Biological cartridge and cell culture method using same

Abstract

A biological cartridge is provided that includes a containment chamber defining an interior region that contains one or more porous hydrogel blocks (PHBs) seeded with at least one cell type of interest. The biological cartridges include inlet and outlet orifices that allow media and other fluids to enter the PHB for culturing the cell types seeded within the PHB. The biological cartridges can be interconnected in series and / or parallel to define a biological cartridge network (BCN), which is capable of mass producing a particular cell type of interest and / or simultaneously culturing multiple cell types of interest and / or producing one or more biologics of interest.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 312,254, filed February 21, 2022, which is expressly incorporated by reference in its entirety herein.

[0002] Inventive embodiments of the present disclosure generally relate to a biological cartridge that includes a containment chamber defining an interior region that contains one or more porous hydrogel blocks (PHBs) seeded with at least one cell type of interest. The biological cartridge includes inlet and outlet orifices that allow media, other fluids, and gases to enter the PHB for culturing the cell type seeded within the PHB. The biological cartridges can be interconnected in series and / or parallel to define a biological cartridge network (BCN), which is capable of mass producing a particular cell type of interest and / or simultaneously culturing multiple cell types of interest and / or producing one or more biologics of interest. [Background technology]

[0003] One of the challenges in mass-producing biological products such as recombinant antibodies, growth factors, exosomes, extracellular vesicles, proteins, and oligonucleotides is that it is logistically, economically, and precisely difficult to replicate physiological systems on a large scale outside of nature. This is because most physiological systems are created to generate a lifetime of biological resources for a single individual. These physiological systems are carefully balanced, and scaling up or down such systems can disrupt homeostasis within the individual. In contrast, when attempting to replicate such physiological systems outside of the body for the production of raw materials such as biologics, they lose the physiological properties and appropriate scale to function well with currently available resources.

[0004] Thus, there remains a need in the art for systems and / or methods that replicate (e.g., operate in parallel) the physiological systems of a living organism (e.g., a mammal) outside the living organism, which would enable large-scale, economical production of biopharmaceuticals. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been made to solve the problems in the conventional techniques described above. [Means for solving the problem]

[0006] One or more embodiments of the present invention may address one or more of the problems described above. Some embodiments according to the present invention provide a biological cartridge including a containment chamber defining an interior region, the containment chamber including an inlet orifice and an outlet orifice. The biological cartridge also includes at least one porous hydrogel block (PHB), e.g., a plurality of PHBs vertically stacked on top of each other, disposed within the interior region of the containment chamber. In another aspect, some embodiments of the present invention provide a system including a plurality of biological cartridges (e.g., a plurality of biological cartridges as described and disclosed herein) connected in series to each other to define a biological cartridge network (BCN). The BCN may include at least (i) a first biological cartridge having a first inlet and a first outlet, and (ii) a second biological cartridge having a second inlet and a second outlet (operably communicating with the first outlet). According to some embodiments of the present invention, each of the plurality of biological cartridges includes a plurality of PHBs seeded with a first cell type of interest housed therein.

[0007] In another aspect, some embodiments of the present invention provide a system including a biological cartridge network (BCN), the BCN including (i) a first set of biological cartridges including a first plurality of biological cartridges (e.g., biological cartridges as described and disclosed herein) connected in series with each other, and (ii) a second set of biological cartridges including a second plurality of biological cartridges (e.g., biological cartridges as described and disclosed herein) connected in series with each other. The first set of biological cartridges and the second set of biological cartridges may be arranged in a parallel relationship with respect to each other and to a common feedstock source.

[0008] In another aspect, some embodiments of the present invention provide a system including a biological cartridge network (BCN) including a plurality of biological cartridges (e.g., a plurality of biological cartridges as described and disclosed herein), the plurality of biological cartridges including (i) a first plurality of biological cartridges operatively connected in series, and (ii) a second plurality of biological cartridges operatively connected in parallel. In this regard, the BCN may include an almost unlimited number of individual biological cartridges in an almost unlimited number of different overall configurations (e.g., various serial and / or parallel interconnections of biological cartridges, where any number of biological cartridges are seeded with a wide variety of cell types of interest).

[0009] In another aspect, some embodiments of the present invention provide a method of culturing one or more cell types of interest, the method comprising the steps of: (i) providing a first biological cartridge (e.g., a biological cartridge as described and disclosed herein), the first biological cartridge containing a first plurality of PHBs; (ii) seeding the first plurality of PHBs with at least a first cell type of interest; (iii) providing a first medium to the at least first cell type of interest and allowing the at least first cell type of interest to grow throughout a first network of microporous channels and / or chambers of the first plurality of PHBs; and (iv) harvesting at least a portion of the first cell type of interest located throughout the first network of microporous channels and / or chambers of the plurality of PHBs.

[0010] In another aspect, some embodiments of the invention provide a method of culturing one or more cell types of interest, the method comprising the steps of: (a) providing or forming a biological cartridge network (BCN), the BCN comprising: (i) a first set of biological cartridges comprising a first plurality of biological cartridges (e.g., biological cartridges as described and disclosed herein) connected in series with each other; and (ii) a second set of biological cartridges comprising a second plurality of biological cartridges (e.g., biological cartridges as described and disclosed herein) connected in series with each other, the first set of biological cartridges and the second set of biological cartridges being arranged in a parallel relationship with respect to each other and with respect to a common feedstock source; and (b-1) providing or forming a BCN comprising: a first set of biological cartridges comprising a first plurality of biological cartridges (e.g., biological cartridges as described and disclosed herein) connected in series with each other; or (b-2) seeding each PHB in a first biological cartridge of each of the first and second sets of biological cartridges with a first cell type of interest, seeding the immediately succeeding biological cartridge of the first set of biological cartridges with a second cell type of interest, and seeding the immediately succeeding biological cartridge of the second set of biological cartridges with a third cell type of interest, wherein the first cell type, the second cell type, and the third cell type are all different from one another; and (c) harvesting at least a portion of the first cell type of interest, and / or at least a portion of the second cell type of interest, and / or at least a portion of the third cell type of interest.

[0011] The present invention will now be described in more detail with reference to the accompanying drawings, in which some, but not all, embodiments of the 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, but rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like reference characters refer to like elements throughout. The drawings are as follows: [Brief description of the drawings]

[0012] [Figure 1] 1 illustrates a biological cartridge housing multiple PHBs in a vertical orientation, according to some embodiments of the present invention. [Diagram 2] FIG. 2 shows the biological cartridge of FIG. 1 with the PHB removed to reveal the alignment column. [Diagram 3] 1 shows a top view rendering of a biological cartridge according to some embodiments of the present invention, showing a top plate and multiple inlet ports for introducing various liquids and / or gases. [Figure 4] 4 shows the inner surface of the top plate of FIG. 3. [Diagram 5] FIG. 1 is a schematic diagram of a PHB according to some embodiments of the present invention. [Figure 6] FIG. 2 is a schematic diagram of another PHB according to some embodiments of the present invention. [Figure 7] ~ [Figure 10] Each illustrates a system including multiple biological cartridges connected in series and / or parallel, according to some embodiments of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention will now be described in more detail with reference to the accompanying drawings, in which some, but not all, embodiments of the 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.

[0014] The disclosed invention generally relates to systems and / or methods for replicating (e.g., operating in parallel) physiological systems in a living organism (e.g., a mammal) outside the living organism, which allows for large-scale and economical production of biologics. Some embodiments of the invention provide devices in which human, animal, plant, bacterial, yeast, and fungal cells can grow naturally and be regulated at industrial levels to mimic physiological conditions, while retaining economic advantages over other manufacturing systems. Some embodiments of the invention may allow multiple physiological systems to be combined in sequence and in parallel to simulate appropriate physiological and biological functions and to produce biological products. Properly replicating and pairing physiological systems outside the body is far from trivial, as evidenced by the continuing need in the art. Nevertheless, the ability to construct devices that properly incorporate and combine physiological systems may be particularly important in producing biological products and cells that can be used for therapeutic applications or for physiological modeling to test and potentially diagnose diseases, cancers, and injuries in living organisms. Thus, some embodiments of the present invention provide a breakthrough in personalized medicine. For example, some embodiments of the present invention provide a biological cartridge, which may also be referred to as a "biological capsule" (biocapsule), a "biological cassette" (biocassette), a "biological pod" (biopod), or a "biological chamber" (biochamber), that contains or houses porous hydrogel blocks (PHBs) configured to mimic various environments within an organism of interest (e.g., the human body). The biological cartridges, which contain one or more porous hydrogel blocks, can be perfused (e.g., by a pumping device) with liquid media, and the biological cartridges can be connected in series or in parallel to mimic the flow of blood, lymph, and other biological fluids through various biological environments, thereby mimicking physiological tissues or organs.

