrates step 220 of seeding the first IPHB with one or more target cells (e.g., a first cell type for culture). The nature of the cells is generally not critical since the IPHB is agnostic and will tolerate any cell line. Step 230 may include feeding the now seeded target cells present within the first IPHB. In this regard, one or more culture media may be used according to a desired protocol for the growth of the seeded cells. Again, the feeding step may be performed by applying culture medium to the top surface of the IPHB and allowing the culture medium to diffuse and / or flow through the IPHB. Additionally or alternatively, the IPHB may be soaked or immersed in the culture medium.As shown in Figure 1, optional step 240 may include expanding the initial scaffold associated with the first IPHB by interlocking a second IPHB and a third IPHB with the first IPHB. Step 250 includes harvesting the cultured cells from within the IPHB. Depending on the nature of the hydrogel material forming a continuous polymeric matrix material, harvesting may include degrading the hydrogel material to expose the cells. Alternatively, the cells may be flushed out by passing a liquid form of a harvesting reagent through the IPHB.

[0015] According to certain embodiments of the invention, the method may further comprise the step of harvesting at least a portion of the one or more cells present in the network of microporous channels and / or chambers of the first IPHB. The step of harvesting at least a portion of the one or more cells present in the network of microporous channels and / or chambers of the first IPHB may comprise flushing them out of the first IPHB with a fluid medium. Depending on the IPHB used, the end user may remove the cells from the network of microporous channels and / or chambers, for example, using products based on trypsin and Accutase. According to certain embodiments of the invention, the enzyme reagent physically breaks the bonds of the hydrogel material that forms the continuous polymer matrix material, degrading the IPHB and gently releasing the cells present in the network of microporous channels and / or chambers. Additionally or alternatively, the step of harvesting at least a portion of the one or more cells present in the network of microporous channels and / or chambers of the first IPHB may comprise disintegrating the 3D macrostructure of the first IPHB. As described herein, degradable materials can include naturally occurring biopolymers such as collagen, hyaluronan, gelatin, and nucleic acids. These types of materials, or combinations of these materials, can be degraded by the corresponding enzymes, such as collagenase (e.g., for collagen and gelatin), hyaluronase, and Dnase / Rnase.

[0016] According to certain embodiments of the present invention, the method may optionally include a step of coating the interface between the continuous polymeric matrix material and the network of microporous channels and / or chambers extending through the continuous polymeric matrix material, for example, with a compatibilizer as described and disclosed herein. The compatibilizer may be selected, for example, to promote adhesion of the primary cells of interest to the IPHB. In this regard, the coating step may occur prior to seeding of the IPHB.

[0017] According to certain embodiments of the present invention, IPHBs can be interdigitated with each other to provide continuous 3D growth of various cells and / or tissues. Hydrogels are insoluble polymer matrices that can be engineered to have up to 96% water by weight, such as up to 40, 50, 60, 70, 80, 90, and 95% water by weight). A variety of different polymers can be used individually or in combination to create unique hydrogels. By crosslinking the polymers with light, temperature shift, or chemical reaction, hydrogels can be tailored to exhibit different mechanical properties, diffusion gradients, osmotic pressures, chemical compositions, and structures, such as pores and fibers of various shapes and sizes. Hydrogels can also be degradable or non-degradable. Hydrogels are versatile in their capabilities and are used in different applications, such as soft contact lenses to provide optics to correct the patient's vision. Hydrogels have been used as dressings in wound healing applications and are also used in life science applications as bioinks to create unique structural scaffolds for microfluidic experiments or to provide a substrate on which cells can be cultured.

[0018] IPHBs according to certain embodiments of the invention can be joined together or mated together via at least one mating male component and at least one mating female component. For example, at least one mating male component of a first IPHB is configured to be received into at least one corresponding mating female component of a second IPHB. In addition to joining IPHBs via their 3D macrostructures, microstructures of networks of microporous channels and / or chambers (e.g., void spaces). Most hydrogels are solid materials. However, by introducing void spaces and microchannels into the hydrogel, migration of liquids, gases, and cells can be directed with the purpose of expanding the hydrogel together to form a continuous matrix. This feature according to certain embodiments of the invention can be particularly beneficial. Because the microstructure of the network of microporous channels and / or chambers (e.g., void spaces) allows a second medium to be used to interlock the IPHB (e.g., hydrogel) together to create a larger or expanded material for cell and / or tissue growth.

[0019] Hydrogels can be formed by crosslinking any synthetic polymer, biopolymer, tissue component (derived from human, animal, plant, or combinations thereof) in the presence of water using a reaction mediated by free radicals (e.g., photoreaction, chemical reaction) or as a result of a change in temperature.

[0020] IPHBs (e.g., hydrogels) according to certain embodiments of the invention may be suitable for a variety of applications, e.g., cell culture, biologics, exosomes, extracellular vesicles, growth factors, monoclonal antibodies, peptides, proteins, viral particles, oligonucleotides, organelle production or growth, organoid formation, plant growth, drug delivery, tissue formation, ex vivo modeling, electrical conduction, wound healing, cell reprogramming, filtration, optics, microfluidics, custom networks of microchannels, custom scaffold architecture construction, custom extracellular matrix derived scaffolds, dissolvable hydrogels, custom tissue formation, allowing for patient cells, custom configuration, modularity, and microenvironment engineering.

[0021] According to certain embodiments of the present invention, the IPHB allows cells to grow in a more natural physiological-like environment compared to culture in 2D plastic cell culture vessels. For example, the IPHB can be tailored or configured to mimic the native tissue environment in which a particular cell originates and grows, unlike plastic cell culture vessels and other technologies that are not customizable and modular. In addition, certain embodiments of the present invention allow a combination of multiple similar or dissimilar IPHB (e.g., hydrogel) matrices to be joined together to form an extended, continuous hydrogel matrix for cell production and / or biologics production. For example, unlike other cell culture vessels or technologies, similar or dissimilar hydrogels (e.g., IPHB) can be joined together to create a unique and custom microenvironment for cells to grow. Moreover, the IPHB can allow the formation of spheroids and organoids without the generation of necrotic cores within the network of microchannels of the IPHB. For example, the IPHB according to certain embodiments of the present invention allows spheroids and organoids to dissolve and form tubes, cylinders, and other sophisticated structures, where nutrients and gases can diffuse uniformly to the cells within the IPHB (e.g., hydrogel). Advantageously, unlike other technologies that claim to mass-produce cells, for example, the IPHB can allow uniform nutrient and gas exchange for healthy cell growth. According to certain embodiments of the present invention, the IPHB can be modified to accommodate a wide variety of different cell types. According to certain embodiments of the present invention, the IPHB allows cells to secrete extracellular matrix and create a natural microenvironment that promotes cell growth, migration, survival, and function. Furthermore, the IPHB advantageously eliminates the need to subculture cells. Moreover, the use of IPHB is easy to use because they can be provided in a preformed format and do not require sophisticated changes in temperature, pH, or chemical exposure to be used.The IPHB may, for example, enable a user to achieve one or more of the following: grow custom cell cultures, grow multiple cell types in parallel or sequentially, combine cell cultures to create complex tissues, mass produce cells without ever stopping cell production, produce cells from benchtop all the way through clinical trials and for industrial production using the same substrate, and use less media and fewer consumables than current technologies, reducing or eliminating the risk of human error and contamination in cell production. According to certain embodiments of the present invention, the IPHB provides a modular platform for any of the above referenced applications.

[0022] Certain embodiments according to the present invention provide an interlocking porous hydrogel block (IPHB) comprising a three-dimensional (3D) macrostructure defined by a continuous polymer matrix material and a network of microporous channels and / or chambers extending through the continuous polymer matrix material. The 3D macrostructure may include a top surface, a bottom surface, and a thickness defined by at least one side edge extending from the top surface to the bottom surface. In this regard, the 3D macrostructure includes at least one mating male component and at least one mating female component. According to certain embodiments of the present invention, at least one mating male component of a first IPHB is configured to be received within a corresponding at least one mating female component of a second IPHB.

[0023] Figure 2, for example, illustrates two separate IPHBs 1, according to certain embodiments of the present invention. Each of these IPHBs includes a top surface 12, a bottom surface 14, and at least one side edge 16. The particular IPHB 1 shown in Figure 2 includes at least one mating male component 50 and at least one mating female component 60. Figure 3 illustrates three mated IPHBs 1, according to certain embodiments of the present invention. The IPHBs 1 shown in Figure 3 each include a first mating male component 51, a second mating male component 52, a first mating female component 61, and a second mating female component 62.

[0024] 4 illustrates a top and side view of an IPHB 1, in accordance with certain embodiments of the present invention, illustrating a continuous polymeric matrix material 10, and a network of microporous channels and / or chambers 30 extending through the continuous polymeric matrix material exposed at the surface of the IPHB. As illustrated by FIG 4, the seeded IPHB may allow cells 33 to grow and migrate in a three-dimensional manner within the network of microporous channels and / or chambers 30.

