Honeycomb scaffold bioreactors for mass cell production

Abstract

A bioreactor support structure and bioreactor system for mass cell production is provided, including for cultivated meat production and for mass production of stem cells. The bioreactor support structure has an outer wall and a plurality of inner walls inside the interior space created by the inner edge of the outer wall. The plurality of inner walls is configured to make channels that house polymeric scaffolds, cells, and fluid. The cross sections of the channels may have an approximately, circular, elliptical, or polygonal cross-sectional area. The configuration of the bioreactor support structure solves the problem of high cell density pressures that hinder large-scale and commercial mass cell production.

Description

HONEYCOMB SCAFFOLD BIOREACTORS FOR MASS CELL PRODUCTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63 / 538,153 filed on September 13, 2023, the content of which is relied upon and incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] A sequence listing in computer readable form, having file name SP23- 254_Sequence_Listing.xml, created September 05, 2024 (8997 bytes) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates to mass cell production (including for cultivated meat and stem cells), bioreactor support structures for mass cell production, and bioreactor systems for mass cell production.BACKGROUND

[0004] Scaffolds used in tissue engineering have been proposed and used for small-scale cell productions. However, the currently available scaffolds for mass cell production (including for cultivated meats and stem cells) are unsuitable for large-scale or commercial scale productions because when scaled, these scaffolds cannot withhold the increasing back pressure over perfusion culture arising from the relatively weak mechanical strength of the highly porous structures. In such back pressure conditions, the scaffolds experience unwanted local compaction, nesting, and even collapse, which, in turn, can lead to poor accessibility to nutrition and dissolved oxygen, and eventually unhealthy cell growth. The increasing back pressure over perfusion culture is a direct result of the reduced porosity of these scaffolds as cell density increases over culture. The present disclosure solves the problem of large-scale or commercial production of cells by improving thestability of polymeric scaffolds during the large-scale or commercial production process with improved supporting structures for the polymeric scaffolds.SUMMARY

[0005] According to some aspects of the present disclosure, an improved bioreactor system for mass cell production (including for cultivated meats and stem cells) is provided. The bioreactor system comprises a first end portion and a second end portion, and at least one support structure comprising a solid outer wall, a plurality of inner walls, a plurality of channels, and polymeric scaffolds. The plurality of channels provides void spaces to host the polymeric scaffolds while the plurality of inner walls provides physical structure to support and constrain the polymeric scaffolds within the plurality of channels. The bioreactor system can culture cells having cell densities up to about 1,000,000 cells / cm2In one embodiment, the bioreactor system is configured to culture cells having densities from about 500,000 cells / cm2to about 1,000,000 cells / cm2. The bioreactor system also cultures cell densities less than about 500,000 cells / cm2. In another embodiment, the bioreactor system cultures cells starting from cell seeding densities of about 2,000 cells / cm2to about 30,000 cells / cm2. In some embodiments, the bioreactor system further comprises a first fluid handling unit at the first end portion of the bioreactor system and a second fluid handling unit at the second end portion. The first fluid handling unit comprises a first port and a first fluid flow guide plate. The second fluid handling unit comprises a second port and a second fluid flow guide plate. The fluid flow guide plate is used to distribute liquid solutions (e.g., media, buffer) evenly once the solution passes through the first port.

[0006] According to other aspects of the present disclosure, an improved bioreactor system for mass cell production is provided. The bioreactor system has a support structure comprising an outer wall having an inner edge, an inner space having an outer edge defined by the inner edge of the outer wall, and a plurality of inner walls configured to create channels in the void spaces between the inner walls. The bioreactor system houses polymeric scaffolds inside the channels of the support structure. The bioreactor system further has a first fluid handling unit comprising a first port and a first fluid flow guide plate. The bioreactor system also has a second fluid handling unit comprising a second port, and a second fluid flow guide plate.

[0007] According to other aspects of the present disclosure, an improved bioreactor system for mass cell production is provided. The bioreactor system comprises a first end portion and a second end portion, at least one support structure comprising a solid outer wall, a plurality of inner walls, a plurality of channels, and polymeric scaffolds. The plurality of channels are void spaces among the inner walls and the polymeric scaffolds are within the channels. In one aspect, the support structure of the bioreactor system withstands a pressure of at least 1,000 Pa without permanent deformation. In other aspects, the support structure of the bioreactor system withstands a pressure of at least 10,000 Pa without permanent deformation. In yet other aspects, the support structure of the bioreactor system withstands a pressure of at least 30,000 Pa without permanent deformation.

[0008] In any of the bioreactor systems of the present disclosure, the plurality of channels of the support structure can be any shape. In some embodiments, the cross-sectional area of each channel is, approximately, one of a circle, ellipse, triangle, quadrilateral, pentagon, hexagon, heptagon, octagon, nonagon, or decagon.

[0009] In any of the bioreactor systems of the present disclosure, the inner walls may be porous. In some embodiments, liquid can traverse the inner walls. In some embodiments, the bioreactor system may be a fixed bed bioreactor. According to one aspect of the present disclosure, more than one support structure may be arranged in a stacked configuration.

[0010] In any of the bioreactor systems of the present disclosure, the outer wall of the support structure may further comprise an outer edge and an inner edge. In some embodiments, the distance between the outer edge and the inner edge is constant along the outer wall. In other embodiments, the distance between the outer edge and the inner edge is not constant along the outer wall.

[0011] In any of the bioreactor systems of the present disclosure, cell expansion and cell differentiation all occur within the same bioreactor system. In some embodiments, the polymeric scaffolds comprise a cell attachment assistor. In one specific embodiment, the cell attachment assistor comprises a cell attachment peptide motif selected from the group consisting of RGD, PHSRN (SEQ ID NO: 1), REDV (SEQ ID NO: 2), YIGSR (SEQ ID NO: 3), IKVAV (SEQ IDNO: 4), DGEA (SEQ ID NO: 5), GxOGER (SEQ ID NO: 6), GxPGER (SEQ ID NO: 7), and RRETAWA (SEQ ID NO: 8), wherein x is a hydrophobic amino acid and O is a hydroxyproline.