[0015] One or more PHBs may be disposed within the biological cartridge, comprising synthetically defined hydrogels, biologically defined hydrogels, or composites of synthetically defined hydrogels and biologically defined hydrogels, and containing a network of microporous channels and / or chambers that mimic arteries and arterioles, extending the entire length of the hydrogel. The biological cartridge may also include additional supporting porous microstructures extending adjacent to the one or more PHBs, each microstructure having a network of microporous channels and / or chambers that direct airflow and gas exchange within the biological cartridge. Within the PHB, arginine, glycine, and aspartate (RGD) residues or other integrin binding residues may permeate the surface of the PHB for cell attachment. The PHB may be encased or housed in a cartridge housing, which may be, for example, a cylindrical structure having a circular cross section, or a polyhedral structure having a non-circular cross section (e.g., a cube, a prism, etc.). According to some embodiments of the invention, the cartridge housing has openings at the top for liquid and / or gas inlet and openings at the bottom for liquid, gas, and / or other substances (e.g., one or more cells of interest, one or more biologics, etc.) outlet. The PHBs may be stacked in the z-direction and may extend the entire length of the cartridge housing. When connected to a fluid delivery device such as a peristaltic pump, various liquids may be perfused throughout the PHBs in the cartridge housing. These liquids may contain cells, nutrients, degradative enzymes, or defined chemicals that can permeate or pass through the stacked PHB structure. According to some embodiments of the invention, the biological cartridge or the cartridge housing alone may be encased in a sealed container, where the environmental temperature may be regulated as desired (e.g., between 4-47°C), e.g., gas containing 1-10% CO2 may be supplied at, e.g., 1-10 PSI. The biological cartridge may be sealed under sterile conditions before being encased in the environmental container (e.g., for temperature control).For example, the encasement creates a sealed environment in which the ends of the biological cartridge or cartridge housing alone may be perforated for liquid and gas movement. The biological cartridge may be oriented vertically so that fluid flows through the device by gravity. The biological cartridge may also generate vortices within the respective networks of microporous channels and / or chambers of each PHB contained therein. According to some embodiments of the invention, a carousel may be placed below the biological cartridge, which may be used to sterilize the filters, separate the components, and collect the liquid contents flowing out of the biological cartridge. Cylindrical devices may be arranged in circular or polygonal arrays to allow sequential or parallel processing of liquids flowing through the PHB. As will be described in more detail below, the PHB can be designed to match the mechanical properties of various biological tissues of interest. According to some embodiments of the invention, specific cells may migrate or pass through the PHB for attachment, proliferation, and biologics production. Once the surface area of ​​the PHB (e.g., the internal surface area associated with the network of microporous channels and / or chambers) is completely covered with cells, the PHB may be optionally dissolved to release the cells and biological contents for collection. Liquids, gases, electromagnetic fields, mechanical gradients, osmotic gradients, mechanical gradients, chemical gradients, and temperature gradients can be permeated through the hydrogel to achieve physiological conditions that mimic native tissue.

[0016] According to some embodiments of the present invention, the biological cartridge provides the industry's first sealed device that can automate cell culture by eliminating the need for direct end-user manipulation. Any type of adherent cells may be perfused into the biological cartridge containing the designated PHB. Media and nutrients can be perfused to culture the cells of interest. According to some embodiments of the present invention, once the cells reach a desired maturity and number, the PHB may be degraded, for example, with an enzymatic reagent that releases the cells and biological contents. The released cells and biological contents may be collected by standard filtration and separation techniques and utilized in various research, diagnostic, and therapeutic applications. Advantageously, the biological cartridge and its system allow for hands-free culture of human, animal, bacterial, yeast, and fungal cells in a standardized format. The outcome of the biological cartridge, according to some embodiments of the present invention, is that cells or biological factors secreted from cells can be easily collected and isolated for use as raw materials in the production of personalized therapeutics, personalized diagnostics, personalized autografts, personalized cosmetics, personalized fillers, personalized grafts, cultured plants, cultured feedstock, and cultured meat products, including, but not limited to, burgers, steaks, ribs, seafood, and shellfish products. The composition of the hydrogel material defining or forming the PHB can be varied to allow further control to create pH, electromagnetic, osmotic, and mechanical gradients to stimulate cell growth and differentiation, and to secrete specific biological factors, including, but not limited to, growth factors, exosomes, extracellular vesicles, nucleic acids, proteins, monoclonal antibodies, enzymes, peptides, and organelles.

[0017] As described above, the PHBs provide a platform that can mimic discrete microenvironments for specific cell types of interest. By connecting the PHBs together in a vertical configuration, the respective networks of microporous channels and / or chambers of each PHB contained in the biological cartridge are aligned to allow liquids, gases, and other aqueous media to flow through them. Each PHB may be formed of a hydrogel material that is synthetic in whole or in part. Without knowing the internal microstructure of the particular native tissue of interest that forms the base (e.g., manufactured by additive manufacturing techniques) for the respective networks of microporous channels and / or chambers of each PHB, it is not clear whether PHBs can be combined vertically to allow continuous flow of fluids. Thus, creating a sealed device that can house PHBs in a vertical configuration can allow control of multiple aspects of the environment in which the cells grow. For example, each PHB may have a top and a bottom surface. The respective top and bottom surfaces may be identical, for example, with respect to the number and / or location of openings therein that serve as micro-inlets to the internal network of microporous channels and / or chambers. In this regard, each PHB may be loaded into the biological cartridge in a vertically stacked manner, where the respective networks of microporous channels and / or chambers of each PHB are purposefully aligned at the interface between each PHB, thereby achieving a continuous aggregate network of microporous channels and / or chambers.

[0018] As will become more apparent later, some embodiments of the present invention provide various applications of culturing and / or producing various biological products. Non-limiting examples of applications and / or methods of producing biological products include various cell cultures, including stem cells (e.g., human or animal), hematopoietic cells (e.g., human or animal), somatic cells (e.g., human or animal), plant cells, bacterial cells, fungi, biologics, recombinant antibodies, engineered antibodies, extracellular vesicles, exosomes, growth factors, nucleic acids, DNA, mRNA, tRNA, rRNA, siRNA, miRNA, shRNA, ncRNA, oligonucleotides, proteins, and enzymes. Furthermore, embodiments of the present invention may be utilized to provide, for example, personalized diagnostics, personalized therapeutics, personalized cosmetics, fillers, implants, plant production, organic matter, organic waste, meat production (e.g., beef products, ham products, chicken products, fish products), shellfish products, bacterial production, protein production, and plasmid production.

[0019] Unlike conventional cell culture vessels or bioreactors, multiple applications and configurations of multiple biological cartridges are possible due to the ability to configure multiple PHBs in a sealed environment, at least in part, where multiple parameters of the environment can be controlled, such as nutrient diffusion, gas exchange, temperature, pH, viscosity, osmolality, electromagnetic conductivity, mechanical stiffness, permeability, and turbidity. In this respect, conventional cell culture vessels or bioreactors cannot achieve such high environmental control compared to the biological cartridges according to some embodiments of the present invention. As described herein, multiple biological cartridges may be connected in series / sequential and / or parallel to introduce a biological program from one biological cartridge to the next under exposure of conditioned medium for manipulating cell growth or stimulating biological secretion.

[0020] The present invention allows multiple biological cartridges to be connected in series / sequential or parallel to stimulate mass production of a cell type of interest. Furthermore, some embodiments of the present invention allow the scale of the produced biological product (which may include, for example, cells, biological factors secreted from the cells, or organelles) to be scaled up or down for various applications. Furthermore, some embodiments of the present invention allow testing of new therapeutic agents to permeate the entire cell population in a biological cartridge containing one cell population and then permeate the entire cell population in a next biological cartridge containing another cell population to measure efficacy and pharmacokinetics. Additional advantages realized by some embodiments of the present invention include, for example, no need for subculturing cells. For example, the cell culture method may be free of cell subculturing procedures or operations.

[0021] Further advantages realized by some embodiments of the invention include, for example, releasing cells from the PHB contained in the biological cartridge for downstream analysis and downstream diagnostics or downstream manufacturing of therapeutics, physiological or disease modeling, mass production of an individual's own cells for diagnostic purposes, custom tissue bioengineering, custom therapeutic bioengineering, or custom organ bioengineering. Some embodiments of the invention may also include stimulating and treating a patient's disease or condition in a sealed environment. Furthermore, some embodiments of the invention may be used to pool entire donor population cells together for the purpose of diversifying mass-produced biological products. According to some embodiments of the invention, biological cartridges loaded with one or more PHBs allow cells to grow in a more natural physiological environment compared to culture in conventional 2D plastic cell culture vessels or other bioreactors. In this respect, unlike plastic cell culture vessels and other technologies that are not customizable or modular, as described herein, the PHB defines a tunable substrate and may be considered to mimic the native tissue environment (e.g., in vivo native tissue) in which a particular cell develops and grows. Additionally, some embodiments of the present invention allow for different cell types to be cultured sequentially or in parallel to produce different desired outcomes for the production of cellular biological factors, a flexibility not currently available with any other technology.

[0022] As described above, some embodiments of the present invention provide a biological cartridge including a storage chamber defining an interior region, the storage chamber including an inlet orifice and an outlet orifice. The biological cartridge also includes at least one porous hydrogel block (PHB), for example, a plurality of PHBs stacked vertically on one another, disposed within the interior region of the storage chamber. According to some embodiments of the present invention, the storage chamber may have a cross section in an xy plane, the cross section including a non-circular cross section in the xy plane, for example, a polygonal cross section having 3-12 sides. Such non-circular cross-sectional embodiments can, for example, facilitate stable positioning of a plurality of PHBs having the same geometric shape housed in the storage chamber, since the non-circular cross section can prevent the individual PHBs from rotating or moving independently of one another. In this way, by intentionally keeping the respective networks of microporous channels and / or chambers of each PHB housed in the storage chamber aligned with one another, discontinuity in the continuous aggregate network of microporous channels and / or chambers described above can be reduced. Stated slightly differently, the non-circular geometric shapes of the PHBs and containment chamber cross-sections advantageously limit relative rotational motion of the PHBs contained within the containment chambers, thereby facilitating the elimination of disturbance of the continuous aggregate network of microporous channels and / or chambers defined by each PHB.