[0025] According to certain embodiments of the present invention, the at least one interlocking male component includes a first interlocking male component extending outward from the at least one side edge. For example, the at least one side edge can include a first side edge and a second side edge, where the at least one interlocking male component includes a first interlocking male component extending outward from the first side edge and a second interlocking male component extending outward from the second side edge. The interlocking male components extending outward from the side edges can be configured to interlock or join with corresponding interlocking female components of other IPHBs, for example, to form an expanding continuous 3D scaffolding system, where the interlocked or joined IPHBs expand outward in the xy plane. According to certain embodiments of the present invention, the at least one interlocking male component can also include a third interlocking male component extending outward from the top surface. In this regard, the interlocking male components extending outward from the top surface may be configured to interlock or join with corresponding interlocking female components on the bottom surface of another IPHB, for example, to form an expanding continuous 3D scaffolding system, with the interlocked or joined IPHBs expanding in the z-direction perpendicular to the xy-plane. According to certain embodiments of the present invention, for example, multiple IPHBs may be interlocked or joined together in the xy-plane and stacked on top of each other in the z-direction. As noted above, the at least one interlocking female component may include a first interlocking female component extending inward from at least one side edge toward an interior portion of the 3D macrostructure. For example, the at least one side edge portion can include a third side edge portion and a fourth side edge portion, wherein the at least one mating female component includes a first mating female component extending inwardly from the third side edge portion toward the inner portion of the 3D macrostructure and a second mating female component extending inwardly from the fourth side edge portion toward the inner portion of the 3D macrostructure.In such an exemplary embodiment, the first and third side edges may define a first pair of opposing side edges, and the second and fourth side edges may define a second pair of opposing side edges. In accordance with certain embodiments of the present invention, and as described above, the at least one mating female component may include a third mating female component extending inwardly from the bottom surface toward an interior portion of the 3D macrostructure. In this regard, multiple IPHBs may be mated or joined together in the xy plane and stacked on top of one another in the z direction.

[0026] According to certain embodiments of the present invention, the interlocking features of the IPHBs allow for the custom formation of a continuous 3D scaffold for growing or growing various cells and / or tissues, where the number of specific cells to be seeded and / or grown in the continuous 3D scaffold is not limited. Such flexibility in the relative positioning and interlocking of different IPHBs allows for the custom growth of multiple cell types, which may form complex interfaces between different types of cells. For example, the multiple interdigitated IPHBs defining a continuous 3D scaffold (e.g., a continuous network of microporous channels and / or chambers extending through the multiple interdigitated IPHBs) may encompass between 1 and 20 different cell and / or tissue types to be grown simultaneously, such as any of the following: at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 different cell and / or tissue types to be grown simultaneously, and / or any of the following: at most about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, and 10 different cell and / or tissue types to be grown simultaneously.

[0027] 5, for example, illustrates an IPHB 1 that includes a mating male component 53 on the top surface 12 and a mating female component 63 that protrudes into the bottom surface 14 of the IPHB, in accordance with certain embodiments of the present invention. In this regard, the mating male component 53 on the top surface 12 and the mating female component 63 that protrudes into the bottom surface 14 of the IPHB may be configured to engage and mate with a separate IPHB, as described herein.

[0028] According to certain embodiments of the invention, the 3D macrostructure may define a cube, a square prism, or a triangular prism, with the exception of at least one interlocking male component and at least one interlocking female component. According to certain embodiments of the invention, the 3D macrostructure may define a polygonal prism having 3-12 side edges, such as at least about 3, 4, 5, 6, 7, and 8 side edges, and / or any of the following: at most about 12, 11, 10, 9, and 8 side edges, with the exception of at least one interlocking male component and at least one interlocking female component. According to certain embodiments of the invention, each side may include either an interlocking male component and / or an interlocking female component. Alternatively, some of the side edges may lack an interlocking male component and an interlocking female component.

[0029] According to certain embodiments of the present invention, the at least one side edge includes a first side edge, a second side edge, and an arcuate side edge between and adjacent to the first side edge and the second side edge. For example, the first side edge can include at least one mating male component extending outwardly from the first side edge, and the second side edge can include at least one mating female component extending inwardly from the second side edge toward an interior portion of the 3D macrostructure. In this regard, each IPHB can define a pie-like shape that, when assembled or mated together, forms a circle (e.g., a cylinder because each IPHB has a thickness). Such a configuration of IPHBs may be desirable for use with circular culture wells. Additionally or alternatively, the at least one interlocking male component includes a second interlocking male component extending outward from the top surface, and / or the at least one interlocking female component includes a second interlocking female component extending inward from the bottom surface toward an interior portion of the 3D macrostructure. In this regard, multiple IPHBs may be stacked on top of each other in the z-direction to form a thicker cylinder or semi-cylinder. According to certain embodiments of the present invention, the 3D macrostructure may define a semi-cylinder, such as 1 / 8 of a cylinder to 1 / 2 of a cylinder, such as 1 / 8, 1 / 4, 1 / 3, or 1 / 2 of a cylinder, with the exception of the at least one interlocking male component and the at least one interlocking female component.

[0030] Figure 6 illustrates a semi-cylindrical shaped IPHB 100 that includes, for example, a mating male component 153 on the top surface 112 and a mating female component 163 that projects into the bottom surface 114 of the IPHB, in accordance with certain embodiments of the present invention. The semi-cylindrical shaped IPHB 100 illustrated by Figure 6 includes a first mating male component 150 extending from the second side edge 118 and a first mating female component 160 that projects into the second side edge 116. The first side edge 116 and second side edge 118 of the IPHB 100 shown in Figure 6 are connected by an arcuate side edge 119 at one end of the IPHB. In this regard, as described herein, the mating male component 153 on the top surface 112 and the mating female component 163 that projects into the bottom surface 114 of the IPHB may be configured to engage and mate with a separate IPHB.

[0031] According to certain embodiments of the present invention, at least one mating male component may occupy or overlap from about 5% to about 50% of the macroscopic surface area of ​​the surface (e.g., side edge, top, or bottom) from which it extends, such as by any of the following: about 10, 15, 20, and 25%, and / or by any of the following: at most about 50, 45, 40, 35, 30, and 25%. Additionally or alternatively, at least one mating female component may occupy or overlap from about 5% to about 50% of the macroscopic surface area of ​​the surface (e.g., side edge, top, or bottom) from which it enters, such as by any of the following: about 10, 15, 20, and 25%, and / or by any of the following: at most about 50, 45, 40, 35, 30, and 25%.

[0032] According to certain embodiments of the present invention, the bottom surface may have a rough texture relative to the top surface. For example, the top surface may be relatively smooth relative to the bottom surface, which may have a textured structure. The textured structure of the bottom surface may promote the flow of culture medium or washing medium throughout the IPHB, for example, by providing a structural spacer to promote the drainage of the culture medium or washing medium from the IPHB. The textured surface of the bottom surface may include, for example, a plurality of mild ridges, such as individual bumps or ridges, which function as short spacers. However, the plurality of mild ridges may be significantly smaller in size compared to the at least one mating male component, for example, at most about 1 / 10 the size of the at least one mating male component. In this regard, the mild ridges may generally not provide any mating functionality according to certain embodiments of the present invention.

[0033] According to certain embodiments of the present invention, the upper surface of the IPHB is approximately 0.25 cm 2 ~about 25cm 2 macroscopic surface area, such as any of the following: at least about 0.25, 0.5, .75, 1, 1.5, 2, 5, 8, 10, and 12 cm 2 , and / or any of the following: approximately 25, 22, 20, 18, 15, and 12 cm 2 Additionally or alternatively, the bottom surface may include a thickness of about 0.25 cm 2 ~about 25cm 2 macroscopic surface area, such as any of the following: at least about 0.25, 0.5, .75, 1, 1.5, 2, 5, 8, 10, and 12 cm 2 , and / or any of the following: approximately 25, 22, 20, 18, 15, and 12 cm 2 Additionally or alternatively, the thickness of the 3D macrostructure can be from about 0.5 cm to about 3 cm, such as any of the following: at least about 0.5, 0.75, 1, 1.25, and 1.5 cm, and / or any of the following: at most about 3, 2.5, 2, and 1.5 cm.

[0034] As noted above, each of the at least one mating female component may be configured to receive a corresponding at least one mating male component of a second IPHB. In this regard, multiple IPHBs may be joined or mated together in an individual, sequential, additive manner to expand the 3D scaffold as desired along the xy plane and z direction alone. For example, cell growth on multiple interconnected IPHBs may be continued in the z direction by stacking additional layers of IPHBs on top of a first layer of IPHBs.

[0035] According to certain embodiments of the invention, the continuous polymer matrix material may be non-degradable. In this regard, cells and / or tissues produced by IPHB may need to be flushed from the inner network of microporous channels and / or chambers for further analysis, purification, or development. Additionally or alternatively, the continuous polymer matrix material may be selectably degradable. For example, the hydrogel formulation may be rendered biodegradable, for example, by the insertion of enzyme-sensitive sequences or the use of compounds derived from the native matrix. For example, the continuous polymer matrix material may comprise a selectably degradable hydrogel material that includes one or more degradable polymers, such as one or more biopolymers derived from an organism. The one or more biopolymers derived from an organism may include, for example, polynucleotides, polysaccharides, polypeptides, or any combination thereof. According to certain embodiments of the invention, the one or more biopolymers may include collagen, gelatin, laminin, alginate, glycosaminoglycans, oligonucleotides (e.g., DNA, RNA), carbohydrates, lipids, cellulose, alginate, and proteins. These may be gently degraded by, for example, the use of protein-specific enzymes, ionic solvents, neutral detergents, weak acids, and peroxides to break the biopolymer chains. According to certain embodiments of the invention, one or more biopolymers may include degradable monomers including esters such as hydroxybutyrate, lactic acid, glycolic acid, and caprolactone, acid anhydrides such as adipic acid and sebacic acid, sugars such as cellulose, alginic acid, pectin, dextrin, chitosan, hyaluronan, chondroitin sulfate, and heparin, proteins, nucleotides (DNA, RNA), peptides such as collagen, gelatin, silk, and fibrin, urethanes, phosphates, carbonates, and vinyl chlorides. According to certain embodiments of the invention, the selectively degradable hydrogel material may further include synthetic polymers such as polyesters, polyanhydrides, polycarbonates, polyurethanes, polyphosphates, or combinations thereof.A continuous polymer matrix material according to certain embodiments of the present invention may comprise a 3D crosslinked polymer network, a non-crosslinked polymer network, or a combination thereof.