[0012] According to an aspect of the present disclosure, the support structure of the bioreactor system is a standalone system. In this aspect, the outer wall of the support structure also serves as the outer wall of the bioreactor system and both the first and second fluid handling units are attached to both ends of the support structure. Approaches to form tight sealing between the end of the support structure and the fluid handling units may be employed (including O-rings, adhesive based bonding, ultrasonic welding, and clamping mechanisms). According to another aspect of the present disclosure, the support structure of the bioreactor system is a modular unit. In this aspect, the outer wall of the support structure is separate from the outer wall that houses of the support structure.

[0013] Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments as described herein, including the detailed description which follows, the claims, as well as the appended drawings.

[0014] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary and are intended to provide an overview or framework to understanding the nature and character of the claims. The accompanying drawings are included to provide a further understanding and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s), and together with the description serve to explain principles and operation of the various embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following is a description of the figures in the accompanying drawings, given purely by way of non-limiting example. The figures are not necessarily to scale, and certain features and certain views of the figures may be shown exaggerated in scale or in schematic in the interest of clarity and conciseness.

[0016] FIG. 1 is a schematic middle-plane side view of a fixed bed bioreactor comprising at least one support structure and polymeric scaffolds for mass cell production according to some aspects of the present disclosure.

[0017] FIG. 2 is a schematic top view of a representative pre-assembled fixed bed bioreactor for mass cell production according to some aspects of the present disclosure.

[0018] FIG. 3 illustrates aspects of a supporting structure for polymeric scaffolds in accordance with embodiments of the present disclosure. FIG. 3A illustrates an exemplary compartment configuration of a supporting structure. FIG. 3B illustrates a supporting structure according to some aspects of the present disclosure.

[0019] FIG. 4 is a middle-plane exploded view of a fixed bed bioreactor comprising a housing vessel, at least one support structure, and polymeric scaffolds for mass cell production according to some aspects of the present disclosure.DETAILED DESCRIPTION

[0020] The various aspects and embodiments will now be fully described herein. These aspects and embodiments may, however, be embodied in many different forms and should not be construed as limiting; rather, these embodiments are provided so the disclosure will be thorough and complete, and will fully convey the scope of the present subject matter to those skilled in the art. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0021] Modifications of the disclosure will occur to those skilled in the art and to those who make or use the disclosure. Therefore, it is understood that the embodiments shown in the drawings and described above are merely for illustrative purposes and not intended to limit the scope of the disclosure, which is defined by the following claims, as interpreted according to the principles of patent law, including the doctrine of equivalents.A. Definitions

[0022] Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, particular methods and materials are now described.

[0023] As used herein the terms “the,” “a,” or “an,” mean “at least one,” and should not be limited to “only one” unless explicitly indicated to the contrary. Thus, for example, reference to “a component” includes embodiments having two or more such components unless the context clearly indicates otherwise.

[0024] Unless otherwise stated, the use of individual numerical values are stated as approximations as though the values were preceded by the word “about” or “approximately.” Similarly, the numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about” or “approximately.” In this manner, variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. As used herein, the terms “about” and “approximately” when referring to a numerical value shall have their plain and ordinary meanings to a person of ordinary skill in the art to which the disclosed subject matter is most closely related or the art relevant to the range or element at issue. The amount of broadening from the strict numerical boundary depends upon many factors. For example, some of the factors which may be considered include the criticality of the element and / or the effect a given amount of variation will have on the performance of the claimed subject matter, as well as other considerations known to those of skill in the art. As used herein, the use of differing amounts of significant digits for different numerical values is not meant to limit how the use of the words “about” or “approximately” will serve to broaden a particular numerical value or range. Thus, as a general matter, “about” or “approximately” broaden the numerical value. Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values plus the broadening of the range afforded by the use of the term “about” or “approximately.” Consequently, recitation of ranges of values herein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein.

[0025] As used herein, the term “and / or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself, or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing components A, B, and / or C, the composition can contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0026] As used herein, “have,” “having,” “include,” “including,” “comprise,” “comprising” or the like are used in their open ended sense, and generally mean “including, but not limited to.”

[0027] “Optional” or “optionally” means that the subsequently described element, component or circumstance may or may not occur, so that the description includes instances where the element, component, or circumstance occurs and instances where it does not.

[0028] ‘Porous” means a porosity greater than zero. Porosity is a measure of the void spaces in a material or a substrate or a scaffold and is a fraction of the volume of voids over the total volume, between 0 and 1, or as a percentage between 0% and 100%. Porosity is determined by dividing the volume of voids by the total volume of a material to determine a percentage and can be measured using conventional techniques (such as Andreola, F. et al., Techniques Used to Determine Porosity, American Ceramic Society Bulletin 2000, 79(7):49-52, which is incorporated herein by reference).

[0029] In this document, relational terms, such as first and second, top and bottom, and the like, are used solely to distinguish one entity or action from another entity or action, without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0030] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.B. Introduction

[0031] The present disclosure relates to the field of mass cell production (including for cultivated meats and stem cells). It also relates to support structures for polymeric scaffolds during large-scale and commercial scale mass cell production, and bioreactor systems using such support structures with mass cell production. The present disclosure further relates to fixed bed reactors comprising a support structure and polymeric scaffolds for mass cell production. The present fixed bed reactors can be used to create large-scale and commercial quantities of cells (i.e. mass cell productions), including but not limited to cultivated meats and stem cells, including mesenchymal stem cells (MSCs, also known as stromal cells), induced pluripotent stem cells (iPSCs), and embryonic stem cells.

[0032] Cultivated meats have been emerging as an alternative food source to meet growing dietary demand and to address concerns associated with animal welfare, climate change, and sustainability. However, large-scale and commercial scale production of cultivated meats is still challenging due to a number of factors, including insufficient developments in cell lines, serum- free media, bioreactor design, and scaffolds.

[0033] A primary challenge for mass cell production is the scaling up of cell production. One challenge relates to the volumetric surface area requirements for a large-scale or commercial production. Another challenge is that the steps of cell expansion and cell differentiation require very distinct physical environments and nutrient conditions, and to do the steps in separate systems is costly in equipment and in processing time. A large-scale or commercial scale production refers to producing at least 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1 kg, 2 kg, 3 kg, 4 kg, 5 kg, 6 kg, 7 kg, 8 kg, 9 kg, 10 kg, 20 kg, 30 kg, 40 kg, 50 kg, 60 kg, 70 kg, 80 kg, 90 kg, 100 kg, or even greater cultivated cells (typically a large-scale production ranges between about 100 to about 1000g of cultivated cells, whereas commercial production masses exceed 1kg). Bioproduction using adherent culture bioreactors typically support cell culture to a surface density of approximately 500,000 cells / cm2To produce 1 kg cell mass (about 3 to 5 x 1011cells) requires an effective surface area of about 60 m2to about 100 m2for cell culture. The typical mass of a single cell is about 2 ng to about 3 ng.