[0023] Alternatively, the containment chamber may have a cross-section in the xy-plane, which cross-section comprises a circular cross-section in the xy-plane. According to some embodiments of the invention, the cross-section of the containment chamber may be the same throughout the working length of the containment chamber, the working length being a section along the z-direction perpendicular to the xy-plane and associated with the region containing the PHB.

[0024] According to some embodiments of the present invention, at least one PHB comprises a plurality of PHBs stacked vertically on top of each other in a z-direction perpendicular to the xy-plane. By way of example only, the plurality of PHBs may comprise from 2 to about 40 individual PHBs, such as at least about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, and 20 individual PHBs, and / or up to about any of 40, 35, 30, 25, and 20 individual PHBs.

[0025] FIG. 1 illustrates a biological cartridge 1 including a chamber housing 1 having a hexagonal prism geometry, by way of example only. FIG. 1 illustrates that the hexagonal cross section may be approximately 2.5 cm in diameter and 15 cm in length, by way of example only. These values ​​are illustrative and not limiting. The chamber housing 1 includes an inlet orifice 20 and an outlet orifice 30. In the particular embodiment illustrated in FIG. 1, the biological cartridge 1 also includes an inlet dispersion filter 29 disposed immediately above the vertically stacked PHBs 50, and an exhaust filter 39 disposed immediately below the vertically stacked PHBs 50.

[0026] According to some embodiments of the present invention, the biological cartridge may also include at least one mounting element extending throughout the interior region. The at least one mounting element may include a single mounting element aligned along the central axis of the receiving chamber and extending along at least a majority of the entire working length of the receiving chamber. The biological cartridge may also include a top plate and / or a bottom plate removably engaged directly or indirectly to the receiving chamber, and the single mounting element may optionally be attached directly or indirectly to the top plate and / or the bottom plate. The top plate and / or the bottom plate may form part of a respective inlet or outlet manifold, for example, to facilitate fluid flow into and out of the biological cartridge. FIG. 2 shows, by way of example, the biological cartridge of FIG. 1 with the PHB removed to reveal the alignment column 15. In this regard, multiple PHBs stacked vertically on one another may include respective female mount components including respective PHB orifices that extend completely through respective thicknesses in the z-direction of the respective PHBs, each configured to allow at least one mount element to extend through the respective PHB orifice. FIG. 5 shows, by way of example, a PHB 50 including a top surface 51, a bottom surface 52, and a thickness 53.

[0027] The PHB 50 may also include a female mounting component 54 including an orifice that extends completely through the entire thickness 53 of the PHB, with a top opening 55 and a bottom opening 56 aligned with one another along the z-direction. In this regard, the PHB 50 may be mounted on an alignment column 15 that extends into and / or through each female mounting component 54 of each PHB. According to some embodiments of the invention, at least one mounting element may have a non-circular cross-section, and each PHB orifice has a corresponding non-circular cross-section. In this regard, the mounting element and the female mounting component have the same non-circular shape to prevent the respective PHBs from rotating relative to one another about the z-direction. The vertically stacked PHBs are thus constrained from rotating relative to one another in their respective xy-planes.

[0028] Alternatively, the at least one mounting element may include at least one fin component attached to the receiving chamber and projecting inwardly into the interior region of the receiving chamber. The at least one fin component may extend, for example, along at least a majority of the entire working length of the receiving chamber, for example, along at least about 55, 60, 65, and 75% of the entire working length of the receiving chamber, and / or along at most about any of 100, 98, 95, 90, 85, 80, and 75% of the entire working length of the receiving chamber. In such an embodiment, multiple PHBs stacked vertically on top of each other may include respective female mounting components including respective PHB cutout portions that extend completely through respective thicknesses in the z-direction of the respective PHBs, each female mounting component configured to allow at least one mounting element to extend through a respective PHB orifice. FIG. 6 shows, by way of example, a PHB 50 including a top surface 51, a bottom surface 52, and a thickness 53. The PHB 50 may also include one or more female mount components 57 that include cutout portions that extend completely through the thickness 53 of the PHB. In this regard, the at least one fin component may act as a guide for loading and aligning the PHB within the chamber housing. Additionally, the male-female coupling between the at least one fin component and the at least one cutout portion of each PHB prevents the respective PHBs from rotating relative to each other about the z-direction. Each vertically stacked PHB is thus constrained from rotating relative to each other in the respective xy-planes.

[0029] According to some embodiments of the invention, the at least one mounting element may be made of various materials. According to some embodiments of the invention, for example, the at least one mounting element may include a fiber optic column, which may be used to image the interior of the PHB from within the biological cartridge. In such exemplary embodiments, the at least one mounting element may include a durable glass, such as borosilicate (Pyrex) or polystyrene (plastic), both of which are transparent and rigid. However, according to some embodiments of the invention, the at least one mounting element may include a conductive metal, such as copper, silver, and gold, for conducting a weak current, or ferrous material, for inducing an electromagnetic field through the PHB in the biological cartridge. These metals may be used to assist with temperature regulation, according to such embodiments. The at least one mounting element may also include a synthetic or biologically derived hydrogel material different from the PHB to form a more advanced osmotic, diffusive, or mechanical gradient (e.g., a silicone gradient) through the PHB. The hydrogel material forming or defining at least one mounting element may be used as a delivery means for releasing growth factors and other agents into the PHB with or against the direction of fluid flow through the PHB.

[0030] As described above and shown in FIG. 1, the biological cartridge may also include an inlet dispersion filter disposed directly or indirectly above all of the at least one PHB. In this regard, the incoming fluid passes through the inlet dispersion filter before entering any of the at least one PHB. The inlet dispersion filter may be formed of a variety of materials, such as non-woven and woven membranes with average pore sizes between 0.1 microns and 20 microns (e.g., 0.2 micron pore sizes are used for sterile filtration of contents). Common membrane materials include, but are not limited to, polyethersulfone, cellulose esters (cellulose acetate and cellulose nitrate), polyvinylidene fluoride (PVDF), polypropylene, and polytetrafluoroethylene (PTFE). Additionally or alternatively, the biological cartridge may also include an exhaust filter disposed directly or indirectly below all of the at least one PHB. In this regard, the outgoing fluid passes through the exhaust filter before exiting the biological cartridge. The exhaust filter may also include a non-woven or woven membrane and may be formed of the same material as the inlet distribution filter. The main difference between the inlet distribution filter and the exhaust filter is that, according to some embodiments, the inlet distribution filter may have a smaller average pore size (e.g., 0.2 microns) for sterilization purposes, while the exhaust filter may have a larger average pore size to allow cells or biological agents to be easily discharged from the biological cartridge and pass on to the next biological cartridge or to a final filtration system downstream of the biological cartridge, depending on the location and application of the biological cartridge.

[0031] As briefly mentioned above, the biological cartridge may also include an inlet manifold including at least one inlet port in operative communication with an inlet orifice of the receiving chamber. The at least one inlet port may include, for example, one to about five individual inlet ports, each in operative communication with an inlet orifice of the receiving chamber. Each inlet port may also include a one-way so-called "check valve" to prevent backflow in the respective inlet port. The inlet ports may be operatively connected, for example, with the respective cell culture, gas, pH adjustment solution, buffer, etc. In this regard, various fluids may easily flow into and through the chamber housing and each PHB. The inlet manifold may further include, for example, a top plate removably engaged, directly or indirectly, to a portion of the inlet manifold. In this regard, the top plate may be engaged to provide a "closed" or "sealed" state, and may be removed to provide an "open" state. FIG. 3 shows, by way of example, a top view of the biological cartridge, with the inlet manifold top plate 25 in sealing engagement with the inactive (e.g., no PHB in this compartment) top end of the chamber housing 10. The inlet manifold includes three separate inlet ports 21, 22, 23. FIG. 4 shows the inner surface 26 of the top plate of FIG. 3, with the mounting element 15 attached thereto. As mentioned above, the mounting element may alternatively be attached to the inner surface of the bottom plate. According to some embodiments of the present invention, the biological cartridge may also include an outlet manifold including at least one outlet port in operative communication with the outlet orifice. For example, the at least one outlet port may include one to about five separate outlet ports, each in operative communication with an outlet orifice of the containment chamber. The outlet manifold may further include a bottom plate removably engaged, directly or indirectly, with a portion of the outlet manifold, similar to that described with respect to the top plate.