[0036] According to certain embodiments of the present invention, the continuous polymer matrix material may include a swellable hydrogel material. The swellable hydrogel material may include a radical-mediated reaction product of at least a first monomer including an acrylate or methacrylate functional group and a second monomer or oligomer including at least two free radically polymerizable functional groups. For example, the at least two free radically polymerizable functional groups may, independently of one another, include an acrylate or methacrylate group, an allyl group, an alkynyl group, a vinyl nitrile, a vinyl ether, a vinyl ester, a vinyl amide, a styrene group, a maleic acid group, a fumaric acid group, or a norbornene group. According to certain embodiments of the present invention, at least one of the first monomer or the second monomer may include, incorporated into the backbone of the monomer and / or grafted to the monomer as a side chain or a component of a side chain, a polyethylene glycol functionality (e.g., -O(C2H4O) n H (where n has a value of 1 to 100), polypropylene glycol functionality (e.g., -O(CHO) nH (where n has a value of 1-100), and / or glycerol functionality. By way of example only, at least one of the first monomer or the second monomer includes 2-hydroxyethyl acrylate (HEA), poly(ethylene glycol) methyl ether acrylate (MPEGA), N-methylacetamide (NMA), or poly(ethylene glycol) diacrylate (PEGDA). Non-limiting examples of non-degradable monomers that may be utilized in the hydrogel material according to certain embodiments of the present invention may include polyolefins (e.g., ethylene, propylene), styrene, nylons (e.g., amides), and / or acrylics. According to certain embodiments of the present invention, non-limiting examples of degradable monomers that may be utilized in the hydrogel material may include esters (e.g., hydroxybutyrate, lactic acid, glycolic acid, caprolactone), acid anhydrides (e.g., adipic acid, sebacic acid), sugars (e.g., cellulose, alginic acid, pectin, dextrin, chitosan, hyaluronan, chondroitin sulfate, heparin), proteins, nucleotides (e.g., DNA, RNA), peptides (e.g., collagen, gelatin, silk, fibrin), urethanes, phosphates, carbonates, and vinyl chlorides. Additionally or alternatively, a third monomer comprising a crosslinker may be incorporated into the continuous polymer matrix material. Additionally or alternatively, the swellable hydrogel material may include one or more natural polymers, such as polymers derived from plants (e.g., cellulose polymers) and polymers derived from animals.

[0037] According to certain embodiments of the invention, the continuous polymeric matrix material may mimic a natural tissue of a subject by incorporating one or more physical properties within about 20%, e.g., within about 15%, 10%, 8%, 5%, 3%, or 1%, of the natural tissue of the subject, where the one or more physical properties of the subject include flexibility and tensile strength. For example, the one or more physical properties may include elastic and / or compressive modulus, storage modulus at 1 Hz, loss modulus at 1 Hz, and / or protein / chemical coating (e.g., collagen I, II, III, IV, laminin I, II, hyaluronan, gelatin, fibrin, fibronectin, etc.). By way of example only, adipose tissue in its natural state has a storage modulus at 1 Hz of 50-100 kPa, a loss modulus at 1 Hz of 10-20 kPa, and an elastic and / or compressive modulus of 3 kPa. In this regard, for example, the IPHB may have a 1 Hz storage modulus of about 110 kPa, a 1 Hz modulus loss of about 22 kPa, and an elastic and / or compressive modulus of about 3 kPa. For example, the IPHB may be analyzed using a Dynamic Mechanical Analyzer (DMA; TA Instruments, RSA3) to evaluate mechanical and physical properties. To prevent points of concentrated force, a 5 mm biopsy punch may be used to separate circular hydrogel samples. DMA is performed by dynamic cylindrical compression analysis with a 0.005-mm / sec compression rate and a 1-Hz frequency sweep. For example, the specific chemical constituents and / or degree of crosslinking may be modulated to tailor one or more physical and / or mechanical properties of the resulting continuous polymer matrix material to mimic or reflect those associated with the natural tissue of interest. Additionally or alternatively, the network of microporous channels and / or chambers and / or the outer surface topography / texture of the IPHB may be engineered. These surfaces may be largely smooth, or grooves, bumps, ridges, divots, and other surface irregularities may be introduced to modulate liquid or gas flow in the network of microporous channels and / or chambers.Such surface irregularities may, for example, help to introduce turbulence to slow the flow of liquid or gas through the network of microporous channels and / or chambers. By way of example only, the surface irregularities may be significantly smaller in size compared to the average diameter of the network of microporous channels and / or chambers, for example at most about 1 / 4 to about 1 / 10 the size of the average diameter of the network of microporous channels and / or chambers.

[0038] According to certain embodiments of the present invention, the continuous polymer matrix material is formed by additive manufacturing techniques such as 3D printing or digital photosynthetic printing. In this regard, the network of microporous channels and / or chambers is a structure that mimics the morphology of the target's natural tissue, for example, by changing the shape and dimensions of the network of microporous channels and / or chambers to reflect the morphology of the target's natural tissue. For example, the morphology of the target's natural tissue can be easily ascertained by one skilled in the art, and this morphology can be reproduced by a 3D printing or digital photosynthetic printing process to form an IPHB having a network of microporous channels and / or chambers that mimics the morphology of the target's natural tissue.

[0039] According to certain embodiments of the invention, the average diameter of the network of microporous channels and / or chambers may include from about 100 to about 800 microns, such as any of the following: at least about 100, 120, 150, 180, 200, 220, and 250 microns, and / or any of the following: at most about 800, 780, 750, 720, 700, 680, 650, 620, 600, 580, 550, 520, 500, 480, 450, 420, 400, 380, 350, 320, 300, 280, and 250 microns. Additionally or alternatively, the network of microporous channels and / or chambers may comprise at least about 40% by volume of the 3D macrostructure, such as any of the following: from at least about 40, 50, 60, and 70% by volume of the 3D macrostructure, and / or any of the following: from at most about 90, 85, 80, 75, and 70% by volume of the 3D macrostructure.

[0040] According to certain embodiments of the invention, the interface between the network of microporous channels and / or chambers and the continuous polymer matrix material may include a coating of a compatibilizer selected to promote adhesion of primary cells of interest. This coating may be applied after formation of the IPHB. By way of example, the coating including the compatibilizer may include a biological coating, such as collagen I (e.g., human mesenchymal stem cells [from adipose, bone marrow, umbilical cord], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human muscle cells, human osteoblasts, human bone cells, human chondrocytes, bovine muscle cells, porcine hepatocytes, porcine chondrocytes, porcine bone cells, equine muscle-derived stem cells), laminin I (e.g., human induced pluripotent stem cells, mouse dorsal root ganglia), hyaluronan (e.g., porcine hepatocytes, human adult dermal fibroblasts), gelatin (e.g., human mesenchymal stem cells [from adipose, bone marrow, umbilical cord], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human muscle cells, human osteoblasts, human bone cells, human chondrocytes, bovine muscle cells, porcine hepatocytes, porcine bone cells, equine muscle-derived stem cells), laminin I (e.g., human induced pluripotent stem cells, mouse dorsal root ganglion), hyaluronan (e.g., porcine hepatocytes, human adult dermal fibroblasts), gelatin (e.g., human mesenchymal stem cells [from adipose, bone marrow, umbilical cord], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human muscle cells, human osteoblasts, human osteo fibroblasts, human keratinocytes, human muscle cells, human osteoblasts, human bone cells, human chondrocytes, human T cells (CD8+), human T cells (CD4+), human macrophages, bovine muscle cells, porcine hepatocytes, porcine chondrocytes, porcine bone cells, equine muscle-derived stem cells), fibrin (e.g., human keratinocytes), fibronectin (e.g., human mesenchymal stem cells [from adipose, bone marrow, umbilical cord], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human osteoblasts, human bone cells, human chondrocytes), or any combination thereof.

[0041] In another aspect, the present invention provides a scaffolding system comprising a plurality of IPHBs, such as those described and disclosed herein. According to certain embodiments of the present invention, for example, the plurality of IPHBs comprises a first IPHB including a first mating male component and a second IPHB including a second mating female component, where the second mating female component is configured to receive the first mating male component. According to certain embodiments of the present invention, for example, the first mating male component can be inserted into the second mating female component, where the first IPHB and the second IPHB are each provided in a swollen state, thereby improving the mating of the first IPHB and the second IPHB. In this regard, the swollen state can be provided due to the absorbency of a liquid, such as water or culture medium. As the first and second IPHBs swell in their mated state, the frictional forces between the mated mating male and female components increase, increasing the force required to separate the IPHBs.

