Honeycomb scaffold bioreactor for mass cell production

Abstract

Provided are a bioreactor support structure and a bioreactor system for mass production of cells, including for production of cultured meat and for mass production of stem cells. The bioreactor support structure has an outer wall and a plurality of inner walls within an interior space created by an inner edge of the outer wall. The plurality of inner walls are configured to form a channel that accommodates a polymeric scaffold, cells, and a fluid. The cross-section of the channel may have approximately circular, elliptical, or polygonal cross-sectional areas. The configuration of the bioreactor support structure addresses the problem of high cell density pressures that hinder large-scale and commercial mass cell production.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 538,153, filed September 13, 2023, pursuant to 35 USC §119, the contents of which are incorporated herein by reference in their entirety.

[0003] Reference to the electronic sequence list

[0004] The computer-readable sequence list (8997 bytes) with the filename SP23-254_Sequence_Listing.xml, created on September 5, 2024, is incorporated herein by reference in its entirety. Technical Field

[0005] This disclosure relates to mass cell production (including for cultured meat and stem cells), bioreactor support structures for mass cell production, and bioreactor systems for mass cell production. Background Technology

[0006] Scaffolds used in tissue engineering have been proposed and applied to small-scale cell production. However, currently available scaffolds for large-scale cell production (including for culturing meat and stem cells) are unsuitable for large-scale or commercial production because, when scaled up, these scaffolds cannot suppress the increasing back pressure during perfusion culture due to the relatively weak mechanical strength of their highly porous structure. Under such back pressure conditions, the scaffolds undergo undesirable localized compaction, clumping, and even collapse, which in turn leads to difficulty in obtaining nutrients and dissolved oxygen, and ultimately results in unhealthy cell growth. The increasing back pressure during perfusion culture is a direct consequence of the decreasing porosity of these scaffolds as cell density increases during culture. This disclosure addresses the problem of large-scale or commercial cell production by improving the stability of polymer scaffolds during large-scale or commercial production processes using an improved support structure. Summary of the Invention

[0007] According to some aspects of the present disclosure, an improved bioreactor system for mass cell production, including for cultured meat and stem cells, is provided. The bioreactor system includes a first end portion and a second end portion, and at least one support structure including a solid outer wall, a plurality of inner walls, a plurality of channels, and a polymer scaffold. The plurality of channels provide void space to accommodate the polymer scaffold, while the plurality of inner walls provide physical structure to support and constrain the polymer scaffold within the plurality of channels. The bioreactor system can culture cells at a cell density of up to about 1,000,000 cells / cm 2 In one embodiment, the bioreactor system is configured to culture cells at a density of about 500,000 cells / cm 2 to about 1,000,000 cells / cm 2 The bioreactor system also cultures cell densities of less than about 500,000 cells / cm 2 In another embodiment, the bioreactor system begins culturing cells from a cell seeding density of about 2,000 cells / cm 2 to about 30,000 cells / cm 2 In some embodiments, the bioreactor system further includes a first fluid handling unit located at the first end portion of the bioreactor system and a second fluid handling unit located at the second end portion. The first fluid handling unit includes a first port and a first fluid flow guide plate. The second fluid handling unit includes a second port and a second fluid flow guide plate. The fluid flow guide plates are used to evenly distribute liquid solutions (e.g., media, buffers) after the solutions pass through the first port.

[0008] 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 including an outer wall having an inner edge, an interior 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 space between the inner walls. The bioreactor system accommodates a polymer scaffold within the channels of the support structure. The bioreactor system further has a first fluid handling unit including a first port and a first fluid flow guide plate. The bioreactor system also has a second fluid handling unit including a second port and a second fluid flow guide plate.

[0009] According to other aspects of the present disclosure, an improved bioreactor system for large scale 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 a polymer scaffold. The plurality of channels are interstitial spaces between the inner walls, and the polymer scaffold is located 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.

[0010] 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: circular, elliptical, triangular, quadrilateral, pentagonal, hexagonal, heptagonal, octagonal, nonagonal, or decagonal.

[0011] In any of the bioreactor systems of the present disclosure, the inner walls can be porous. In some embodiments, a liquid can traverse the inner walls. In some embodiments, the bioreactor system can be a fixed bed bioreactor. According to one aspect of the present disclosure, more than one support structure can be arranged in a stacked configuration.

[0012] In any of the bioreactor systems of the present disclosure, the outer wall of the support structure can 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.

[0013] 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 scaffold comprises a cell attachment auxiliary factor. In one particular embodiment, the cell attachment auxiliary factor 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 ID NO: 4), DGEA (SEQ ID NO: 5), GxOGER (SEQ ID NO: 6), GxPGER (SEQ ID NO: 7), and RRETAWA (SEQ ID NO: 8), where x is a hydrophobic amino acid and O is hydroxyproline.

[0014] According to one 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 fluid handling unit and the second fluid handling unit are attached to both ends of the support structure. Methods to form a tight seal between the ends of the support structure and the fluid handling units can 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 the support structure.

[0015] Additional features and advantages will be set forth in the detailed description that follows, and in part will be apparent to those skilled in the art from that description, or can be learned by practice of the embodiments described herein, including the detailed description that follows, the claims, and the appended drawings.

[0016] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are intended to provide a framework or a basis for 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 DRAWINGS

[0017] The following is a description of the drawings in the figures, which are purely by way of non-limiting example. The drawings are not necessarily to scale, and certain features and certain views of the drawings can be shown exaggerated in scale or in schematic in the interest of clarity and conciseness.

[0018] Figure 1is a schematic mid-plane side view of a fixed-bed bioreactor comprising at least one support structure and a polymer scaffold for large-scale cell production according to some aspects of the present disclosure.