[0032] According to some embodiments of the present invention, PHB can provide continuous 3D growth of various cells and / or tissues. Hydrogels are insoluble polymer matrices that can be designed to retain up to 96% water content by mass (e.g., up to 40, 50, 60, 70, 80, 90, and 95% water content by mass). Various polymers can be used alone 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 pressure, chemical composition, and structure (e.g., pores and fibers of different shapes and sizes). Hydrogels can also be degradable or non-degradable. Hydrogels are versatile in that they can be used for a variety of applications (e.g., used in soft contact lenses to provide optics to correct the patient's vision). Hydrogels have been used in wound care applications as dressings and in life science applications as bioinks to create unique structural scaffolds for microfluidic experiments or to prepare substrates on or in which cells can be cultured. Most hydrogels are solid materials. However, by introducing spaces and microchannels into the hydrogel, it is possible to direct the movement of liquids, gases, and cells to expand the hydrogel and form a continuous substrate. Formation of hydrogels may be achieved by crosslinking any synthetic polymer, biopolymer, tissue component (extracted from human, animal, plant, or combination thereof) in the presence of water using free radical mediated reactions (e.g., photoreactions, chemical reactions) or reactions as a result of temperature changes, as described above.

[0033] PHBs (e.g., hydrogels), according to some embodiments of the invention, can be suitable for a variety of applications, including 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, soluble hydrogels, custom tissue formation, hosting of patient cells, custom configuration, modularity, and microenvironment manipulation.

[0034] According to some embodiments of the present invention, the PHB allows cells to grow in a more natural physiological environment compared to culturing in 2D plastic cell culture vessels. For example, the PHB can be tuned or configured to mimic the native tissue environment in which a particular cell develops and grows, unlike plastic cell culture vessels and other technologies that are not customizable or modular. Furthermore, the PHB can allow the formation of spheroids and organoids without the development of a necrotic core inside the microchannel network of the PHB. For example, the PHB according to some embodiments of the present invention allows spheroids and organoids to unravel and form tubes, cylinders, and other sophisticated structures where nutrients and gases can diffuse uniformly to cells within the PHB (e.g., hydrogel). Advantageously, for example, the PHB can allow uniform exchange of nutrients and gases for healthy cell growth, unlike other technologies that claim to mass-produce cells. According to some embodiments of the present invention, the PHB can be modified to suit a wide variety of cell types. According to some embodiments of the present invention, the PHB allows cells to secrete extracellular matrix to create a natural microenvironment that promotes cell growth, migration, survival, and function. Furthermore, the PHB advantageously eliminates the need for subculture of cells. Furthermore, the PHB is easy to use because it can be provided in a preformed format to be loaded into a containment chamber and does not require extensive modification of temperature, pH, or chemical exposure for use. The PHB can, for example, enable users to achieve one or more of the following: growing custom cell cultures, growing multiple cell types in parallel or sequentially, combining cell cultures to create complex tissues, mass-producing cells without stopping cell development, consistently producing cells from benchtop through clinical trials to industrial production using the same substrate, using less media and consumables than current technologies, and reducing or eliminating points of human intervention in cell production, thereby reducing human error and contamination risks.According to some embodiments of the present invention, a biological cartridge containing a PHB provides a modular platform for any of the above mentioned applications.

[0035] According to some embodiments of the present invention, the PHB has a three-dimensional (3D) macrostructure defined by a continuous polymer matrix material and a network of microporous channels and / or chambers extending throughout 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. At least one PHB has a PHB cross-section in an xy-plane, and the PHB cross-section may include a non-circular cross-section in the xy-plane, for example, a polygonal cross-section having 3-12 sides. Alternatively, at least one PHB has a PHB cross-section in an xy-plane, and the PHB cross-section includes a circular cross-section in the xy-plane. Additionally, at least one PHB has a PHB thickness in a z-direction perpendicular to the xy-plane. According to some embodiments of the invention, at least one PHB has a dimension ratio of the longest cross-sectional width to the thickness in the xy plane of about 1:5 to about 1:0.1, e.g., at least about any of 1:5, 1:2, 1:1.75, 1:1.5, 1:1.25, and 1:1, and / or at most about any of 1:0.1, 1:0.25, 1:0.5, 1:0.75, and 1:1. Additionally or alternatively, the 3D macrostructure defines a cylinder, a regular square prism, or a triangular prism. Additionally or alternatively, the 3D macrostructure defines a polygonal prism having 3 to 12 side edges, e.g., at least about any of 3, 4, 5, 6, 7, and 8 side edges, and / or at most about any of 12, 11, 10, 9, and 8 side edges.

[0036] According to some embodiments of the invention, as described above, the 3D macrostructure includes or defines a female mount component including a PHB orifice that extends through the entire thickness of the PHB, the female mount component being larger than the average diameter of the network of microporous channels and / or chambers. Additionally or alternatively, the 3D macrostructure includes or defines a female mount component including a PHB cutout portion that extends through the entire thickness of the PHB, the female mount component being larger than the average diameter of the network of microporous channels and / or chambers. According to some embodiments of the invention, the average diameter of the network of microporous channels and / or chambers is from about 100 to about 800 microns, e.g., at least about any of 100, 120, 150, 180, 200, 220, and 250 microns, and / or at most about any of 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.

[0037] According to some embodiments of the present invention, the continuous polymer matrix material may be non-degradable. In this regard, cells and / or tissues generated in the PHB contained in the containment chamber may need to be discharged from the internal network of microporous channels and / or chambers for further analysis, purification, or development. Additionally or alternatively, the continuous polymer matrix material may be selectively degradable. For example, the hydrogel formulation may be rendered biodegradable, for example, by inserting enzyme-sensitive sequences or utilizing natural matrix-derived compounds. For example, the continuous polymer matrix material may comprise a selectively degradable hydrogel material comprising one or more degradable polymers (e.g., one or more biopolymers of biological origin). The one or more biopolymers of biological origin may comprise, for example, polynucleotides, polysaccharides, polypeptides, or any combination thereof. According to some 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, which can be gently degraded, for example, by disrupting the biopolymer chains using protein-specific enzymes, ionic solvents, mild detergents, mild acids, or peroxides. According to some embodiments of the invention, the one or more biopolymers may include degradable monomers, including esters (e.g., hydroxybutyrate, lactic acid, glycolic acid, and caprolactone), anhydrides (e.g., adipic acid and sebacic acid), saccharides (e.g., cellulose, alginate, pectin, dextrin, chitosan, hyaluronic acid, chondroitin sulfate, and heparin), proteins, nucleotides (DNA, RNA), peptides (e.g., collagen, gelatin, silk, and fibrin), urethanes, phosphates, carbonates, and vinyl chlorides. According to some embodiments of the present invention, the selectively degradable hydrogel material may further comprise a synthetic polymer (eg, a polyester, a polyanhydride, a polycarbonate, a polyurethane, a polyphosphate, or a combination thereof).The continuous polymer matrix material, according to some embodiments of the present invention, may comprise a 3D crosslinked polymer network, a non-crosslinked polymer network, or a combination thereof.

[0038] The continuous polymer matrix material may, according to some embodiments of the present invention, comprise a swellable hydrogel material. The swellable hydrogel material may comprise at least a radical-mediated reaction product of a first monomer comprising an acrylate or methacrylate functional group and a second monomer or oligomer comprising at least two free-radically polymerizable functional groups. For example, the at least two free-radically polymerizable functional groups may, independently of each other, comprise 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 some embodiments of the present invention, at least one of the first monomer or the second monomer comprises a polyethylene glycol functional group (e.g., -O(C2H4O) n H, where n has a value of 1 to 100), polypropylene glycol functional groups (e.g., -O(CHO) nH, where n has a value from 1 to 100), and / or glycerol functional groups incorporated into the backbone of the monomer and / or grafted onto the monomer as a side chain or side chain component. 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 can be utilized in the hydrogel material according to some embodiments of the present invention can include polyolefins (e.g., ethylene, propylene), styrene, nylon (e.g., amide), and / or acrylic. According to some embodiments of the present invention, non-limiting examples of degradable monomers that can be used in the hydrogel material include esters (e.g., hydroxybutyrate, lactic acid, glycolic acid, caprolactone), anhydrides (e.g., adipic acid, sebacic acid), saccharides (e.g., cellulose, alginates, pectins, dextrins, chitosan, hyaluronic acid, 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 (e.g., plant-derived polymers (e.g., cellulose polymers) and animal-derived polymers).

[0039] According to some embodiments of the invention, the continuous polymer matrix material may mimic a natural tissue of interest by including one or more physical properties within about 20% (e.g., within about 15%, 10%, 8%, 5%, 3%, or 1%) of the natural tissue of interest, where the one or more physical properties of interest include softness and tensile strength. For example, the one or more physical properties may include elastic modulus and / or compressibility, storage modulus at 1 Hz, decrease in elastic modulus at 1 Hz, and / or protein / chemical coating (e.g., collagen type I, II, III, IV, laminin type I, II, hyaluronic acid, gelatin, fibrin, fibronectin, etc.). By way of example only, natural adipose tissue has a storage modulus at 1 Hz of 50-100 kPa, a decrease in elastic modulus at 1 Hz of 10-20 kPa, and an elastic modulus and / or compressibility of 3 kPa. In this regard, for example, the IPHB may have a storage modulus of about 110 kPa at 1 Hz, a modulus drop of about 22 kPa at 1 Hz, and a modulus and / or compressibility of 3 kPa. Analysis of the IPHB may be performed, for example, on a Dynamic Mechanical Analyzer (DMA) (TA Instruments RSA3) set up to evaluate mechanical and physical properties. A 5 mm biopsy punch may be used to isolate the circular hydrogel sample to avoid force concentration points. Dynamic mechanical analysis (DMA) may be performed by dynamic cylindrical compression analysis at a compression speed of 0.005 mm / sec and a frequency sweep of 1 Hz. For example, by modifying certain chemical components and / or the degree of crosslinking, one or more physical and / or mechanical properties of the resulting continuous polymer matrix material may be tailored to mimic and / or reflect the physical and / or mechanical properties associated with the native tissue of interest. Additionally or alternatively, it is possible to engineer the network of microporous channels and / or chambers and / or the outer surface topography / texture of the PHB.While most of these surfaces may be smooth, grooves, ridges, hills, depressions, and other surface irregularities may be introduced to alter the flow of liquid or gas through the network of microporous channels and / or chambers. Such surface irregularities may, for example, generate turbulence that helps to slow the flow of liquid or gas throughout the network of microporous channels and / or chambers. By way of example only, the surface irregularities may be significantly smaller in size than the average diameter of the network of microporous channels and / or chambers, for example, up to about ¼ to about 1 / 10 the size of the average diameter of the network of microporous channels and / or chambers.