[0042] According to certain embodiments of the present invention, the first IPHB has a first network of microporous channels and / or chambers and the second IPHB has a second network of microporous channels and / or chambers, where a first portion of the first network of microporous channels and / or chambers at least partially overlaps a first portion of the second network of microporous channels and / or chambers when the first and second IPHBs are interdigitated to define a collective continuous network of microporous channels and / or chambers. In this regard, as described above, the density of microporous channels at the surface of the IPHB is sufficiently large. As a result, the partial overlap of at least a portion of the microporous channels at the surface of each IPHB allows the formation of a continuous collective continuous network of microporous channels and / or chambers extending through each interdigitated IPHB. As described above, multiple IPHBs can be interdigitated together along their xy plane and / or along the z direction.

[0043] According to certain embodiments of the invention, a first IPHB may be seeded with a first primary cell and a second IPHB may be seeded with a second primary cell, where the first primary cell is different from the second primary cell. As noted above, each of the IPHBs may be seeded with different and / or unique primary cells or a combination of multiple primary cells. Alternatively, for mass production of a given cell of interest, each IPHB may be seeded with the same primary cells.

[0044] By way of example only, one or more of the IPHBs may be seeded and allowed to grow any of the following cells: (1) human stem cells, such as human Wharton's jelly cells (MSCs), human bone marrow derived mesenchymal stem cells (MSCs), human adipose derived mesenchymal stem cells (MSCs), human skin derived induced pluripotent stem cells (iPSCs), human blood derived induced pluripotent stem cells (iPSCs), human CD4+ T cells, human CD8+ T cells; (2) primary mammalian cells, such as HepG2 cells (liver cancer cells), human adult skin fibroblasts (primary cells), human neonatal skin fibroblasts (primary cells), human adult keratinocytes (primary cells), mouse dorsal root ganglia (primary neurons), bovine muscle cells (primary cell line), primary porcine hepatocytes, porcine chondrocytes, porcine bone cells, equine muscle derived stem cells (primary MSCs), primary snail cells, human macrophages; (3) primary mammalian cells, such as human erythrocytes (primary cells), human keratinocytes (primary cells), human dorsal root ganglia (primary neurons), bovine muscle cells (primary cell line), primary porcine hepatocytes, porcine chondrocytes, porcine osteocytes, equine muscle derived stem cells (primary MSCs), primary snail cells, human macrophages; Mortalized mammalian cell lines, such as UB-OC2 cells (mouse cochlear epithelium), human myoblastoma (muscle carcinoma), PC3 (prostate cancer), CHO (Chinese hamster ovary cells), HEK293 (human embryonic kidney cells), SHSY5Y (neuronoma), PANC-1 (human pancreatic cancer), HeLa (cervical cancer), A549 (lung cancer), A673 (muscle carcinoma), and (4) primary plant cells, such as rosemary, tobacco, and tomato.

[0045] Figure 7 illustrates the migration of cells 33 from a first IPHB 1 to a second IPHB 2 when they are meshed together, according to certain embodiments of the present invention. As illustrated in Figure 7, a first IPHB 1 may be seeded with cells 33 and then mated to an empty (e.g., devoid of cells) second IPHB 2. Once mated together, cells 33 will migrate from the first IPHB 1 into the second IPHB 2 until equilibrium is reached. After equilibrium is reached in both IPHBs, cells 33 will continue to proliferate.

[0046] As noted above, a method of culturing cells according to certain embodiments of the present invention utilizes one or more IPHBs. By way of example, a method of culturing cells may include providing an initial scaffold comprising a first IPHB and seeding the first IPHB with one or more cells of interest (e.g., a first cell type). Once seeded, the one or more cells may be provided with appropriate nutrients by one or more culture media, which may reach the seeded cells by way of a microporous network formed in the first IPHB. In this regard, old or exhausted culture media may be drained and / or aspirated from the IPHB and / or the well in which the IPHB may be housed. After removal of the old or exhausted culture media, new culture media may be applied to the IPHB. For example, the first culture medium may be desired in the early stages of growth after seeding, but once cell growth is well established and expansion of cells within the IPHB is well established, an altered (different) culture medium may be preferred. In this regard, the method may include providing one or more target cells to facilitate or allow the one or more target cells to grow and expand into the network of microporous channels and / or chambers. Once the cells have grown and expanded into the first IPHB, the initial scaffold may be expanded by interdigitating a second IPHB with the first IPHB. In this regard, the first IPHB has a first network of microporous channels and / or chambers and the second IPHB has a second network of microporous channels and / or chambers that at least partially overlap at an interface between the first IPHB and the second IPHB. In this regard, the one or more target cells are allowed to grow and expand from the first IPHB into the second IPHB, where a culture medium may be provided to facilitate the growth and expansion of the cell line in the second IPHB.

[0047] According to certain embodiments of the present invention, one or more target cells seeded in the microporous channels and / or chambers of the network may grow and grow in various patterns within the network, as illustrated in Figure 8A, for example, one or more target cells 33 (e.g., seeded in an IPHB) grow and grow within the network of microporous channels and / or chambers 30 to define a winding pattern, where as the one or more target cells grow and grow from an inner portion of the first IPHB toward one or more outer surfaces, e.g., the top surface, the bottom surface, and / or at least one side edge, the one or more target cells bend back and forth in a 3D configuration across at least a portion of the individual microporous channels. As illustrated in Figure 8B, for example, one or more target cells 33 (e.g., seeded in the IPHB) grow and proliferate within the network of microporous channels and / or chambers 30, and as the one or more target cells proliferate from the inner portion of the first IPHB toward one or more outer surfaces, e.g., the top surface, the bottom surface, and / or at least one side edge, they define a spiral pattern attached to the walls of the network of microporous channels and / or chambers. As illustrated in Figure 8C, for example, one or more target cells 33 (e.g., seeded in the IPHB) grow and proliferate within the network of microporous channels and / or chambers 30, and as the one or more target cells proliferate from the inner portion of the first IPHB toward one or more outer surfaces, e.g., the top surface, the bottom surface, and / or at least one side edge, they define a network of nodes and branches that define open areas between the nodes and branches. As illustrated in FIG. 8D , for example, one or more target cells 33 (e.g., seeded in the IPHB) grow and proliferate within the network of microporous channels and / or chambers 30, and as the one or more target cells proliferate and proliferate from an interior portion of the first IPHB toward one or more exterior surfaces, e.g., the top surface, the bottom surface, and / or at least one side edge, they define a continuous cell sheet adhered to the walls of the network of microporous channels and / or chambers.As illustrated in Figure 8E, for example, one or more target cells 33 (e.g., seeded in the IPHB) grow and proliferate within the network of microporous channels and / or chambers 30 to define a spheroid. As illustrated in Figure 8F, for example, one or more target cells 33 (e.g., seeded in the IPHB) grow and proliferate within the network of microporous channels and / or chambers 30 to define a comet-like structure having a spheroid structure and a spiral structure arising from the spheroid structure as the one or more target cells proliferate from an inner portion of the first IPHB toward one or more outer surfaces, e.g., a top surface, a bottom surface, and / or at least one side edge.

[0048] As noted above, methods according to certain embodiments of the present invention uniquely allow for the formation of 3D cell growth and proliferation expansions that resemble in vivo growth and proliferation expansions. In this regard, FIG. 9 is a schematic diagram illustrating the nomenclature and conceptual cell migration of multiple IPHBs according to certain embodiments of the present invention. As shown in FIG. 9, the initially seeded IPHB 300 may be referred to as the primary or seeded block (I). Once the seeded block 300 reaches the desired confluency, which is cell phenotype dependent and may take 1-2 weeks, it may be expanded, for example, by mating two secondary (II) IPHBs 302a, 302b directly to the seeded block 300. The number of secondary, tertiary, etc. may depend on the final goal for cell and / or tissue growth. FIG. 9 illustrates a single tertiary (III) IPHB 304a directly attached to both of the secondary (II) 302a, 302b IPHBs. Figure 10 is a schematic diagram illustrating addition options for adding various numbers and configurations of additional IPHBs to the IPHB of Figure 9. Figure 10 illustrates three exemplary additions, including a variety of configurations, including one example with a quaternary (IV) IPHB 308. As illustrated in Figures 9-10, the method of utilizing multiple IPHBs provides an infinite number of different continuous growth scaffolds for customizing growth protocols.

[0049] According to certain embodiments of the present invention, the method includes a sequential culture method including sequential addition of a plurality of secondary blocks to a first IPHB. The plurality of secondary blocks may include a second IPHB and a third IPHB directly interconnected to the second IPHB, such that the second IPHB is directly between the first IPHB and the third IPHB, where the plurality of secondary IPHBs are initially devoid of cells. In this regard, the target cells present in the first IPHB are harvested after expansion of the cells from the first IPHB to the second IPHB, and the target cells present in the second IPHB are harvested after expansion of the cells from the second IPHB to the third IPHB. Such a method thus allows for continuous growth and harvest of the target cells, particularly without the need for reseeding or subculturing. According to certain embodiments of the invention, the number of secondary blocks (i.e., secondary IPHBs) can be at least 2, for example, any of the following: at least about 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 blocks.