[0034] The present disclosure provides a support system for scaffolds for mass cell production to solve the large-scale and commercial scale production problems. The support structures of the present disclosure are advantageous as the presently available scaffold structures are unsuitable for large-scale and commercial scale productions. The present disclosure also provides a fixed bed reactor comprising a support structure and polymeric scaffolds.

[0035] The bioreactor system of the present disclosure is advantageous as it simplifies large-scale and commercial manufacturing processes for mass cell production, reduces the amount of capital investment required for large-scale and commercial scale mass cell production, and decreases the processing time for mass cell production since the bioreactor systems use perfusion culture. Advantageously, it is possible to switch from cell proliferation (also called expansion) media to cell differentiation media and leverage the capability of porous scaffolds (e.g., polymeric scaffolds) to support both cell proliferation and differentiation within the same system. Cell proliferation relates to the stages of cell growth and cell division. Cell differentiation relates to the stages of a cell becoming a more specific cell type (e.g., a mesenchymal stem cell differentiating into myocytes (muscle cells)). It also solves the additional problem of non-uniform flow and thus unhealthy cell growth and even death arising from the compaction, nesting, or collapsing of the porous scaffold because of increasing back pressure upon perfusion as cells continue growing within the mechanically weak scaffold structure when there is no support structure to hold the scaffold in place.

[0036] Additional features and advantages will be set forth in the detailed description which follows and will be apparent to those skilled in the art from the description or recognized by practicing the embodiments as described in the following description, together with the claims and appended drawings.C. Support Structures

[0037] As disclosed herein, support structures for polymeric scaffolds that aid in large- scale and commercial scale mass cell production have been developed. As illustrated in FIGS. 1- 4, a bioreactor system 100 comprises a support structure 110. The support structure 110 comprises an outer wall 125, inner walls 160, and a plurality of channels 135 defined by the inner walls 160.In some embodiments, the outer wall is a solid. A solid outer wall means it is liquid impermeable. The support structure, including its outer wall, may be cell adhesive or cell non-adhesive. In a preferred embodiment, inner walls 160 that surround each channel are not cell adhesive, thus preventing cell attachment so that cells will only grow within the polymeric scaffolds.

[0038] According to one embodiment, the outer wall of the support structure has an outer edge and an inner edge. A non-limiting example of this embodiment is shown in FIG. 2. In some embodiments, the distance between the outer edge and inner edge of the outer wall of the support structure remains approximately constant around the outer wall. A non-limiting example of this embodiment is found in FIG. 3A. In some embodiments, the distance between the outer edge and inner edge of the outer wall of the support structure may vary around the outer wall to accommodate the shapes and placement of individual channels of the plurality of channels in the support structure. A non-limiting example of this embodiment is found in FIG. 3B. In one embodiment, the cross-sectional shape of the outer wall 125 is approximately circular, as shown in FIGS. 2, and 3B. The cross-sectional shape of the outer wall can be any desired shape, such as approximately circular, approximately rectangular, approximately square, approximately ellipsoidal, or approximately polygonal.

[0039] The support structure comprises inner walls. The inner walls may be thin such that the inner walls will occupy a small volume of the total support structure, leaving a high void volume (void space) to contain polymeric scaffolds. In one embodiment, the outer wall of the support structure is thicker than the inner walls. The thickness of the inner wall may be from about 0.1 mm to about 4 mm, from about 0.1 mm to about 3 mm, from about 0.1 mm to about 2 mm, from about 0.1 mm to about 1 mm, from about 0.1 mm to about 0.5 mm, from about 0.2 mm to about 2 mm, from about 0.2 mm to about 1 mm, or from about 0.2 mm to about 0.5 mm. In one embodiment, the support structure is a modular structure, as illustrated as an example in FIG. 4, and the outer wall 125 of the support structure 110 has a thickness from about 0.5 mm to about 5 mm, from about 0.5 mm to about 4 mm, from about 0.5 mm to about 3 mm, from about 0.2 mm to about 2 mm, from about 1 mm to about 5 mm, from about 1 mm to about 4 mm, from about 1 mm to about 3 mm, or from about 1 mm to about 2 mm. In another embodiment, the support structure is a standalone structure, as illustrated as an example in FIG. 1, and the outer wall 125 ofthe support structure 110 may be thicker than when the support structure is a modular structure to meet the mechanical stability and integrity requirements for bioreactors and production operation. In such a standalone structure, the outer wall 125 of the support structure 110 has a thickness from about 2 mm to about 20 mm, about 2 mm to about 10 mm, about 5 mm to about 20 mm, or about 10 mm to about 20 mm. The thickness of the outer wall 125 of the support structure 110 is dependent on the size of the reactor. The larger size the reactor is, the greater thickness the outer wall.

[0040] The diameter of the support structure is larger than the diameter of the channels within the support structure. In one embodiment, the diameter of the support structure is from about 20 cm to about 500 cm, from about 20 cm to about 400 cm, from about 20 cm to about 300 cm, from about 20 cm to about 200 cm, from about 30 cm to about 300 cm, or from about 30 cm to about 200 cm. In one embodiment, the diameter of each channel of the plurality of channels of the support structure is from about 0.5 cm to about 10 cm, from about 0.5 cm to about 8 cm, from about 0.5 cm to about 5 cm, from about 1 cm to about 10 cm, from about 1 cm to about 8 cm, from about 1 cm to about 5 cm, from about 2 cm to about 10 cm, from about 2 cm to about 8 cm, from about 2 cm to about 5 cm, from about 5 cm to about 50 cm, or from about 5 cm to about 40 cm. As the scaffold structures (such as polymeric scaffolds) themselves are highly porous (due to the presence of interconnected pore networks and because they are made of soft polymeric or biopolymeric materials), any variability in dimension (e.g., the thickness and diameter) of the inner channels of the support structure can generally be well tolerated.

[0041] When scaling up the production (e.g., from 1 kg to 10 kg of cultivated cells), for the support structure (either as a modular structure or as a standalone structure), it is preferable to keep the height of the support structure and the dimensions of each of the channels in the plurality of channels approximately the same but to increase, proportionally, the diameter of the support structure. In this way, both operational parameters and total surface areas for cell attachment to the polymeric scaffolds can be linearly scaled up.