[0040] According to some embodiments of the present invention, the continuous polymer matrix material is formed by additive manufacturing techniques (e.g., 3D printing or digital photosynthetic printing). In this regard, a network of microporous channels and / or chambers is constructed to mimic the morphology of the natural tissue of interest, for example, by modifying the geometry and dimensions of the network of microporous channels and / or chambers to reflect the morphology of the natural tissue of interest. For example, the morphology of the natural tissue of interest can be easily determined by those skilled in the art, and this morphology can be replicated by 3D printing or digital photosynthetic printing operations to form a PHB having a network of microporous channels and / or chambers that mimics the morphology of the natural tissue of interest.

[0041] According to some embodiments of the invention, the average diameter of the microporous channel and / or chamber network may be from about 100 to about 800 microns, for example, at least about any of 100, 120, 150, 180, 200, 220, and 250 microns, and / or at most about any of 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 occupy at least about 40% by volume of the 3D macrostructure, for example, at least about any of 40, 50, 60, and 70% by volume of the 3D macrostructure, and / or may occupy up to about any of 90, 85, 80, 75, and 70% by volume of the 3D macrostructure.

[0042] According to some embodiments of the present invention, the interface between the network of microporous channels and / or chambers and the continuous polymeric 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 comprising a compatibilizer may include a biological coating, such as collagen type I (e.g., human mesenchymal stem cells [from adipose, bone marrow, umbilical cord], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human myocytes, human osteoblasts, human bone cells, human chondrocytes, bovine myocytes, porcine hepatocytes, porcine chondrocytes, porcine osteocytes, equine muscle derived stem cells), laminin type I (e.g., human induced pluripotent stem cells, mouse dorsal root ganglia), hyaluronic acid (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 myocytes, human osteoblasts, human bone cells, human chondrocytes, bovine myocytes, porcine hepatocytes, porcine osteocytes, equine muscle derived stem cells), laminin type I (e.g., human induced pluripotent stem cells, mouse dorsal root ganglia), hyaluronic acid (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 ]human mesenchymal stem cells, human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human myocytes, human osteoblasts, human bone cells, human chondrocytes, human T cells (CD8+), human T cells (CD4+), human macrophages, bovine myocytes, 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.

[0043] In another aspect, some embodiments of the present invention provide a system including a plurality of biological cartridges (e.g., a plurality of biological cartridges as described and disclosed herein) connected in series to define a biological cartridge network (BCN). The BCN may include at least (i) a first biological cartridge having a first inlet and a first outlet, and (ii) a second biological cartridge having a second inlet and a second outlet (operably communicating with the first outlet). According to some embodiments of the present invention, each of the plurality of biological cartridges includes a plurality of PHBs seeded with a first cell type of interest housed therein. By way of example only, the BCN may include between 2 and about 50 biological cartridges, e.g., at least about any of 2, 5, 8, 10, 12, 15, 18, 20, 22, and 25 biological cartridges, and / or up to about any of 50, 45, 40, 35, 30, and 25 biological cartridges.

[0044] According to some embodiments of the invention, the BCN may include a fresh feed inlet and a product outlet. The fresh feed inlet may include a fresh feed manifold having one or more ports in operative communication with a first biological cartridge (e.g., as described above). The first biological cartridge may be the first biological cartridge of the BCN that receives fluid from the fresh feed manifold. Additionally or alternatively, one or more of the plurality of biological cartridges may include a respective inlet manifold operatively connected to the immediately preceding biological cartridge via a respective conduit. Each inlet manifold may include, for example, one or more make-up ports operatively configured with a respective one of the plurality of PHBs for the supply of make-up fluids. For example, make-up fluids (e.g., gases, nutrients, pH adjusting reagents, etc.) may be selectively added directly to one or more biological cartridges of the BCN without the need to pass these make-up fluids through each preceding biological cartridge.

[0045] According to some embodiments of the invention, the BCN also includes a shroud and the BNC is located within the shroud. The shroud may include, for example, a temperature control mechanism configured to control a temperature of a space within the shroud and outside the plurality of biological cartridges.

[0046] FIG. 7 illustrates an example system 100 including a BCN 90, in which fresh feed material flows sequentially through a first biological cartridge 1a, a second biological cartridge 1b, and then a third biological cartridge 1c before exiting the system. Each of the biological cartridges 1a, 1b, and 1c may be seeded with the same cell type of interest. Such a system utilizing a serially interconnected configuration may be particularly suitable for mass culture of a cell type of interest. Although FIG. 7 illustrates only three biological cartridges in series, some embodiments of the invention may include from about 2 to about 100 serially interconnected biological cartridges, for example, at least about any of 2, 5, 8, 10, 12, 15, 18, 20, 30, 40, and 50 serially interconnected biological cartridges, and / or up to about any of 100, 90, 80, 70, 60, and 50 serially interconnected biological cartridges.

[0047] In another aspect, some embodiments of the present invention provide a system including a biological cartridge network (BCN), the BCN including (i) a first set of biological cartridges including a first plurality of biological cartridges (e.g., biological cartridges as described and disclosed herein) connected in series with each other, and (ii) a second set of biological cartridges including a second plurality of biological cartridges (e.g., biological cartridges as described and disclosed herein) connected in series with each other. The first set of biological cartridges and the second set of biological cartridges may be arranged in a parallel relationship with each other and with a common feedstock source. According to some embodiments of the present invention, the first biological cartridge of each of the first and second sets of biological cartridges may be seeded with a first cell type of interest, and the immediately succeeding biological cartridge of each may be seeded with a second cell type of interest, the first cell type and the second cell type being different.

[0048] FIG. 8 shows, by way of example, a system 105 including a BCN 90 having a first and second set of biological cartridges (e.g., first set=1a, 2a, 3a, second set=1b, 2b, 3b) arranged in a parallel relationship with respect to each other and to a common feedstock source. As shown in FIG. 8, the first biological cartridges 1a, 1b of the first and second sets of biological cartridges may each be seeded with a first cell type of interest, the immediately succeeding biological cartridges 2a, 2b may each be seeded with a second cell type of interest, and the immediately succeeding biological cartridges 3a, 3b may each be seeded with a third cell type of interest. The first cell type, the second cell type, and the third cell type may all be different from each other.

[0049] According to some embodiments of the invention, the BCN may include at least three sets of biological cartridges connected in series with each other, for example, at least about any of 3, 5, 8, 10, 15, and 20 such sets, and / or up to about any of 100, 90, 80, 70, 60, 50, 40, 30, and 20 such sets.

[0050] According to some embodiments of the invention, one or more of the first and / or second plurality of biological cartridges include a respective inlet manifold operatively connected to a preceding biological cartridge via a respective conduit. Each inlet manifold may include one or more make-up ports operatively configured with a respective one of the plurality of PHBs for delivery of make-up fluid, for example, as described above. The system may also include a shroud, with the BNC located within the shroud, as described above.

[0051] 9 illustrates another example system 107 including a BCN 90 in which a first set of biological cartridges 1a, 2 (in a series configuration) are arranged in parallel with a second set of biological cartridges 1b, 3 (in a series configuration). In this regard, the first biological cartridges 1a, 1b of each of the first and second sets of biological cartridges may each be seeded with a first cell type of interest, while the immediately succeeding biological cartridges 2, 3 may each be seeded with a different cell type of interest. Biological cartridge 2 may be seeded with a second cell type of interest and biological cartridge 3 may be seeded with a third cell type of interest, where the first cell type, second cell type, and third cell type may all be different from each other.

[0052] In another aspect, some embodiments of the present invention provide a system including a biological cartridge network (BCN) including a plurality of biological cartridges (e.g., a plurality of biological cartridges as described and disclosed herein), the plurality of biological cartridges including (i) a first plurality of biological cartridges operatively connected in series, and (ii) a second plurality of biological cartridges operatively connected in parallel. In this regard, the BCN may include an almost unlimited number of individual biological cartridges in an almost unlimited number of different overall configurations (e.g., various serial and / or parallel interconnections of biological cartridges, where any number of biological cartridges are seeded with a wide variety of cell types of interest). According to some embodiments of the present invention, the BCN may include at least one fresh feed inlet (e.g., 1 to about 10 fresh feed inlets) and at least one product outlet (e.g., 1 to about 10 product outlets).