[0050] According to certain embodiments of the invention, the method includes a multiplex cell culture method, in which the one or more target cells seeded into a first IPHB include a first cell type, and a second IPHB is seeded with a second cell type. In this regard, the first cell type is different from the second cell type. In this regard, cells of the first cell type (e.g., bone cells) and cells of the second cell type (e.g., chondrocytes) are allowed to proliferate toward one another and form a first interface between the first cell type and the second cell type. According to certain embodiments of the invention, the method may further include disassembling each of the IPHBs to expose each cell type and the first interface. In this regard, each cell type and the first interface may be isolated for further analysis and / or use. According to certain embodiments of the invention, the method may further include intermeshing a third IPHB directly with the second IPHB, where the second IPHB is directly between the first and third IPHB, and seeding the third IPHB with a third cell type different from the first and second cell types. The method may also include allowing and / or growing the cells of the second cell type and the cells of the third cell type to proliferate toward each other and form a second interface between the second and third cell types. According to certain embodiments of the invention, the method may further include disassembling each of the IPHBs to expose each cell type, the first interface, and the second interface.

[0051] As described above, one or more of the cells of interest may produce or secrete a therapeutic agent, e.g., a biologic, of interest. For example, the therapeutic agent may include an exosome, an extracellular vesicle, a growth factor, a monoclonal antibody, a peptide, a protein, a viral particle, an oligonucleotide, an organelle, or a combination thereof. In this regard, various configurations of multiple IPHBs may be interdigitated to provide a collective scaffold network, in which multiple cell lines, e.g., cell lines capable of producing or secreting a therapeutic agent, are seeded on different IPHBs.

[0052] As described above, methods according to certain embodiments of the present invention may include interdigitating multiple IPHBs in a myriad of different configurations to provide a wide variety of continuous growth scaffolds for customizing growth protocols for a wide variety of different cell lines. Figures 11-21 illustrate only a few non-limiting exemplary configurations according to certain embodiments of the present invention. Figure 11 is a schematic diagram of an invasion assay configuration, for example, of a series of interdigitated empty IPHBs 410 (e.g., IPHBs initially lacking seeded cells), with four seeded IPHBs 420 interdigitated as side chains to the series of interdigitated empty IPHBs. All of the IPHBs are contained in a tray for containing IPHBs and / or cell culture medium. In this regard, the seeded cells of the seeded IPHBs 420 grow and proliferate within the series of interconnected empty IPHBs. 12 is a schematic diagram of a dual invasion assay configuration of a series of interdigitated empty IPHBs 410, where a first set of four IPHBs 420 seeded with a first cell type are interdigitated as side chains to the series of interdigitated empty IPHBs, and a second set of four IPHBs 430 seeded with a second cell type capable of producing or secreting a therapeutic agent are interdigitated as side chains on the opposite side of the series of interdigitated empty IPHBs. In this regard, the series of initially interdigitated empty IPHBs 410 (e.g., lacking seeded cells) can be simultaneously infiltrated from cell lines from IPHBs 420 and IPHBs 430. In this particular configuration, the first and second cell types can form multiple interfaces and / or regions of interaction with each other in the initially empty IPHBs 410. FIG. 13, for example, illustrates a multiple invasion assay configuration. Here, eight IPHBs 420, 425, 430, 435, 440, 445, 450, 455 are attached as side chains to a series of interdigitated, empty IPHBs 410, where each attached side chain is seeded with a different cell type, each of which may produce or secrete a different therapeutic agent. In this regard, the growth and expansion of multiple cell lines may be simultaneously grown and expanded within a series of interdigitated, initially empty IPHBs 410.In this regard, multiple distinct interfaces between different cell types or cell lines may also be formed in a series of interdigitated initially empty IPHBs.

[0053] FIG. 14 illustrates a migration assay configuration, in which a series of seeded IPHBs 420 have eight side chains of unseeded or initially empty IPHBs 410 attached to them. Such a configuration can provide, for example, a multi-chain culture method, as described above. Thus, the target cells seeded on the IPHBs 420 grow and proliferate outwardly within each of the initially empty IPHBs 410. In this way, the earlier IPHBs (those closest to the seeded IPHBs 420) infiltrated by cell growth can be harvested while cell line growth simultaneously grows in subsequent IPHBs (e.g., IPHBs further from the seeded IPHBs 420). Such an approach can provide, for example, mass production of target cells from which the cells can be harvested without stopping the continuous 3D growth of the target cells.

[0054] FIG. 15 illustrates a dual migration assay configuration, in which a series of seeded IPHBs includes a first group of IPHBs seeded with a first cell type 420, and a second group of IPHBs seeded with a second cell type 430, in which the eight side chains of unseeded or initially empty IPHBs 410 are attached to them. In this regard, such a configuration can provide for the mass production of multiple different cell types of interest, in which each cell type can be harvested without stopping the continuous 3D growth of multiple different target cells. FIG. 16 illustrates a multiple migration assay configuration. Here, a series of seeded IPHBs 420, 425, 430, 435, 440, 445, 450, 455, each IPHB being seeded with a different cell type, and eight side chains of unseeded or initially empty IPHB 410 are attached thereto, etc. As shown in Figure 16, each of the different cell types can produce or secrete a therapeutic agent of interest.

[0055] FIG. 17 illustrates an 8-chamber omni-tray loaded with multiple IPHBs 420, 425, 430, 435, 440, 445, 450, 455 and spacers, where each chamber is loaded with a group of four interlocking IPHBs seeded with a different cell type (e.g., each chamber has its own cell type therein). Such a configuration allows for the production of multiple cell lines that can produce or secrete a therapeutic agent of interest for isolation on the same tray over the same period of time. FIG. 18 illustrates an 8-chamber omni-tray loaded with multiple IPHBs and spacers, where the IPHBs are configured as a one-way paracrine signaling assay. FIG. 19 illustrates an 8-chamber omni-tray loaded with multiple IPHBs and spacers, where the IPHBs are configured as a two-way paracrine signaling assay. Figure 20 illustrates an 8-chamber omni-tray loaded with multiple IPHBs and spacers, where the IPHBs are configured as a paracrine signaling assay. Figure 21 illustrates an 8-chamber omni-tray loaded with multiple IPHBs and spacers, where the IPHBs are configured as a 4-way paracrine signaling assay.

[0056] The example configurations described above are merely illustrative of a few of the possible ways in which certain embodiments of the present invention can be practiced. Thus, the example configurations and diagrams described above are merely illustrative and not limiting. EXAMPLES

[0057] The present disclosure is further illustrated by the following examples, which should not be construed as limiting in any way. Thus, the specific features described in the following examples are merely illustrative and not limiting.

[0058] In this example, we demonstrate a 3D hydrogel system that improves the outcome of expansion of MSC populations, such as adipose-derived MSCs (ASCs). A customizable 3D hydrogel system was bioprinted and formulated into a bioinert matrix that closely mimics the mechanics of native adipose tissue. Ultimately, it acts like a tailored bioreactor for MSC expansion and biologic harvesting. A 3D hydrogel system with a unique "puzzle-piece" macroarchitectural design was constructed (e.g., multiple IPHBs), which allows for easy addition of additional hydrogels (e.g., one IPHB). In addition, the unique porous microarchitecture design allows for mass transport, promoting cell migration and proliferation, and eliminates the need to subculture cells by relying on cell migration between hydrogels in microchannels. The initial utilization of a bioinert matrix allowed us to explore and observe the potential role of the mechanical and dimensional properties of the 3D system on ASC senescence and stem cell-like phenotypic characteristics without the introduction of a bioactive matrix. It was believed that the more flexible 3D hydrogel substrate would provide a more natural mechanical environment for ASCs relative to conventional 2D culture methods, leading to the maintenance of a non-senescent stem cell-like ASC population. Moreover, the unique architecture design would allow for continuous expansion of ASCs by cell migration between attached hydrogels (e.g., attached IPHBs), thus eliminating the exposure of cells to negative 2D subculture procedures and their subsequent sequelae.

[0059] Materials and Methods cell culture Human ASCs (Lot No. 18TL212639, 23 year old female, black) and human keratinocytes (KC; Lot No. 18TL318559, 62 year old male, white) obtained from Lonza (Basel, Switzerland) were utilized in this study. MSC-GM Mesenchymal Stem Cell Growth Medium BulletKit™ was obtained from Lonza (#PT-3001) and used for ASCs. MesenCult™-ACF Plus Medium Kit (Stem Cell Technologies, British Columbia, Canada; Catalog No. 05445) was used as serum-free medium. DermaLife K Keratinocyte Medium Complete Kit was obtained from Lifeline Cell Technologies (Maryland, USA; #LL-0007) and used for KCs.

[0060] 3D Printed Hydrogel Cell Culture System The bioinert 3D hydrogel system (e.g., IPHB) is approximately 1 x 1 x 1 cm and is a polyethylene glycol (PEG)-based system that contains a unique microarchitecture design. It was engineered to mimic the mechanics of native adipose soft tissue and demonstrated no significant change in mechanical properties over a 3-month period. The 3D hydrogels were plated onto glass 6-well culture plates for culture. Fibronectin is a commonly chosen coating substrate for ASCs due to their natural fibronectin secretion. Because cells do not efficiently adhere / attach to PEG-based polymers, both the 2D culture plastic / glass and the 3D hydrogels were plated with 5 μg / cm 2 To enhance initial cell attachment, the fibronectin concentration was normalized to the surface area due to the inherent surface area to volume difference between the 2D and 3D systems. The approximate surface area of ​​the 3D hydrogel was calculated from the 3D model used for bioprinting.