[0042] The support structure may be manufactured from plastics, ceramics, glass, or other materials such as stainless steel. In one embodiment, the support structure is plastic or ceramic. Insome embodiments, the support structure can be made in a single process or technique (e.g., injection molding, extrusion, or 3D printing). In some embodiments, the outer wall of the support structure may be manufactured from a different type of material than the inner walls of the support structure. For example, the outer wall may be made from plastic materials and the inner wall may be made from ceramic materials. In such an example, the outer wall and the inner structure can be made separately.

[0043] The support structure provides compartmentalization for polymeric (including biopolymeric) scaffolds. The outer wall of the support structure creates an inner space that is the space entrapped by the inner edge of the outer wall. Said differently, the inner space has an outer edge defined by the inner edge of the outer wall. The inner space has a plurality of inner walls. The spaces between inner walls define the individual channels of the plurality of channels. In some embodiments the inner walls may be solid. In some embodiments, the inner walls may be porous. In some embodiments, the inner walls have a porosity of less than about 30%, about 20%, about 10%, about 5%, about 2%, or about 1%. In other embodiments, the inner walls (entire walls or alternatively sections of walls) may be a mixture of porous and non-porous walls. For example, one inner wall may be porous and the next inner wall may be non-porous. In another example, one section of an inner wall may be porous and another section of the same inner wall may be non- porous.

[0044] The individual channels of the plurality of channels may be any shape, such as circular (or approximately circular), hexagonal (or approximately hexagonal), octagonal (or approximately octagonal), pentagonal (or approximately octagonal), rectangular (or approximately rectangular), square (or approximately square), triangular (or approximately triangular), or other shapes, including parallelograms, decagons, ellipses, etc. In some embodiments, each channel of the plurality of channels are open at each end of the channel so it is possible to collect all cells after culturing. In some embodiments, the inner walls are porous, and a certain percentage of channels have one closed end at the end nearest to where fluid flows into the bioreactor system. In such embodiments, the percentage of channels having a closed end may be less than about 50%, about 40%, about 30%, about 20%, about 10%, or about 5%. In such embodiments, polymeric scaffolds are placed inside the channels having both ends open, and once media flows into channelshaving two ends open, the media can diffuse into nearby channels having only one end open through the porous inner walls, which helps minimize or eliminate the back pressure during the high-density cell culture. In these embodiments, cells are preferably introduced into the bioreactor system through a port located at the outlet from the bioreactor system, so that cells are only distributed within the channels having two open ends. Once the cells adhere within the polymeric scaffolds and start growing, medium can be introduced from the open end of the support structure (that is, through a port located at the inlet to the bioreactor system).

[0045] In FIGS. 3A-3B, the opening of the channels of the support structure 110 are shown with a hexagonal shape (FIG. 3A) or an approximately hexagonal shape (FIG. 3B). As illustrated in FIG. 3A, the opening of the channels of support structure 110 have a diameter D. In some embodiments of the present disclosure, the diameter of the opening of the channels are from about 0.5 mm to about 50 cm, depending on the size of the bioreactor desired. In other embodiments, the diameter of the opening of the channels is from about from about 0.5 cm to about 10 cm, from about 0.5 cm to about 8 cm, from about 0.5 cm to about 5 cm, from about 1 cm to about 10 cm, from about 1 cm to about 8 cm, from about 1 cm to about 5 cm, from about 2 cm to about 10 cm, from about 2 cm to about 8 cm, from about 2 cm to about 5 cm, from about 5 cm to about 50 cm, or from about 5 cm to about 40 cm. The preferred diameter of the channels of the support structure is dependent on the types and mechanical properties of the scaffold structures (e.g., polymeric scaffolds). Generally, the less stiff the scaffold structure is, the smaller the diameter of the inner channels of the support structure can be. In some embodiments, the diameter along the length of the channel is about the same diameter of the opening of the channel.

[0046] The support structure of the present disclosure provides additional strength in a bioreactor system for large-scale or commercial scale mass cell productions, and for high cell density perfusion cell cultures in a large-scale or commercial scale production. Polymeric (including biopolymeric) scaffolds are typically soft, highly porous, and often deform under mild pressures. As cells grow into high density within a bioreactor, the media flow rate needs to increase over time to meet the demand of nutrition and oxygen due to cell metabolism, thus presenting an increasing pressure up to several folds (e.g., 5 times atmospheric pressure) to the soft scaffolds upon perfusion, which, in turn, results in the compaction, nesting, or collapsing of the polymericscaffolds. For high-density cell culture using polymeric scaffolds, the back pressure induced by media flow can be high as 10 Pa to 1000 Pa, which can cause the polymeric scaffolds deform. The support structure described herein presents a solution to such problems by creating supports for the polymeric scaffolds. The use of the support structure in the bioreactor system allows for smaller and more uniform spaces for the cells to grow in, such that cells can be more uniformly distributed inside the polymeric scaffolds within the plurality of channels and without the issues just described. Furthermore, the inner walls can guide fluid flow movement and assist in maintaining the structural integrity of the polymeric scaffolds during perfusion cell culture. When inner walls are porous, this can further direct the fluidic movement and reduce the back pressure during the high-density cell culture.

[0047] In certain embodiments, the support structure will withstand pressures of at least about 67,000 Pa (~10 psi). The pressure can be monitored or tested for using state-of-the-art sensors (e.g., PendoTECH single-use pressure sensors). As used herein, a support structure withstanding pressure means it does not permanently collapse or have permanent deformation. In one embodiment, the support structure will withstand pressures of 60,000 Pa, 50,000 Pa, 40,000 Pa, 30,000 Pa, 20,000 Pa, 10,000 Pa, 7,500 Pa, 5,000 Pa, 2,500 Pa, or 1,000 Pa. In another embodiment, the support structure will withstand pressures from about 10 Pa to about 60,000 Pa, from about 10 Pa to about 30,000 Pa, from about 10 Pa to about 10,000 Pa, from about 10 Pa to about 5,000 Pa, or from about 10 Pa to about 1,000 Pa.D. Polymeric Scaffolds