[0053] FIG. 10 shows, by way of example, a system 103 including a BCN 90 that includes multiple individual biological cartridges 1, 2a, 2b, 3a, 3b, 4a, 4b, 5, 6 in an overall configuration utilizing both serial interconnections between some of the biological cartridges as well as parallel arrangements between some of the biological cartridges. The system 103 of FIG. 10 shows one fresh feed and three product outlets. According to some embodiments of the invention, biological cartridge 1 is seeded with a first cell type, biological cartridges 2a, 2b (arranged in parallel) are seeded with a second cell type, biological cartridges 3a, 3b (arranged in series) are seeded with a third cell type, biological cartridges 4a, 4b are seeded with a fourth cell type, biological cartridge 5 is seeded with a fifth cell type, and biological cartridge 6 is seeded with a sixth cell type.

[0054] The above example configurations of multiple biological cartridges are merely illustrative of some possible systems according to some embodiments of the present invention, i.e., the above example configurations and drawings of multiple biological cartridges are illustrative only and non-limiting.

[0055] In another aspect, some embodiments of the present invention provide a method of culturing one or more cell types of interest, the method comprising the steps of: (i) providing a first biological cartridge (e.g., a biological cartridge as described and disclosed herein), the first biological cartridge containing a first plurality of PHBs; (ii) seeding the first plurality of PHBs with at least a first cell type of interest; (iii) providing a first medium to the at least first cell type of interest and allowing the at least first cell type of interest to grow throughout a first network of microporous channels and / or chambers of the first plurality of PHBs; and (iv) harvesting at least a portion of the first cell type of interest located throughout the first network of microporous channels and / or chambers of the plurality of PHBs.

[0056] According to some embodiments of the invention, harvesting at least a portion of the first cell type of interest located throughout the first network of microporous channels and / or chambers of the first plurality of PHBs comprises flushing them from the first plurality of PHBs with a fluid medium. Alternatively, harvesting at least a portion of the first cell type of interest located throughout the first network of microporous channels and / or chambers of the first plurality of PHBs comprises disintegrating each 3D macrostructure of each of the first plurality of PHBs.

[0057] According to some embodiments of the invention, the method may further include providing a second biological cartridge (e.g., a biological cartridge as described and disclosed herein), the second biological cartridge containing a second plurality of PHBs seeded with a first cell type of interest, and operatively connecting the first biological cartridge and the second biological cartridge in series. In this regard, a fluid stream exiting the first biological cartridge may be provided to the second biological cartridge. For example, a medium provided to the first biological cartridge may pass through the first biological cartridge, enter the second biological cartridge, and pass through the second biological cartridge.

[0058] According to some embodiments of the invention, the method may further include providing a second biological cartridge (e.g., a biological cartridge as described and disclosed herein) containing a second plurality of PHBs seeded with a first cell type of interest, operatively connecting the first biological cartridge and the second biological cartridge in series, and optionally providing a second medium to the second cell type of interest and allowing the second cell type of interest to grow throughout the second network of microporous channels and / or chambers of the second plurality of PHBs. According to some embodiments of the invention, the method may further include harvesting at least a portion of the second cell type of interest located throughout the second network of microporous channels and / or chambers of the second plurality of PHBs. Harvesting at least a portion of the second cell type of interest located throughout the second network of microporous channels and / or chambers of the second plurality of PHBs comprises flushing them from the second plurality of PHBs with a fluid medium. Alternatively, harvesting at least a portion of the second cell type of interest located throughout the second network of microporous channels and / or chambers of the second plurality of PHBs comprises disintegrating each 3D macrostructure of each of the second plurality of PHBs.

[0059] In another aspect, some embodiments of the invention provide a method of culturing one or more cell types of interest, the method comprising the steps of: (a) providing or forming a biological cartridge network (BCN), the BCN comprising: (i) a first set of biological cartridges comprising a first plurality of biological cartridges (e.g., biological cartridges as described and disclosed herein) connected in series with each other; and (ii) a second set of biological cartridges comprising a second plurality of biological cartridges (e.g., biological cartridges as described and disclosed herein) connected in series with each other, the first set of biological cartridges and the second set of biological cartridges being arranged in a parallel relationship with respect to each other and with respect to a common feedstock source; and (b-1) providing or forming a BCN comprising: a first set of biological cartridges comprising a first plurality of biological cartridges (e.g., biological cartridges as described and disclosed herein) connected in series with each other; or (b-2) seeding each PHB in a first biological cartridge of each of the first and second sets of biological cartridges with a first cell type of interest, seeding the immediately succeeding biological cartridge of the first set of biological cartridges with a second cell type of interest, and seeding the immediately succeeding biological cartridge of the second set of biological cartridges with a third cell type of interest, wherein the first cell type, the second cell type, and the third cell type are all different from one another; and (c) harvesting at least a portion of the first cell type of interest, and / or at least a portion of the second cell type of interest, and / or at least a portion of the third cell type of interest. According to some embodiments of the invention, the method may further comprise the step of providing a second culture medium to a second cell type of interest and providing a third culture medium to a third cell type of interest.

[0060] According to some embodiments of the present invention, as described above, one or more cell types of interest may produce or secrete a therapeutic agent (e.g., a biologic) of interest. For example, the therapeutic agent may be 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, to prepare a BCN, multiple biological cartridges of various configurations may be interconnected in various configurations, and multiple cell lines (e.g., cell lines that can produce or secrete a therapeutic agent) are seeded on separate PHBs housed in separate biological cartridges.

[0061] According to some embodiments of the invention, a PHB seeded with a first cell type of interest and contained in a first biological cartridge may have a first network of microporous channels and / or chambers having a first structure, and a PHB seeded with a second cell type of interest and contained in a second biological cartridge may have a second network of microporous channels and / or chambers, the first structure and the second structure being different. As described above, the PHB of each biological cartridge seeded with a particular cell type of interest is physically constructed to mimic the native tissue of interest. By way of example, the first network of microporous channels and / or chambers, the second network of microporous channels and / or chambers, or both, independently of one another, may have an average diameter of from about 100 to about 800 microns, e.g., at least about any of 100, 120, 150, 180, 200, 220, and 250 microns, and / or at most about any of 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 first network of microporous channels and / or chambers, the second network of microporous channels and / or chambers, or both, may, independently of one another, occupy at least about 40% by volume of the respective 3D macrostructure, such as at least about any of 40, 50, 60, and 70% by volume of the respective 3D macrostructure, and / or may occupy up to about any of 90, 85, 80, 75, and 70% by volume of the respective 3D macrostructure.

[0062] According to some embodiments of the invention, the first PHB (contained in the first biological cartridge) may comprise a first continuous polymeric matrix material of the first PHB, which comprises a non-degradable hydrogel material or a selectively degradable hydrogel material (e.g., as described above). Additionally or alternatively, the second PHB (contained in the second biological cartridge) may comprise a second continuous polymeric matrix material of the second PHB, which comprises a non-degradable hydrogel material or a selectively degradable hydrogel material (e.g., as described above). According to some embodiments of the invention, the first continuous polymeric matrix material of the first PHB, the second continuous polymeric matrix material of the second IPHB, or both, comprise a selectively degradable hydrogel material, which comprises a degradable polymer (e.g., naturally occurring biopolymers such as collagen, hyaluronic acid, gelatin, and nucleic acids). These types of materials, or combinations of these materials, are degradable by corresponding enzymes (e.g., collagenase (e.g., effective for collagen and gelatin), dispase, pepsin, hyaluronase, and deoxyribonuclease / ribonuclease). The selectively degradable hydrogel material may, for example, include one or more degradable polymers (e.g., one or more biopolymers of biological origin (as described above). For example, the one or more biopolymers of biological origin may include polynucleotides, polysaccharides, polypeptides, or any combination thereof. According to some 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, which can be gently degraded, for example, by disrupting the biopolymer chains using protein-specific enzymes, ionic solvents, mild detergents, weak acids, or peroxides.According to some embodiments of the invention, the one or more biopolymers comprise degradable monomers, including esters (e.g., hydroxybutyrate, lactic acid, glycolic acid, and caprolactone), anhydrides (e.g., adipic acid, and sebacic acid), saccharides (e.g., cellulose, alginates, pectins, dextrins, chitosan, hyaluronic acid, chondroitin sulfate, and heparin), proteins, nucleotides (DNA, RNA), peptides (e.g., collagen, gelatin, silk, and fibrin), urethanes, phosphates, carbonates, and vinyl chlorides. According to some embodiments of the invention, the selectively degradable hydrogel material further comprises a synthetic polymer (e.g., polyesters, polyanhydrides, polycarbonates, polyurethanes, polyphosphates, or combinations thereof). According to some embodiments of the invention, the hydrogel material may comprise at least 50% by weight of one or more degradable polymers (e.g., one or more biopolymers of biological origin), e.g., at least about 50, 60, 70, and 75% by weight, and / or up to about any of 100, 95, 90, 85, 80, and 75% by weight. In this regard, cells cultured according to some methods of the invention can be readily harvested and expelled from the respective biological cartridge by degrading or dissolving the hydrogel material of the PHB.