[0061] Expansion of ASCs and KCs ASCs and KCs were seeded in T-150 flasks and grown to approximately 80% confluence before subculture (passage). Cells were subcultured by removing the culture medium, washing three times with Hanks Balanced Salt Solution (HBSS, Massachusetts, USA; calcium-free, magnesium-free), and incubating with 0.05% trypsin / EDTA (Lonza; Cat. No. CC-3232) for 5 min at 37°C. Trypsin was neutralized with serum, and cells were centrifuged at 500×g for 5 min, then pelleted and resuspended for reseeding into new 2D tissue culture plastic vessels. ASCs at passage 1 (P1) were reseeded onto 2D culture plastic or onto / into bioprinted 3D hydrogel (e.g., IPHB) systems by dropwise application of concentrated cell solution onto the surface of the hydrogel. This process was repeated five times with the remaining cell solution to ensure efficient cell seeding. This repeated seeding process allowed the cells to distribute among the microporous structures within the hydrogel system. The passage equivalent time points were utilized to allow a similar comparison to 2D cultures, as the attachment of additional hydrogel and increased surface area eliminated the need for subculture in this study. Therefore, passaging events were typically performed every 4–5 days for 2D cultures of ASCs, but not for 3D cultures. After 4–5 days of 2D culture of P2 ASCs, the cells were passaged and considered to be P3 in 2D, and ASCs in 3D were considered to be passage P3 equivalent. To normalize cell numbers, culture expansion was determined for ASCs based on known 2D and 3D surface areas, initial cell seeding density, and average population doubling time of 2.25 days (determined experimentally in 2D, data not shown). ASCs were assayed at 5,000 cells / cm 2 The medium supplement was 150 μl / cm for ASC expansion cultures. 2 to account for dilutional differences in surface area to volume ratios between 2D and 3D cultures.

[0062] ASC phenotype characterization MSC stem cell-like phenotype was assessed for P1 / 2 / 6 / 10 ASCs by immunolabeling characterization of three major MSC surface markers (CD73 / 90 / 105). ASCs were serially passaged in T-150 flasks or allowed to expand in 3D hydrogel systems. At each respective passage time, cells were cultured at approximately 5000 cells / cm in 2D. 2The cells were seeded at a standardized density of 1000×1000 cells / well into 96-well glass culture plates (Cellvis, CA, USA; Cat. No. P96-1.5HN) for 2 days, fixed, and then assessed for ASC phenotype by immunolabeling of surface CD markers. For ASCs in the 3D culture system, cells were fixed and stained in situ. In this study, positive staining for CD73 / 90 / 105 and negative staining for CD34 / 45 were used to indicate a stem cell-like MSC phenotype. After fixation with 4% paraformaldehyde, cells were washed three times with HBSS and placed in blocking buffer (1% donkey serum in HBSS) for 1 h. After blocking, primary antibodies for CD73 (Abcam, MA, USA; Cat. No. 133582; 1:100), CD90 (Abcam; Cat. No. 181469; 1:100), CD105 (Abcam; Cat. No. 231774; 1:100), CD34 (Abcam; Cat. No. 81289; 1:200), or CD45 (Abcam; Cat. No. 40763; 1:200) were added and cells were incubated overnight at 4°C. The following day, cells were washed three times and secondary antibodies were applied for 1 h, followed by three additional washes. Cells were counterstained with Hoechst 33342 (Invitrogen, Massachusetts, USA; Cat. No. H3570; 1:1000) and Alexa Fluor® 647 phalloidin (Invitrogen; Cat. No. A22287; 1:1000). Immunofluorescence was assessed by a Revolve microscope using filters for 4',6-diamidino-2-phenylindole (DAPI; EX-380 / 30, EM-450 / 50), fluorescein isothiocyanate (EX-470 / 40, EM-525 / 50), Texas Red (EX-560 / 40, EM-630 / 75), and Cy5 (EX-630 / 40, EM-700 / 75) (Echo, CA, USA) and a 20x objective lens (Olympus Corporation, Tokyo, Japan; UPlanSApo, 0.75NA).ASC stem cell-like phenotype quantification was performed in quadruplicates (n=4) and images were taken from a total of 10 random fields per biological replicate (2D=per ​​well, 3D=per ​​hydrogel) to achieve a maximum of 40 total measurements for each sample. Total nuclei were counted and total positive cells were assessed. Phalloidin counterstaining was used to aid in identifying positive staining. ASCs characterized at P1 were used as a baseline for comparison.

[0063] Characterization of ASC senescence Assessment of ASC senescence was performed similarly to the ASC phenotyping method above. ASCs were serially subcultured in T-150 culture flasks until each respective assay time point when they were seeded into 96-well glass culture plates. ASCs on 96-well plates were allowed to acclimate in serum-based medium for 2 days, fixed, and then assessed for senescence activity by immunofluorescent labeling of β-galactosidase activity with the CellEvent™ Senescence Green detection kit (Invitrogen; Cat. No. C10850) according to the manufacturer's instructions. ASCs in the 3D system were simultaneously assessed at the P2 / 6 / 10 passage equivalent time point. Cells were counterstained with Hoechst and phalloidin as in the above method. Senescence characterization was performed in quadruplicates (n=4), and images were taken from a total of 10 random fields per biological replicate to achieve a maximum of 40 total measurements per sample. Total nuclei were counted and total senescent positive and negative cells were determined.

[0064] Isolation of ASC-conditioned medium Media addition for ASC expansion was standardized to account for dilution differences in surface area to volume ratio between 2D and 3D cultures. Media was changed every 2 days. For ASC-conditioned medium (ASC-CM) collection, MSC-GM was removed and cells were washed 3 times with HBSS and then cultured in serum-free MSC medium for 48 hours before collection (for both 2D and 3D cultures). The collected ASC-CM was then centrifuged at 1500 × g for 10 minutes to eliminate cell debris, steriflip filtered through a 0.22 μm filter, and stored at -80 °C until use.

[0065] KC activity after ASC-CM treatment ASC-CM was collected at each respective time point according to the above protocol. ASC-CM was then placed on KCs for up to 24 hours to assess its ability to modulate metabolic, proliferative, or migratory activity to evaluate the wound healing performance of ASC-CM. For these studies, ASC-CM was added to KC growth medium at a 1:1 ratio. Experimental assays were performed according to the manufacturer's instructions. PrestoBlue fluorescence was obtained at 560 / 590 nm (n=4) and used to assess metabolic activity after 24 hours of ASC-CM treatment. To control for possible differences in cell number and obtain approximate metabolic activity per cell, Hoechst was added to PrestoBlue samples and values ​​were expressed as the average relative fluorescence unit value of the PrestoBlue / Hoechst signal. PicoGreen fluorescence was obtained at 485 / 535 nm (n=4) and used to assess cell number as a surrogate measure of KC proliferation after 24 hours of ASC-CM culture. Total cell numbers per 96-well plate were calculated based on an average DNA content of 7.7 pg / cell. KC scratch assays were performed to assess changes in wound size as a surrogate measure of KC migration (n=3). Migration images were captured using an ImageXpress micro XLS imaging system (Molecular Devices, CA, USA). The entire wound was imaged for each wound triplicate, and wound area was calculated using three different wound areas per wound triplicate. The three wound area values ​​were averaged per triplicate and time point for each group and expressed as percentage wound area recovered (n=3).

[0066] statistical analysis All data are reported as mean with standard error of the mean. Characterization of ASC populations by immunolabeling of senescence and CD markers was assessed by two-way analysis of variance. KC metabolism, proliferation, and migration activity were assessed by two-way analysis of variance. Data were tested for normality by Shapiro-Wilk and Kolmogorov-Smirnov tests and plotted in QQ plots. GraphPad-Prism 9.0.2 software (GraphPad, CA, USA) was used for analysis and a p-value <0.05 was considered significant.

[0067] result Unique 3D hydrogel (IPHB) design eliminates subculture Approximately 1cm 3The 3D printed hydrogel system contains a unique “puzzle piece” macrostructure that allows for the sequential addition of additional hydrogels (Figure 22A) and promotes cell migration from the primary seeded hydrogel into the newly attached hydrogel (Figures 22A and B). ASCs were cultured in / on a single hydrogel system for 2 weeks and allowed to migrate into the hydrogel. After that, additional hydrogels were added for 5 days and the cells were allowed to migrate into the newly attached hydrogel. The cells were then stained and assessed for migration and proliferation between the two hydrogels (Figure 22B). ASCs were seen to line the porous channels beyond the superficial surface within the internal structure and can be seen to form a network within the hydrogel pores (Figures 22B and C). Cells were able to migrate both on the surface of the attached hydrogel and within the microchannels and can be seen to have numerous cell processes and focal adhesions protruding from the cells to aid in migration (Figure 22C). 3D ASC interactions and the formation of a 3D network within the hydrogel microarchitecture were readily identified. Notably, ASCs could be seen lining the surface of the hydrogel and migrating into one of the pore channels, eventually interacting with other cell populations within the pores in three dimensions.