[0048] A bioreactor system of the present disclosure comprises polymeric (including biopolymeric) scaffolds. A polymeric scaffold is a substrate that supports cell attachment, expansion, or differentiation during a large-scale or commercial scale mass cell production. In embodiments when the polymeric scaffolds are made of edible biomaterials, the entire final cell culture product can be pushed out of the bioreactor system using high pressure or mechanical force, and then used for downstream food processing. In embodiments when the polymeric scaffolds are made of biocleavable materials for cultivated meat production, the cells can be harvested and collected after directly dissolving away the scaffolds inside the bioreactor via enzymatic reactions,or by first pushing the scaffold structures out of the bioreactor followed by dissolving away the scaffolds via enzymatic reactions. The cells are collected and then used for downstream food processing. With stem cell cultures, scaffolds made of biocleavable materials are preferred since it is desired to harvest cells with great viability (at least 90%). Automated in-situ cell harvesting may be performed using the present reactor. Specifically, after cell culturing is complete, cells are washed using a buffer media, followed by an enzymatic dissolvation of the scaffolds, followed by cell collection. All these steps can be performed using the bioreactor system. The collected cells may then be subjected to downstream washing and concentration steps. Stem cells may grow into 3-dimensional structure (e.g., spheroids, aggregates, organoids), dependent on the types of stem cells and culture conditions, within the porous polymeric scaffolds.

[0049] In one embodiment, the polymeric scaffolds are made of biocleavable materials. A biocleavable material refers to that material can be degraded or broken down by enzymes. Biocleavable biomaterials that are suitable to make dissolvable scaffolds include, but are not limited to, animal derived collagen, recombinant collagen, gelatin, polygalacturonic acid (PGA), pectin, dextran, or other polypeptides, and cellulose. Polymeric scaffolds formed from collagen can be degraded by collagenase, while polymeric scaffolds formed from PGA or pectin can be rapidly degraded by combining EDTA (ethylenediaminetetraacetic acid) with pectinase, scaffolds formed from gelatin or other polypetides can be rapidly degraded by pronase. Pronase is a mixture of several nonspecific endoproteases and exoproteases that digest proteins down to single amino acids. Polymeric scaffolds formed from dextran can be degraded by dextranase, while polymeric scaffolds formed from cellulose can be rapidly degraded by cellulases. The amount and concentration of enzymes used to degrade the polymeric scaffolds is dependent on several factors, including, but not limited to, the size and structure of scaffolds, the cell density, protocol (e.g, temperature, solution composition), and the desired duration to complete the degradation of scaffold structure. The amount and concentration of enzymes required can be optimized experimentally. Scaffold-cell product complexes are generally washed first with buffer solution, followed by enzymatic reactions. For faster degradation, higher concentration of enzyme solution can be used.

[0050] In one embodiment, the polymeric scaffolds can be made of edible biomaterials. Common edible materials suitable to make polymeric scaffolds include, but are not limited to, natural or synthetic biomaterials such as polysaccharides (e.g., starch, alginate, carrageenan, chitosan, agarose, cellulose, nanocellulose fibers, carboxymethylcellulose, soy proteins, zein, pectin), polypeptides (e.g., collagen, gelatin, gluten), lipids (e.g., paraffin, shellac), and compos ites / synthetics (e.g., polygalacturonic acid (PGA), polyethylene glycol (PEG)) (reviewed in Chen, L. et al., Large-scale cultured meat production: trends, challenges and promising biomanufacturing technologies, Biomaterials 2022, 280, 121274).

[0051] In one embodiment, the polymeric scaffolds can be made of non-edible biomaterials. Common non-edible materials suitable to make polymeric scaffolds include, but are not limited to, synthetic polymers or hydrogels such as poly(lactic-co-glycolic acid) (“PLGA”), poly(glycolic acid), polyethylene, polypropylene, polyester, or any other polymers that are not used for animal food consumption.

[0052] In some embodiments, the polymeric scaffolds are made from decellularized natural materials, which are highly porous and edible. In one embodiment, decellularized spinach polymeric scaffolds made from decellularized spinach leaves can be formed using solvent extraction and buffer washing of the spinach leaves, as has been described elsewhere, for example, by Jordan D. Jones, Alex S.Rebello, Glenn R Gaudette. Decellularized spinach: An edible scaffold for laboratory-grown meat, Food Bioscience, 2021, 41, 100986, which is incorporated herein by reference. Such decellularized spinach scaffolds can be further shaped mechanically (e.g., cutting) and used to fill in the plurality of channels within the support structure.

[0053] In one embodiment, the polymeric scaffolds are porous. Porous polymeric scaffolds can be purchased commercially (for instance, textured soy proteins are widely available) or premade in batches and in some embodiments, the porous polymeric scaffolds are then shaped mechanically (e.g., cutting) to fill the plurality of channels of the support structure.

[0054] In another embodiment, the porous polymeric scaffolds can be formed directly inside the plurality of channels of the support structure. Common techniques that can be used to form porous polymeric scaffolds directly inside the plurality of channels include, but are notlimited to, particle leaching, melt molding, freeze-drying, and gas foaming. For example, high porosity scaffolds can be made by first providing a solution of gelatin and hyaluronic acid can be poured to fill the channels of the support structure, followed by rapidly freezing the filed support structure to form ice crystals within the polymeric scaffold structures being formed at the same time, followed by thawing the frozen support structure. Pore size and porosity of porous polymeric scaffolds can be fine-tuned by using different concentrations of gelatin and hyaluronic acid, as well as controlling crosslinking kinetics. Detailed protocols about the freeze-drying approach can be found in, for example, Brougham, C.M. et al. Freeze-drying as a novel biofabrication method for achieving a controlled microarchitecture within large, complex natural biomaterial scaffolds. Adv. Healthc. Mater. 2017 Nov;6(21). doi: 10.1002 / adhm.201700598, which is incorporated herein by reference. The exact conditions for the freeze-drying processes are dependent on the desired structure and porosity of scaffolds as well as the materials used to make the scaffold. In general, for mass cell production applications, polymeric scaffolds with high porosity (i.e., porosity of >90%, >80%, >70%, >60%, or >50%) with large dimension of pores (e.g. >500 microns, >250 microns, >100 microns, >50 microns) are desired.