[0063] These and other modifications and variations to the present invention may be implemented by those skilled in the art without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. Moreover, it will be understood that aspects of the various embodiments may be interchanged in whole or in part. Moreover, those skilled in the art will appreciate that the above description is merely illustrative 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 exemplary description of the forms contained herein.

Claims

1. a containment chamber defining an interior region, the containment chamber including an inlet orifice and an outlet orifice; at least one porous hydrogel block (PHB) located within the interior region of the containment chamber; A biological cartridge comprising:

2. The biological cartridge of claim 1, wherein the containment chamber has a cross-section in an xy plane, the cross-section including a non-circular cross-section in the xy plane, for example a polygonal cross-section having 3 to 12 sides.

3. 2. The biological cartridge of claim 1, wherein the containment chamber has a cross-section in an xy plane, the cross-section comprising a circular cross-section in the xy plane.

4. A biological cartridge according to any one of claims 2 to 3, wherein the cross-section is the same over the entire working length of the containment chamber.

5. The biological cartridge of any one of claims 1 to 4, wherein the at least one PHB comprises a plurality of PHBs stacked vertically on top of each other in a z-direction perpendicular to the xy plane.

6. The biological cartridge of claim 5, wherein the plurality of PHBs comprises from 2 to about 40 individual PHBs, for example, at least about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, and 20 individual PHBs, and / or up to about any of 40, 35, 30, 25, and 20 individual PHBs.

7. The biological cartridge of any one of claims 5 to 6, further comprising at least one mounting element extending throughout said interior region.

8. The biological cartridge of claim 7 , wherein the at least one mounting element comprises a single mounting element aligned along a central axis of the containment chamber and extending along the entire working length of the containment chamber.

9. The biological cartridge of claim 7, further comprising a top plate and / or a bottom plate removably engaged directly or indirectly to the storage chamber, and wherein the single mounting element may optionally be attached directly or indirectly to the top plate and / or the bottom plate.

10. A biological cartridge as described in any one of claims 8 to 9, wherein the multiple PHBs stacked vertically on one another include respective female mount components including respective PHB orifices that extend completely through respective thicknesses in the z-direction of the respective PHBs, and the respective female mount components are configured to allow the at least one mount element to extend through the respective PHB orifices.

11. The biological cartridge of claim 10, wherein the at least one mounting element may have a non-circular cross-section and each of the PHB orifices has a corresponding non-circular cross-section, and each of the vertically stacked PHBs is restrained from rotating relative to each other in their respective x-y planes.

12. The biological cartridge of claim 7 , wherein the at least one mounting element comprises at least one fin component attached to the containment chamber and projecting inwardly into the interior region of the containment chamber.

13. The biological cartridge of claim 12, wherein at least one fin component extends along at least a majority of the entire working length of the storage chamber, e.g., extending along at least about 55, 60, 65, and 75% of the entire working length of the storage chamber, and / or extending along at most about any of 100, 98, 95, 90, 85, 80, and 75% of the entire working length of the storage chamber.

14. A biological cartridge as described in any one of claims 12 to 13, wherein the multiple PHBs stacked vertically on one another include respective female mount components including respective PHB cut-out portions extending completely through respective thicknesses in the z-direction of the respective PHBs, the respective female mount components being configured to allow the at least one mount element to extend through the respective PHB orifice.

15. The biological cartridge of any one of claims 7 to 14, wherein the at least one mounting element comprises a transparent and rigid substrate (e.g., glass or plastic), a conductive metal (e.g., copper, silver, or gold), or a hydrogel material different from the hydrogel material of the PHB.

16. A biological cartridge as described in any one of claims 1 to 15, further comprising an inlet dispersion filter disposed directly or indirectly over all of the at least one PHB, wherein the incoming fluid passes through the inlet dispersion filter before entering any of the at least one PHB.

17. 17. A biological cartridge as described in any one of claims 1 to 16, further comprising an exhaust filter disposed directly or indirectly below all of said at least one PHB, wherein exiting fluid passes through said exhaust filter before exiting said biological cartridge.

18. 18. The biological cartridge of any one of claims 1 to 17, further comprising an inlet manifold including at least one inlet port in operative communication with the inlet orifice of the containment chamber.

19. 20. The biological cartridge of claim 18, wherein said at least one inlet port comprises from 1 to about 5 individual inlet ports, each in operative communication with said inlet orifice of said containment chamber.

20. The biological cartridge of any one of claims 18 to 19, wherein the inlet manifold further comprises a top plate removably engaged, directly or indirectly, to a portion of the inlet manifold.

21. 21. The biological cartridge of any one of claims 1 to 20, further comprising an outlet manifold including at least one outlet port in operative communication with said outlet orifice.

22. 22. The biological cartridge of claim 21, wherein said at least one exit port comprises from 1 to about 5 individual exit ports, each in operative communication with said exit orifice of said containment chamber.

23. 23. The biological cartridge of any one of claims 21-22, wherein the outlet manifold further comprises a bottom plate removably engaged, directly or indirectly, to a portion of the outlet manifold.

24. The biological cartridge of any one of claims 1 to 24, wherein the at least one PHB has a three-dimensional (3D) macrostructure defined by a continuous polymeric matrix material and the network of microporous channels and / or chambers extending throughout the continuous polymeric matrix material.

25. The biological cartridge of claim 24, wherein the at least one PHB has a PHB cross-section in an xy plane, the PHB cross-section comprising a non-circular cross-section in the xy plane, for example comprising a polygonal cross-section having 3 to 12 sides.

26. 25. The biological cartridge of claim 24, wherein said at least one PHB has a PHB cross-section in an xy plane, said PHB cross-section comprising a circular cross-section in said xy plane.

27. A biological cartridge according to any one of claims 24 to 25, wherein the at least one PHB has a PHB thickness in a z-direction perpendicular to the xy plane.

28. The biological cartridge of claim 27, wherein the at least one PHB has a dimensional ratio of its longest cross-sectional width in the xy plane to its thickness of about 1:5 to about 1:0.1, for example at least about any of 1:5, 1:2, 1:1.75, 1:1.5, 1:1.25, and 1:1, and / or at most about any of 1:0.1, 1:0.25, 1:0.5, 1:0.75, and 1:

1.

29. A biological cartridge according to any one of claims 24 to 28, wherein the 3D macrostructure defines a cylinder, a regular square prism, or a triangular prism.

30. The biological cartridge of any one of claims 24 to 28, wherein the 3D macrostructure defines a polygonal prism having between 3 and 12 side edges, for example at least about 3, 4, 5, 6, 7, and 8 side edges, and / or up to about any of 12, 11, 10, 9, and 8 side edges.

31. A biological cartridge as described in any one of claims 24 to 30, wherein the 3D macrostructure includes or defines the female mount component including the PHB orifice extending throughout the thickness of the PHB, the female mount component being larger than the average diameter of the network of microporous channels and / or chambers.

32. A biological cartridge as described in any one of claims 24 to 30, wherein the 3D macrostructure includes or defines the female mount component including the PHB cut-out portion extending throughout the thickness of the PHB, and the female mount component is larger than the average diameter of the network of microporous channels and / or chambers.

33. The biological cartridge of any one of claims 24 to 32, wherein the continuous polymeric matrix material is non-degradable.

34. The biological cartridge of any one of claims 24 to 32, wherein the continuous polymeric matrix material is selectively degradable.

35. The biological cartridge of any one of claims 24 to 34, wherein the continuous polymeric matrix material comprises a 3D crosslinked polymer network, a non-crosslinked polymer network, or a combination thereof.

36. The biological cartridge of any one of claims 24 to 35, wherein the continuous polymeric matrix material comprises a swellable hydrogel material.

37. 37. The biological cartridge of claim 36, wherein the swellable hydrogel material comprises a radical-mediated reaction product of at least a first monomer comprising an acrylate or methacrylate functional group and a second monomer or oligomer comprising at least two free-radically polymerizable functional groups.

38. 38. The biological cartridge of claim 37, wherein the at least two free radically polymerizable functional groups may, independently of each other, comprise an acrylate or methacrylate group, an allyl group, an alkynyl, 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.

39. At least one of the monomer or the second monomer has a polyethylene glycol functional group (e.g., —O(C ２ H ４ O) ｎ H, where n has a value of 1 to 100; polypropylene glycol functional groups (e.g., -O(C ３ H ６ O) ｎ H, where n has a value from 1 to 100), and / or a glycerol functional group incorporated in the backbone of said monomer and / or grafted onto said monomer as a side chain or component of a side chain.

40. 40. The biological cartridge of any one of claims 37 to 39, wherein at least one of the monomer or the second monomer comprises 2-hydroxyethyl acrylate (HEA), poly(ethylene glycol) methyl ether acrylate (MPEGA), N-methylacetamide (NMA), or poly(ethylene glycol) diacrylate (PEGDA).

41. 41. The biological cartridge of claim 40, wherein the swellable hydrogel material comprises one or more natural polymers (e.g., plant-derived polymers and animal-derived polymers).

42. The biological cartridge of any one of claims 24 to 41, wherein the continuous polymer matrix material mimics a natural tissue of interest by comprising one or more physical properties within about 20% (e.g., within about 15%, 10%, 8%, 5%, 3%, or 1%) of the natural tissue of interest, the one or more physical properties of interest including softness and tensile strength.

43. The biological cartridge of any one of claims 24 to 42, wherein the continuous polymer matrix material is formed by an additive manufacturing technique (e.g. 3D printing or digital photosynthetic printing).