[0068] Retention of stem cell-like MSC surface markers in 3D hydrogels over time Assessment of the stem cell-like ASC population over time was performed by immunolabeling quantification of CD markers CD73 / 90 / 105 (Figure 23A). The abundance of stem cell-like CD markers decreased over time in both 2D and 3D systems. However, 2D cultures had a significantly enhanced loss rate of these markers relative to the 3D system (Figure 23B). Similarly, there was a clear decrease in the intensity of stained positive cells over time in both 2D and 3D systems. Culture in the 3D system resulted in significant retention of all three positive stem cell-like markers at all passage points, with the exception of the initial P2 comparison of CD105. A significant loss of stem cell-like phenotype in 2D cultures can be clearly seen when comparing 3D ASCs at P6 and P10 to 2D ASCs at P2 (Figure 23B). Over the course of the equivalent of 5 or 9 passages in 3D (i.e., 3 or 6 weeks in the 3D system for P6 or P10, respectively) and only one passage event in 2D (seeded at P1), ASCs had similar expression patterns for multiple CD markers. There was only a significant difference between 3D expression of CD105 at P10 relative to 2D CD105 at P2 (Figure 23B). Both systems maintained low populations (<5%) of cells staining positive for CD34 and CD45 markers.

[0069] Delayed induction of aging in 3D hydrogels over time The prevalence of senescent ASC populations over time was assessed by fluorescent labeling of β-galactosidase activity, a commonly used surrogate measure of cellular senescence (Figure 24A). Baseline expression of β-galactosidase activity was approximately 5% in both 2D and 3D ASCs at P2. Over the course of multiple passaging events in 2D, senescence increased significantly in 2D, with 11.7 and 22.5% senescent cells at P6 and P10, respectively (Figure 24B). In contrast, in the 3D system, there was no significant change in the prevalence of senescence over time in the ASC populations over the course of this study (6 weeks) (Figure 24B). Similarly, there were striking differences in cell morphology in the 2D versus 3D systems. More specifically, 2D ASCs appeared more flattened, with a more heterogeneous morphological distribution and a clear increase in cell size over time in culture, whereas 3D ASCs maintained a more homogeneous morphology with no observable change in cell size or morphology.

[0070] Retention of wound healing potential of ASC-conditioned medium Assessment of the functional ability of ASC-CM to regulate secondary cell populations was used to demonstrate a dynamic correlation between ASC population phenotype and the ability to promote wound healing activity of KCs (Figure 25D). KCs were treated with either 2D or 3D ASC-CM from each respective time point for up to 24 hours. KCs treated with ASC-CM from 2D cultures showed a significant drop in repaired wound area when treated with ASC-CM from P6 and P10 relative to P2. There was also a mild decrease in KC migration activity observed with 3D ASC-CM from P10 relative to P2 (Figures 25A and B). Overall, 3D ASC-CM maintained a significantly higher ability to enhance KC migration and close their respective wounds relative to their 2D ASC-CM counterparts. The metabolic activity of KCs demonstrated a decreasing trend when treated with ASC-CM from 2D expanded cells, with a significant decrease at P10 relative to ASC-CM from P2 (Figure 25C). No significant differences were observed with ASC-CM from 3D culture over time. Similarly, the proliferation activity of KCs treated with ASC-CM from 2D culture demonstrated a decreasing trend, with a significant difference between ASC-CM from P10 and P2, but no significant change was observed when KCs were treated with ASC-CM from 3D culture (Figure 25D). Moreover, the proliferation activity of KCs treated with 3D ASC-CM from P6 and P10 was significantly higher than their 2D ASC-CM counterparts.

[0071] Consideration The intrinsic regenerative properties of MSCs have attracted great interest from advancing the technological fields of regenerative medicine and tissue engineering. However, MSCs must typically first be removed from donor tissue sources and cultured outside the body in an artificial environment that is not the natural state of human tissue. To date, commercially available in vitro expansion culture systems are almost exclusively 2D in nature. The rigid 2D system is non-physiological for the cells and rapidly leads to a loss of multipotent stem cell-like characteristics of MSCs with subsequent loss of viability and induction of senescence. These changes lead to MSC populations with significantly reduced regenerative capabilities. This is complicated by the lack of standardized cell culture conditions, creating a significant bottleneck in the growth and development of regenerative therapies. Therefore, there is a critical need to develop a culture system for MSC expansion that is 3D and whose mechanical, architectural, and matrix compositional properties are more tissue mimicking, and ultimately can circumvent many of the limitations of traditional 2D culture, such as the need for continuous subculture.

[0072] Recent advances in 3D systems have demonstrated progress toward producing more stem cell-like MSC populations. However, 3D systems, such as spheroids, organoids, microspheres, and many scaffold systems, typically do not closely mimic the natural state mechanics of their cell / tissue sources (e.g., fat mechanics for ASCs) and often require large bioreactor systems and continuous subculture to achieve large cell numbers for clinical use. However, tissue-engineered hydrogel systems appear to be advantageous toward producing tunable tissue-mimetic systems for cell culture systems. More specifically, the mechanotransduction response to the more flexible substrate of hydrogels is believed to aid in the retention of stem cell-like characteristics. Unfortunately, most current hydrogel systems are manufactured to promote controlled differentiation of stem cells toward specific tissue lineages rather than allowing cells to maintain a stem cell-like phenotype for long-term expansion. Ultimately, an ideal MSC hydrogel expansion system would improve retention of the regenerative stem cell-like phenotype while protecting against senescence, and enable long-term expansion with minimal subculture or user intervention.

[0073] Although MSC-based therapies have demonstrated promise, with over 1000 clinical trials listed by the US FDA to date, they have not progressed as quickly as previously thought. This is believed to be due, at least in part, to the detrimental impact that aged MSC populations may have on tissue regeneration. Senescence is typically a progressive form of cell cycle arrest due to DNA and / or oxidative damage, which results in MSCs with impaired DNA repair modalities. They no longer proliferate and exhibit loss of multipotency. Moreover, aged MSCs have been shown to secrete factors that negatively impact tissue regeneration and wound healing by impairing angiogenesis, increasing oxidative stress, and exacerbating inflammation through the secretion of factors, known as the senescence-associated secretory phenotype. The composition of this phenotype may be heterogeneous and is dependent on the mechanism of senescence induction and environmental stimuli. This likely contributes to the heterogeneity of patient outcomes seen in clinical trials with both cell-based and cell-free therapies. Therefore, developing an in vitro expansion system that limits / prevents the induction of senescence of healthy allogeneic or autologous MSC populations intended for patients would improve the efficacy and consistency of MSC-based clinical therapies.

[0074] In this current example, culturing ASCs in a conventional 2D culture system resulted in a significant increase in senescence, as previously established in the literature. In contrast, the 3D hydrogel system according to certain embodiments of the present invention did not result in a significant change in the senescence of the ASC population over the course of a 6-week study (i.e., the equivalent of 10 passages). Furthermore, changes in cell morphology and size can be indicative of phenotypic changes. The apparent increase in cell size seen with 2D ASCs may be associated with the induction of senescence, thus further supporting the β-galactosidase imaging data and previous literature. Overall, these data support the previous hypothesis that 3D culture and matrix mechanics maintain a protective role against senescence. However, in the 3D hydrogel system (IPHB) according to certain embodiments of the present invention, the need for subculture is eliminated and secretory by-products are more readily available than in more traditional cast / molded hydrogels, which lack a porous microarchitecture.

[0075] Similarly, the stem cell-like phenotype of MSCs is crucial for their regenerative potential and can change rapidly depending on the cell culture environment. MSC populations that differentiate and lose their stem cell-like characteristics result in variability in cell phenotype and modulation of secretome composition, ultimately reducing the consistency of both cell-based and cell-free regenerative MSC therapies. Therefore, MSC populations such as ASCs are often used within only a few passaging events in an attempt to avoid loss of regenerative potential. However, as seen in this example, even within one additional passaging event in 2D culture, ASCs significantly modulate their expression of stem cell-like markers. Moreover, ASCs super-expanded in 3D culture for 6 weeks for the equivalent of 6 or 10 passages (i.e., P6 or P10) maintained similar expression levels of several markers relative to baseline P2 ASCs and higher expression relative to their respective 2D counterparts, further highlighting the detrimental effects of 2D culture systems and the potentially protective effects of 3D culture on MSC populations. The ability to improve the retention of stem cell-like properties within MSC populations over longer periods of time is desirable for a number of applications, including cell therapy, tissue engineering, immunotherapy, and production of secreted biologics.

[0076] As mentioned earlier, recent MSC treatments have expanded to explore biologics as potential regenerative therapies. MSC populations are highly adaptive in nature and are known to sense their surrounding environmental cues and secrete factors accordingly. Hence, the secreted bioactive compounds act to coordinate and bridge various tissue repair processes in an autocrine, paracrine, or endocrine manner. In this study, conditioned media from ASCs cultured in 2D versus 3D systems was collected over time and utilized as a means to functionally evaluate the phenotype of the ASC secretome towards promoting wound healing activity as a treatment for the secondary cell population KC. Working under the hypothesis that the ASC phenotype deteriorates over time in 2D culture but persists in 3D systems, we expected to see a gradual decline in the regenerative potential of ASC-CM from 2D culture, but minimal changes in ASC-CM from 3D culture. This hypothesis was supported by metabolic, proliferation, and migration data of KCs treated with ASC-CM: ASC-CM from 2D cultures demonstrated a steadily declining ability to increase KC activity and was consistently outperformed by its 3D counterpart over the course of the experiment.