[0055] In some embodiments, the polymeric scaffolds are modified to have cell attachment assistors that can be incorporated into the scaffolds for cell culture. A cell attachment assistor can be introduced using conventional approaches, for instance, physical absorption or covalent coupling. In one embodiment, the cell attachment assistor may include an extracellular matrix protein (e.g., fibronectin, collagen, fibrinogen, laminin), or a peptide containing at least one of the cell attachment peptide motifs, for instance, RGD or YIGSR (SEQ ID NO: 3). Such cell attachment assistors can be obtained commercially, such as Synthemax® from Corning® for use with the polymeric scaffolds.

[0056] In some embodiments, the polymeric scaffolds may comprise covalently attached assistors that include cell attachment peptide motifs. In one embodiment, the cell attachment assistor may be a peptide comprising at least one of the amino acid sequence Arg-Gly-Asp (known as “RGD”), the amino acid sequence Pro-His-Ser-Arg-Asn (known as “PHSRN”) (SEQ ID NO: 1), the amino acid sequence Arg-Glu-Asp-Val (known as “REDV”) (SEQ ID NO: 2), the amino acid sequence Tyr-Ile-Gly-Ser-Arg (known as “YIGSR”) (SEQ ID NO: 3), the amino acidsequence Ile-Lys-Val-Ala-Val (known as “IKVAV”) (SEQ ID NO: 4), the amino acid sequence Asp-Gly-Glu-Ala (known as “DGEA”) (SEQ ID NO: 5), the amino acid sequence Gly-x-Hyp- Gly-Glu-Arg (known as “GxOGER”, wherein the “O” amino acid is hydroxyproline, and “x” is a hydrophobic amino acid, for example, an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan, preferably phenylalanine) (SEQ ID NO: 6), the amino acid sequence Gly-x-Pro-Gly-Glu-Arg (known as “GxPGER”, wherein “x” is a hydrophobic amino acid, for example, an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan, preferably phenylalanine) (SEQ ID NO: 7), and the amino acid sequence Arg-Arg-Glu-Thr-Ala-Trp-Ala (known as “RRETAWA”) (SEQ ID NO: 8).

[0057] In one embodiment, the polymeric scaffolds can be crosslinked with agents that are Generally Recognized as Safe (GRAS) by the Food and Drug Administration (FDA). In one specific embodiment, the natural product genipin can be used to crosslink amine containing polymeric scaffolds. In another embodiment, the amine containing polymeric scaffold that is crosslinked with genipin includes zein, soy proteins, collagen, gelatin, chitosan, gluten, or other proteins. In another embodiment, food-safe enzymes that perform crosslinking reactions with polymeric scaffolds may be used, including transglutaminase. In yet another embodiment, photoactivated crosslinking agents may be used to crosslink the polymeric scaffolds, including sodium benzoate. In another embodiment, physical crosslinking methods can also be used to crosslink the polymeric scaffolds, including heat treatment for soy proteins and zein. Common crosslinking agents such as l-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) and glutaraldehyde do not have the FDA Generally Recognized as Safe (GRAS) status, and cannot be used in food production and should be avoided.

[0058] The stiffness of the polymeric scaffolds is increased when the polymeric scaffolds are crosslinked. In one embodiment, the stiffness is improved by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, when the polymeric scaffolds are crosslinked. Young’s modulus values as measured by a tensile test can be used to determine the stiffness.E. Bioreactor System

[0059] The bioreactor system of the present disclosure comprises at least one support structure and polymeric scaffolds. The bioreactor system may further comprise at least one fluid handling unit. Fluid handling units allow fluid to pass into and out of the bioreactor system more uniformly. Fluid handling units comprise at least one port to allow fluid to pass and may have a fluid flow guide plate (also termed a fluid flow distributor plate) to assist flow inside the bioreactor system. The fluid flow guide plate may be made of any material that allows fluids to flow across the plate and more evenly disperse fluid into all the channels of the support structure. It may have any shape that assists in evenly dispersing fluid more evenly across the all channels of the support structure than if no fluid flow guide plate was used with the bioreactor system.

[0060] In some embodiments, the bioreactor system may be a fixed bed (also called a packed bed) bioreactor. In one specific embodiment, the bioreactor system is a fixed bed bioreactor and the fixed bed bioreactor is configured for perfusion.

[0061] FIG. 1 shows a middle-plane side-view schematic drawing of a standalone bioreactor system having a fixed bed for mass cell production. The bioreactor system 100 comprises a first end portion 120 and a second end portion 130. The support structure 110 has a plurality of channels 135 and polymeric scaffolds 140 within the channels. The first end portion 120 may comprise at least one inlet port 112, and the second end portion 130 may comprise at least one outlet port 115. In this non-limiting embodiment, fluid enters the bioreactor system 100 from the bottom of the system and exits from the top of the system. It should be understood that the inlet and outlet ports may be reversed; that is, fluid may enter from the top and exit from the bottom.

[0062] In a standalone bioreactor system, the support structure 110 can be assembled into a bioreactor system 100 by attaching a first fluid flow handling unit to a first end portion 120 of the support structure 110 and a second fluid flow handling unit to a second end portion 130 of the support structure 110. The first fluid flow handling unit comprises an inlet port 112 and a fluid flow guide plate, the latter of which may optionally be in contact with the first end portion 120 of the support structure 110. The fluid flow guide plate of the first fluid flow handling unit is used todistribute fluids or media evenly across the area inside the support structure 110. The second fluid flow handling unit comprises an outlet port 115 and a second fluid flow guide plate, the latter of which may optionally be in contact with the second end portion 130 of the support structure 110. Spent fluid may exit the support structure 110 through outlet port 115 and the second fluid flow guide plate. When fluid flow guide plates are present with both the first and second fluid handling units, both fluid flow guide plates may have the same design, or each one may have different designs. In the standalone bioreactor system, the outer wall 125 of the support structure 110 acts as the housing vessel for the polymeric scaffolds 140, which are housed within the plurality of channels 135 inside the support structure 110. When media flows into a fluid flow guide plate through inlet port 112, the media will more evenly distribute to the plurality of channels 135 of the support structure 110, will pass through porous scaffolds 140 inside the plurality of channels 135, and will eventually reach the second fluid flow guide plate and flow out of the bioreactor through outlet port 115. Again, it should be understood that the inlet and outlet ports may be reversed; that is, fluid may enter from the top and exit from the bottom.

[0063] FIG. 2 shows a top view of a schematic drawing of a bioreactor system having a fixed bed for mass cell production. The bioreactor system comprises a support structure 110 having an outer wall 125 and inner walls 160, wherein the inner walls 160 are configured to create a plurality of channels, and wherein the channels are filled with polymeric scaffolds 140. The outer wall 125 comprises an outer edge 145 and an inner edge 150.