44. A biological cartridge according to any one of claims 24 to 43, wherein the network of microporous channels and / or chambers is constructed to mimic the morphology of the native tissue of interest, for example by modifying the geometry and dimensions of the network of microporous channels and / or chambers to reflect the morphology of the native tissue of interest.

45. 45. The biological cartridge of any one of claims 24 to 44, wherein the average diameter is from about 100 to about 800 microns, for example at least about any of 100, 120, 150, 180, 200, 220, and 250 microns, and / or at most about any of 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.

46. 46. ​​A biological cartridge according to any one of claims 24 to 45, wherein the network of microporous channels and / or chambers occupies at least about 40% by volume of the 3D macrostructure, such as at least about any of 40, 50, 60 and 70% by volume of the 3D macrostructure, and / or occupies up to about any of 90, 85, 80, 75 and 70% by volume of the 3D macrostructure.

47. A biological cartridge according to any one of claims 24 to 46, wherein the interface between the network of microporous channels and / or chambers and the continuous polymeric matrix material comprises a coating of a compatibilizer selected to promote adhesion of primary cells of interest.

48. 50. A plurality of biological cartridges according to any one of claims 1 to 47, connected in series with one another to define a biological cartridge network (BCN), said BCN including at least: (i) a first biological cartridge having a first inlet and a first outlet; and (ii) a second biological cartridge having a second inlet and a second outlet (in operative communication with the first outlet). Including, each of said plurality of biological cartridges includes a plurality of PHBs seeded with a first cell type of interest housed therein; system.

49. 49. The system of claim 48, wherein the BCN includes a fresh feed inlet and a product outlet.

50. The system of any one of claims 48 to 49, wherein the BCN comprises from 2 to about 50 biological cartridges, for example, at least about any of 2, 5, 8, 10, 12, 15, 18, 20, 22, and 25 biological cartridges, and / or up to about any of 50, 45, 40, 35, 30, and 25 biological cartridges.

51. The system of any one of claims 48 to 50, wherein the fresh feed inlet includes a fresh feed manifold having one or more ports in operative communication with the first biological cartridge, the first biological cartridge being a first biological cartridge of the BCN that receives fluid from the fresh feed manifold.

52. The system of any one of claims 48 to 51, wherein said one or more of said plurality of biological cartridges includes a respective inlet manifold operatively connected to a preceding biological cartridge via a respective conduit.

53. 53. The system of claim 52, wherein the respective inlet manifold includes one or more make-up ports operatively configured with the respective plurality of PHBs for the supply of make-up fluid.

54. The system of any one of claims 48 to 53, further comprising a shroud, the BNC being located within the shroud.

55. 55. The system of claim 54, wherein the shroud includes a temperature control mechanism configured to control a temperature of a space within the shroud and outside the plurality of biological cartridges.

56. 10. A biological cartridge network (BCN) comprising: (i) a first set of biological cartridges comprising a first plurality of biological cartridges according to any one of claims 1 to 47 connected in series with each other; and (ii) a second set of biological cartridges comprising a second plurality of biological cartridges according to any one of claims 1 to 47 connected in series with each other, wherein the first set of biological cartridges and the second set of biological cartridges are arranged in a parallel relationship with each other and with respect to a common feedstock source. A system including:

57. 57. The system of claim 56, wherein an initial biological cartridge of each of the first and second sets of biological cartridges is seeded with a first cell type of interest and each immediately succeeding biological cartridge is seeded with a second cell type of interest, the first cell type and the second cell type being different.

58. 57. The system of claim 56, wherein a first biological cartridge of each of the first and second sets of biological cartridges is seeded with a first cell type of interest, a successive biological cartridge of the first set of biological cartridges is seeded with a second cell type of interest, and a successive biological cartridge of the second set of biological cartridges is seeded with a third cell type of interest, wherein the first cell type, the second cell type, and the third cell type are all different from one another.

59. The system of any one of claims 56 to 58, wherein the BNC comprises at least three sets of biological cartridges connected in series with each other, for example, at least about any of 3, 5, 8, 10, 15, and 20 of said sets, and / or up to about any of 100, 90, 80, 70, 60, 50, 40, 30, and 20 of said sets.

60. The system of any one of claims 56 to 59, wherein one or more of the first plurality of biological cartridges and / or the second plurality of biological cartridges include a respective inlet manifold operatively connected to a preceding biological cartridge via a respective conduit.

61. 61. The system of claim 60, wherein the respective inlet manifold includes one or more make-up ports operatively configured with the respective plurality of PHBs for the supply of make-up fluid.

62. The system of any one of claims 56 to 61, further comprising a shroud, the BNC being located within the shroud.

63. 63. The system of claim 62, wherein the shroud includes a temperature control mechanism configured to control a temperature of a space within the shroud and outside the plurality of biological cartridges.

64. A system comprising a biological cartridge network (BCN) comprising a plurality of biological cartridges described in any one of claims 1 to 47, wherein the plurality of biological cartridges comprises: (i) a first plurality of biological cartridges operatively connected in series; and (ii) a second plurality of biological cartridges operatively connected in parallel.

65. 65. The system of claim 64, wherein the BCN comprises at least one fresh feed inlet (e.g., 1 to about 10 fresh feed inlets) and at least one product outlet (e.g., 1 to about 10 product outlets).

66. 1. A method of culturing one or more cell types of interest, comprising the steps of: (i) providing a first biological cartridge according to any one of claims 1 to 47, said first biological cartridge containing a first plurality of PHBs seeded with a first cell type of interest; (ii) seeding said first plurality of PHBs with at least a first cell type of interest; (iii) providing a first culture medium to the at least a first cell type of interest and allowing the at least a first cell type of interest to grow throughout the first network of microporous channels and / or chambers of the first plurality of PHBs; (iv) harvesting at least a portion of the first cell type of interest located throughout the first network of microporous channels and / or chambers of the plurality of PHBs; The method includes:

67. 67. The method of claim 66, wherein the step of harvesting at least a portion of the first cell type of interest located throughout the first network of microporous channels and / or chambers of the first plurality of PHBs comprises vigorously flushing them from the first plurality of PHBs with a fluid medium.

68. 67. The method of claim 66, wherein the step of harvesting at least a portion of the first cell type of interest located throughout the first network of microporous channels and / or chambers of the first plurality of PHBs comprises disintegrating the 3D macrostructure of each of the first plurality of PHBs.

69. The method of any one of claims 66 to 68, further comprising the steps of: providing a second biological cartridge as described in any one of claims 1 to 47, the second biological cartridge containing a second plurality of PHBs seeded with the first cell type of interest; and operatively connecting the first biological cartridge and the second biological cartridge in series.

70. 69. The method of any one of claims 66 to 68, further comprising the steps of: providing a second biological cartridge according to any one of claims 1 to 47, the second biological cartridge containing a second plurality of PHBs seeded with a second cell type of interest; and operatively connecting the first biological cartridge and the second biological cartridge in series, optionally supplying a second culture medium to the second cell type of interest, and allowing the second cell type of interest to grow throughout the second network of microporous channels and / or chambers of the second plurality of PHBs.

71. 71. The method of claim 70, further comprising the step of harvesting at least a portion of the second cell type of interest located throughout the second network of microporous channels and / or chambers of the second plurality of PHBs.

72. 72. The method of claim 71 , wherein harvesting at least a portion of the second cell type of interest located throughout the second network of microporous channels and / or chambers of the second plurality of PHBs comprises vigorously flushing them from the second plurality of PHBs with a fluid medium.

73. 72. The method of claim 71 , wherein the step of harvesting at least a portion of the second cell type of interest located throughout the second network of microporous channels and / or chambers of the second plurality of PHBs comprises disintegrating the 3D macrostructure of each of the second plurality of PHBs.

74. 1. A method of culturing one or more cell types of interest, comprising the steps of: (a) providing or forming a biological cartridge network (BCN), the BCN comprising: (i) a first set of biological cartridges comprising a first plurality of biological cartridges according to any one of claims 1 to 47 connected in series with each other; and (ii) a second set of biological cartridges comprising a second plurality of biological cartridges according to any one of claims 1 to 47 connected in series with each other, the first set of biological cartridges and the second set of biological cartridges being arranged in a parallel relationship with respect to each other and to a common feedstock source; (b-1) seeding each PHB in a first biological cartridge of each of the first and second sets of biological cartridges with a first cell type of interest and seeding each immediately succeeding biological cartridge with a second cell type of interest, wherein the first cell type and the second cell type are different; or (b-2) seeding each PHB in a first biological cartridge of each of the first and second sets of biological cartridges with a first cell type of interest, seeding the immediately succeeding biological cartridge of the first set of biological cartridges with a second cell type of interest, and seeding the immediately succeeding biological cartridge of the second set of biological cartridges with a third cell type of interest, wherein the first cell type, the second cell type, and the third cell type are all different from one another; (c) harvesting at least a portion of the first cell type of interest, and / or at least a portion of the second cell type of interest, and / or at least a portion of the third cell type of interest; The method includes:

75. 75. The method of claim 74, further comprising providing a second culture medium to the second cell type of interest.

76. 76. The method of any one of claims 74-75, further comprising the step of providing a third culture medium to said third cell type of interest.

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