[0077] The limitations of this example include the formulation of a hydrogel system that was intentionally bioinert to preclude any contribution of bioactive matrix. Therefore, it was not degradable. As a result, full removal of cells from this formulation was not feasible. Therefore, immunofluorescence labeling was utilized as an alternative to flow cytometry or RNA analysis to demonstrate the senescence and phenotype of the MSC population. However, the ability to perform in situ visualization of adherent populations such as MSCs without the need to resuspend them is an advantage of immunolabeling over cytometry. Moreover, the relative comparison between 2D and 3D cultures helps provide a supportive and holistic perspective that reinforces the immunolabeling data, paired with the functional data of ASC-CM. However, a more comprehensive analysis of the secretome is needed to evaluate both qualitative and quantitative changes.

[0078] conclusion This example illustrates the successful use of a 3D hydrogel system to demonstrate the benefits of culturing MSCs in a system that more closely resembles the mechanics of their native tissue. The 3D system contains a unique architectural design that provides a continuous 3D culture system that does not impede effective mass and fluid transport while also allowing cell migration within and between attached hydrogels, virtually eliminating the need to subculture cells. Continuity is achieved by the addition of additional hydrogels to a previously seeded hydrogel, like attaching two puzzle pieces together. The porous microarchitecture creates a "tunnel" system for cells to interact in the x, y, and z planes and migrate within and between the hydrogels in addition to surface migration at the attachment points.

[0079] These and other modifications and variations of the present invention may be implemented by those skilled in the art without departing from the spirit and scope of the present invention as more particularly set forth in the appended claims. In addition, it should be understood that aspects of the various embodiments may be interchanged in whole or in part. Furthermore, those skilled in the art will appreciate that the foregoing description is by way of example only and is not intended to limit the invention as further set forth in such appended claims. Thus, the spirit and scope of the appended claims should not be limited to the illustrative description of the variations contained herein. [Explanation of symbols]

[0080] 1. First IPHB 2. Second IPHB 10 Continuous polymer matrix materials 12 Top side 14 Bottom 16 Side edge 30 Network of microporous channels and / or chambers 33 cells 50 Interlocking male components 51 first mating male component 52 Second mating male component 53 Upper mating male component 60 Interlocking Female Components 61 First interlocking female component 62 Second interlocking female component 63 mating female component protruding into the bottom surface 100 Semi-cylindrical IPHB 112 Top surface 114 Bottom 116 First side edge 118 Second side edge 119 Arched side edge 150 First mating male component extending from second side edge 153 Upper mating male component 160 a first mating female component protruding into the second side edge portion 163 Interlocking female components protruding into the bottom surface 210 Steps 230 Steps 240 steps 250 steps 300 IPHB initially sown 302 Secondary IPHB 304 Tertiary IPHB 308 Quaternary IPHB 410 Interlocking Empty IPHB 420 IPHB seeded 425 IPHB seeded 430 IPHBs sown 435 IPHB seeded 440 IPHBs sown 445 IPHB seeded 450 IPHBs sown 455 IPHB seeded

Claims

1. 1. A method for culturing cells comprising: (i) providing an initial scaffold comprising a first interlocking porous hydrogel block (IPHB), wherein said first IPHB comprises a three-dimensional (3D) macrostructure defined by a continuous polymer matrix material and a network of microporous channels and / or chambers extending through said continuous polymer matrix material, wherein said 3D macrostructure comprises a top surface, a bottom surface, and a thickness defined by at least one lateral edge extending from said top surface to said bottom surface, wherein the structure of said 3D macrostructure includes at least one interlocking male component and at least one interlocking female component; (ii) seeding the first IPHB with one or more cells of interest; (iii) providing a first culture medium to the one or more target cells and allowing the one or more target cells to grow and expand in the network of microporous channels and / or chambers; (iv) expanding the initial scaffold by mating a second IPHB to the first IPHB, wherein the at least one mating male component or at least one mating female component of the first IPHB is joined to a corresponding mating male component or corresponding mating female component of the second IPHB; and (v) allowing the one or more cells of interest to expand from the first IPHB into the second IPHB and providing the one or more cells of interest in the second IPHB with the first culture medium or the second culture medium; A method for culturing and propagating cells, comprising:

2. The method of claim 1, wherein the one or more target cells grow and proliferate within the network of microporous channels and / or chambers to define a bending pattern, wherein as the one or more target cells proliferate from an inner portion of the first IPHB toward one or more outer surfaces, such as the top surface, the bottom surface, and / or the at least one side edge, the one or more target cells bend back and forth in a 3D configuration across at least a portion of individual microporous channels.

3. The method of claim 1, wherein the one or more target cells grow and proliferate within the network of microporous channels and / or chambers, and as the one or more target cells proliferate from an inner portion of the first IPHB toward one or more outer surfaces, such as the top surface, the bottom surface, and / or the at least one side edge portion, they define a spiral pattern attached to the walls of the network of microporous channels and / or chambers.

4. The method of claim 1, wherein the one or more target cells grow and proliferate within the network of microporous channels and / or chambers, and as the one or more target cells proliferate and proliferate from an interior portion of the first IPHB toward one or more exterior surfaces, such as the top surface, the bottom surface, and / or the at least one side edge portion, they define a network of nodes and branches that define open areas therebetween.

5. The method of claim 1, wherein the one or more target cells grow and proliferate within the network of microporous channels and / or chambers, and as the one or more target cells proliferate from an inner portion of the first IPHB toward one or more outer surfaces, such as the top surface, the bottom surface, and / or the at least one side edge portion, they define a continuous cell sheet adherent to the walls of the network of microporous channels and / or chambers.

6. 2. The method of claim 1, wherein the one or more target cells grow and proliferate within the network of microporous channels and / or chambers to define spheroids.

7. 2. The method of claim 1, wherein the one or more target cells grow and proliferate within the network of microporous channels and / or chambers, and when the one or more target cells proliferate from an inner portion of the first IPHB towards one or more outer surfaces, such as the top surface, the bottom surface, and / or the at least one side edge portion, define a comet-like structure having a spheroid structure and a spiral structure arising from the spheroid structure.

8. The method of any one of claims 1 to 7, further comprising the step of harvesting at least a portion of the one or more cells present in the network of microporous channels and / or chambers of the first IPHB.

9. The method of claim 8, wherein the step of harvesting at least a portion of the one or more cells present in the network of microporous channels and / or chambers of the first IPHB comprises flushing them out of the first IPHB with a fluid medium.

10. The method of claim 8, wherein the step of harvesting at least a portion of the one or more cells present in the network of microporous channels and / or chambers of the first IPHB comprises disintegrating the 3D macrostructure of the first IPHB.

11. The method of any one of claims 1 to 10, wherein the method comprises a chain culture method comprising sequentially adding a plurality of secondary blocks to the first IPHB, wherein the plurality of secondary blocks comprises the second IPHB and a third IPHB directly interconnected to the second IPHB, such that the second IPHB is directly between the first IPHB and the third IPHB, and wherein the plurality of secondary IPHBs are initially devoid of cells.

12. The method of claim 11, wherein the target cells present in the first IPHB are harvested after expansion of cells from the first IPHB to the second IPHB, and the target cells present in the second IPHB are harvested after expansion of cells from the second IPHB to the third IPHB.

13. 13. The method of claim 12, wherein the number of secondary blocks is at least 2, such as at least about any of the following: 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 blocks.

14. 14. The method of claims 1-13, wherein the method comprises a multiplex cell culture method, wherein the one or more target cells seeded into the first IPHB comprise a first cell type, and the second IPHB is seeded with a second cell type, wherein the first cell type is different from the second cell type.

15. 15. The method of claim 14, wherein cells of the first cell type and cells of the second cell type are allowed to proliferate toward each other and form a first interface between the first cell type and the second cell type.

16. 16. The method of claim 15, further comprising disintegrating each of said IPHBs to expose each cell type and said first interface.

17. 16. The method of claim 15, further comprising interdigitating a third IPHB directly with the second IPHB, wherein the second IPHB is directly between the first IPHB and the third IPHB, and seeding the third IPHB with a third cell type different from the first cell type and the second cell type.

18. 18. The method of claim 17, wherein the cells of the second cell type and the cells of the third cell type are allowed to proliferate toward each other and form a second interface between the second cell type and the third cell type.

19. 20. The method of claim 18, further comprising disassembling each of the IPHBs to expose each cell type, the first interface, and the second interface.

20. The method of claims 1-19, wherein the one or more cells of the subject produce or secrete a therapeutic agent, e.g. a biologic, of the subject.

21. 21. The method of claim 20, wherein the therapeutic agent comprises an exosome, an extracellular vesicle, a growth factor, a monoclonal antibody, a peptide, a protein, a viral particle, an oligonucleotide, an organelle, or a combination thereof.

22. 22. The method of any one of claims 1 to 21, further comprising the step of coating an interface between the continuous polymeric matrix material and the network of microporous channels and / or chambers extending through the continuous polymeric matrix material with a compatibilizer, wherein the compatibilizer is selected to promote adhesion of primary cells of a subject to the IPHB.

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