[0064] FIG. 3A shows a cross-section of a plurality of channels 135 for a support structure (in FIG. 3A the channels are inside the hexagonal shapes). Each channel has a hexagonal shape with diameter “D”, which is the cross-sectional area defined by inner walls 160. FIG. 3B is a top view of a support structure 110 for a bioreactor system, wherein the support structure 110 has an outer wall 125, and a plurality of channels divided by inner walls, and wherein the cross-sectional area of each channel has an approximately hexagonal shape.

[0065] In another embodiment, a non-limiting example of which is illustrated in FIG. 4, the support structure 110 for polymeric scaffolds 140 is a modular structure, meaning it can be placed inside a housing vessel 190 such that the outer wall 125 of support structure 110 is in closecontact with the inner wall of housing vessel 190 and media does not flow between the housing vessel 190 and modular support structure 110 once assembled into a bioreactor system 100. In some embodiments, a sealing structure (e.g., an O-ring) 192 is added to seal any gaps between the inner wall of the housing vessel and the outer wall 125 of the support structure 110. Approaches to form tight sealing between the end of the support structure and the fluid handling units include O-rings, adhesive based bonding, ultrasonic welding, and clamping mechanisms. The support structure 110 has polymeric scaffolds 140 within a plurality of channels 135. In some embodiments, the bioreactor system 100 also has a first fluid flow handling unit 111 having a fluid flow guide plate 113 and a second fluid flow handling unit 114 having a fluid flow guide plate 116. In those embodiments, the support structure 110 is between a fluid flow guide plate 113 at a first end portion of bioreactor system 100 and the other fluid flow guide plate 116 at a second end portion of the bioreactor system 100. The fluid flow guide plate 113 is placed after an inlet port 112 on the housing vessel 190 at the first end portion and the other fluid flow guide plate 116 is placed before an outlet port 115 on the housing vessel 190 at the second end portion. When media flows into fluid flow guide plate 113 through inlet port 112, the media will evenly distribute to all channels of the support structure 110, and will pass through the polymeric scaffolds 140, and will eventually reach the other fluid flow guide plate 116 and flow out of the housing vessel 190 through outlet port 115. It should be understood that the inlet and outlet ports may be reversed; that is, fluid may enter from the top and exit from the bottom.

[0066] More than one support structure may exist in a single bioreactor system under the present disclosure. In one embodiment, a bioreactor system comprises at least two support structures. In another embodiment, a bioreactor system comprises at least two support structures in a stacked configuration.

[0067] The support structure of the bioreactor system of the present disclosure may have any of the configurations described in the present disclosure. In one embodiment, the support structure of a bioreactor system comprises a honeycomb structure, wherein the inner space of the support structure comprises inner walls that are configured to make channels with a cross-sectional area between the inner walls. The cross-sectional area may be in an approximate shape such as a circle, an ellipse, a triangle, a quadrilateral, a pentagon, a hexagon, or an octagon. Morespecifically, in one embodiment, the support structure has inner walls that are configured to make channels with a cross-sectional area between the inner walls that is approximately a hexagon. A non-limiting example of channels having an approximately hexagon shape is illustrated in FIG. 3B. The hexagon may be either a regular or irregular hexagon. In another embodiment, the support structure of the bioreactor system has channels with a cross-sectional area between the inner walls that is approximately a circle, or that is approximately an ellipse. In another embodiment, the support structure of the bioreactor system has inner walls that are configured to make channels with a cross-sectional area between the inner walls that is approximately the shape of a triangle. Triangle shapes include an equilateral triangle, an isosceles triangle, a right angle triangle, or a scalene triangle. In another embodiment, the support structure of the bioreactor system has inner walls that are configured to make channels with a cross-sectional area that is approximately quadrilateral in shape. Quadrilateral shapes include, a square, a rectangle, a parallelogram, a trapezoid, or a rhomboid. A non-limiting example of channels having an approximately square shape is illustrated in FIG. 2. In another embodiment, the support structure of the bioreactor system has inner walls that are configured to make channels with a cross-sectional area that is approximately a pentagon. The pentagon may be either a regular or irregular pentagon. In another embodiment, the support structure of the bioreactor system has inner walls that are configured to make channels with a cross-sectional area that is approximately a heptagon. The heptagon may be either a regular or irregular heptagon. In another embodiment, the support structure of the bioreactor system has inner walls that are configured to make channels with a cross-sectional area that is approximately an octagon. The octagon may be either a regular or irregular octagon. In another embodiment, the support structure of the bioreactor system has inner walls that are configured to make channels with a cross-sectional area that is approximately a nonagon. The nonagon may be either a regular or irregular nonagon. In another embodiment, the support structure of the bioreactor system has inner walls that are configured to make channels with a cross-sectional area that is approximately a decagon. The decagon may be either a regular or irregular decagon.

[0068] Liquid can be added to and / or removed from the bioreactor system of the present disclosure. Liquid can also flow through the bioreactor system. Fresh liquid can be provided to cell cultures in the bioreactor system through at least one port that allows fluid to flow into the 1bioreactor system. Spent liquid can be removed from the cell cultures in the bioreactor system through at least one port that allows fluid to flow out of the bioreactor system. In some embodiments, liquid can enter bioreactor system through the bottom of the system and exit from the top of the system. In other embodiments, liquid can enter the bioreactor system though the top of the system and exit from the bottom of the system.

[0069] In some embodiments, the liquid is cell culture expansion media. In some embodiments, the liquid is a medium that promotes cell attachment. In another embodiment, the liquid is cell differentiation media. In some embodiments, the liquid is cell harvesting medium, or washing buffer. In one specific embodiment, the cell culture medium is a medium that promotes cell expansion. In another specific embodiment, the cell culture medium is a medium that promotes cell differentiation. At any desired point in time during a cell culture production, the type of liquid that enters the bioreactor system can be switched. In one embodiment, cell expansion medium can be switched to cell differentiation medium. In other embodiment cell expansion medium can be switched to cell harvesting medium. In yet another embodiment, cell differentiation medium can be switched to cell harvesting medium. Switching of cell medium allows cell attachment, cell expansion, and cell differentiation occur in a single bioreactor system. In one embodiment, cell expansion and differentiation occur in a single bioreactor system. In another embodiment, cell attachment and cell expansion occur in a single bioreactor system. In another embodiment, cell attachment, cell expansion, and cell differentiation occur in a single bioreactor system.

[0070] A bioreactor system comprising at least one support structure and polymeric scaffolds improves the density at which cells can be cultured in a mass cell production. The bioreactor systems of the present disclosure can culture cells from a cell culture seeding density of about 2000 cells / cm2to about 30,000 cells / cm2, about 5000 cells / cm2to about 25,000 cells / cm2, about 5000 cells / cm2to about 20,000 cells / cm2, about 10,000 cells / cm2to about 20,000 cells / cm2, about 5000 cells / cm2to about 15,000 cells / cm2, about 5000 cells / cm2to about 10,000 cells / cm2, about 2,500 cells / cm2to about 7,500 cells / cm2, or less than about 5,000 cells / cm2. In one specific embodiment, when a support structure of bioreactor system has an outer wall and either a porous or non-porous plurality of inner walls, the bioreactor system cultures cells from cell seeding densities of about 2000 cells / cm2to about 30,000 cells / cm2, about 5000 cells / cm2to about 25,000cells / cm2, about 5000 cells / cm2to about 20,000 cells / cm2, about 10,000 cells / cm2to about 20,000 cells / cm2, about 5000 cells / cm2to about 15,000 cells / cm2, or about 5000 cells / cm2to about 10,000 cells / cm2, about 2,500 cells / cm2to about 7,500 cells / cm2, or less than about 2,000 cells / cm2.

[0071] The bioreactor systems of the present disclosure can culture cells reaching cell densities of about 500,000 cells / cm2to about 1,000,000 cells / cm2When cells are cultured, the final cell density is dependent on culture duration and conditions. The cell density achieved with the bioreactor systems of the present disclosure, when the culture is finished (i.e., cells are ready to be harvested for further downstream processing), may be up to about 500,000 cells / cm2, up to about 1,000,000 cells / cm2, or at any value from about 500,000 cells / cm2to about 1,000,000 cells / cm2.

[0072] While the present disclosure includes a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the present disclosure.

Claims

CLAIMSWhat Is Claimed Is:

1. A fixed bed bioreactor system, comprising: a first end portion and a second end portion; and at least one support structure comprising a solid outer wall, a plurality of inner walls, a plurality of channels, and polymeric scaffolds; wherein the channels are void spaces defined by the inner walls, and wherein the bioreactor system cultures cells up to about 1,000,000 cells / cm2.

2. The bioreactor of claim 1, wherein the bioreactor system cultures cells of about 500,000 cells / cm2to about 1,000,000 cells / cm2.

3. The bioreactor system of claim 1, wherein the bioreactor system cultures cells from cell culture seeding densities of about 2,000 cells / cm2to about 30,000 cells / cm2.

4. The bioreactor system of claim 1, wherein a cross-sectional area of each channel is chosen from a circle, ellipse, triangle, quadrilateral, pentagon, hexagon, heptagon, octagon, nonagon, or decagon.

5. The bioreactor system of claim 1, further comprising at least two support structures, arranged in a stacked configuration.

6. The bioreactor system of claim 1, wherein the inner walls are porous.

7. The bioreactor system of claim 6, wherein liquid can traverse the inner walls.

8. The bioreactor system of claim 1, wherein cell expansion, and cell differentiation both occur within the same bioreactor system.

9. The bioreactor system of claim 1, wherein the polymeric scaffolds comprise a cell attachment assistor.

10. The bioreactor system of claim 9, wherein the cell attachment assistor has a peptide motif selected from the group consisting of: RGD, PHSRN (SEQ ID NO: 1), REDV (SEQ ID NO: 2), YIGSR (SEQ ID NO: 3), IKVAV (SEQ ID NO: 4), DGEA (SEQ ID NO: 5), GxOGER (SEQ ID NO: 6), GxPGER (SEQ ID NO: 7), RRETAWA (SEQ ID NO: 8), and a mixture thereof; wherein x is a hydrophobic amino acid and O is a hydroxyproline.

11. The bioreactor system of claim 1, further comprising a first fluid handling unit at the first end portion and a second fluid handling unit at the second end portion.

12. The bioreactor system of claim 11, wherein the first fluid handling unit comprises a first port and a fluid flow guide plate, and wherein the second fluid handling unit comprises a second port.

13. The bioreactor system of claim 12, wherein the second fluid handling unit further comprises a fluid flow guide plate.

14. A bioreactor system, comprising: a support structure comprising an outer wall having an inner edge and an inner space having an outer edge defined by the inner edge of the outer wall; a first fluid handling unit comprising a first port and a first fluid flow guide plate; and a second fluid handling unit comprising a second port and a second fluid flow guide plate; wherein the inner space has a plurality of inner walls configured to create channels in spaces between the inner walls, and wherein polymeric scaffolds are housed within the channels.

15. The bioreactor support structure of claim 14, wherein a cross-sectional area of each channel is a hexagon.

16. The bioreactor support structure of claim 14, wherein a cross-sectional area of each channel is chosen from a circle, an ellipse, a triangle, a quadrilateral, a pentagon, an octagon, a nonagon, or a decagon.

17. The bioreactor support structure of claim 14, wherein the inner walls are porous.

18. The bioreactor support structure of claim 17, wherein fluid can traverse the channels through the inner walls.

19. The bioreactor support structure of claim 14, wherein the distance between the outer edge and the inner edge is not constant along the outer wall.

20. The bioreactor support structure of claim 14, wherein the distance between the outer edge and the inner edge is constant along the outer wall.

21. The bioreactor support structure of claim 14, wherein the support structure is a standalone unit.

22. The bioreactor support structure of claim 14, wherein the support structure is a modular unit.

23. A bioreactor system, comprising: a first end portion and a second end portion; and at least one support structure comprising a solid outer wall, a plurality of inner walls, a plurality of channels, and polymeric scaffolds; wherein the channels are void spaces between the inner walls of the plurality of inner walls and the polymeric scaffolds are within the channels, and wherein the support structure withstands pressures of at least 1,000 Pa without permanent deformation.

24. The bioreactor system of claim 23, wherein the support structure withstands pressures of at least 10,000 Pa without permanent deformation.

25. The bioreactor system of claim 23, wherein the support structure withstands pressures of at least 30,000 Pa without permanent deformation.

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