[0019] Figure 2 is a schematic top view of a representative pre-assembled fixed-bed bioreactor for large-scale cell production according to some aspects of the present disclosure.

[0020] Figure 3 illustrates aspects of a support structure of a polymer scaffold according to embodiments of the present disclosure. Figure 3A An exemplary compartment configuration of a support structure is illustrated. Figure 3B A support structure according to some aspects of the present disclosure is illustrated.

[0021] Figure 4 is a mid-plane exploded view of a fixed-bed bioreactor comprising an enclosure vessel, at least one support structure, and a polymer scaffold for large-scale cell production according to some aspects of the present disclosure. DETAILED DESCRIPTION

[0022] Various aspects and embodiments will now be described in full detail herein. However, these aspects and embodiments can be embodied in many different forms and should not be construed as limiting; rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the subject matter to those skilled in the art. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0023] Modifications to the present disclosure can be practiced by those skilled in the art and within the scope of the disclosure. Accordingly, the embodiments described herein are not intended to limit the scope of the disclosure, which is defined by the appended claims, including the full scope of equivalents to which such claims are entitled. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0024] A. DEFINITIONS

[0025] Unless otherwise defined, all terms and phrases used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs in light of the entirety of the disclosure. 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.

[0026] As used herein, the terms “the,” “a,” and “an” mean “at least one,” and should not be limited to only one unless explicitly indicated to the contrary. Thus, for example, reference to “an element” includes embodiments having two or more such elements unless the context clearly indicates otherwise.

[0027] Unless otherwise stated, the use of a single numerical value is stated as an approximation, as if the value were preceded by the word “about” or “approximately.” Similarly, unless explicitly indicated otherwise, numerical values in the various ranges specified in this application are stated as approximations, as if both the minimum and maximum values of the stated range were 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 result as values within the range. As used herein, the terms “about” and “approximately” when referring to a numerical value have their plain and ordinary meaning to one of ordinary skill in the art in the field or fields most closely related to the disclosed subject matter or the field related to the range or element being discussed. The amount of expansion from the strict numerical boundaries depends on many factors. For example, some of the factors that can be considered include the criticality of the element and / or the effect that a given amount of variation will have on the performance of the claimed subject matter, as well as other considerations known to those skilled in the art. As used herein, the use of different amounts of significant digits for different numerical values does not imply that the use of the term “about” or “approximately” will be restricted to expanding a particular value or range in a particular manner. Thus, generally, “about” or “approximately” will expand the numerical value. Moreover, the disclosure of a range is intended to be a continuous range, including each and every value between the minimum and maximum values, plus the expansion of the range provided by the use of the term “about” or “approximately.” Thus, reference to a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated in the specification as if it were individually recited herein.

[0028] 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 combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B, and C.

[0029] As used herein, “have,” “having,” “include,” “including,” “comprise,” “comprising,” and the like are used in their open-ended, conventional sense, and typically mean “including, but not limited to.”

[0030] “Optional” or “optionally” means that the subsequently described element, component, or condition can or can not be present, and that the description includes instances where the element, component, or condition is present and instances where it is not.

[0031] “Porous” means having a porosity greater than zero. Porosity is a measure of void space in a material or substrate or scaffold and is the fraction of the volume of voids to the total volume, between 0 and 1, or expressed as a percentage between 0% and 100%. Porosity is determined by dividing the volume of voids by the total volume of the material to determine the 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).

[0032] In this document, relational terms such as first and second, top and bottom, and the like can be 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.

[0033] Unless otherwise indicated, all scientific and technical terms used herein have the meanings that are commonly understood by one of ordinary skill in the art. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of the disclosure.

[0034] B. INTRODUCTION

[0035] The present disclosure relates to the field of mass cell production, including for cultured meat and stem cells. It also relates to support structures for polymer scaffolds during large-scale and commercial-scale mass cell production, and bioreactor systems with mass cell production using such support structures. The present disclosure further relates to fixed bed reactors for mass cell production, the fixed bed reactors comprising a support structure and a polymer scaffold. The fixed bed reactors of the present invention can be used to produce large-scale and commercial quantities of cells, i.e., mass cell production, including but not limited to cultured meat and stem cells, including mesenchymal stem cells (MSCs, also known as stromal cells), induced pluripotent stem cells (iPSCs), and embryonic stem cells.

[0036] Cultured meat has emerged as an alternative food source to meet the growing dietary demand and address concerns related to animal welfare, climate change, and sustainability. However, large-scale and commercial-scale production of cultured meat remains challenging due to a number of factors, including underdevelopment of cell lines, serum-free media, bioreactor design, and scaffolds.

[0037] A major challenge for large-scale cell production is scaling up cell production. One challenge relates to the volumetric surface area requirements for large-scale or commercial production. Another challenge is that the steps of cell expansion and cell differentiation require very different physical environments and nutrient conditions, and performing these steps in separate systems is expensive in terms of equipment and in terms of processing time. Large-scale or commercial-scale production refers to production of 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 more of cultured cells (typically large-scale production ranges from about 100 g to about 1000 g of cultured cells, while commercial production is in quantities over 1 kg). Biological production using adherent culture bioreactors typically supports cell culture to a surface density of about 500,000 cells / cm 2 To produce 1 kg of cell mass (about 3 x 10 11 to 5 x 10 11 cells), about 60 m 2 to about 100 m 2 of effective surface area is required for cell culture. The typical mass of a single cell is about 2 ng to about 3 ng.

[0038] The present disclosure provides a support system for a scaffold for large-scale cell production to address large-scale and commercial-scale production issues. The support structure of the present disclosure is advantageous because currently available scaffold structures are not suitable for large-scale and commercial-scale production. The present disclosure also provides a fixed bed reactor comprising a support structure and a polymer scaffold.

[0039] The bioreactor system of the present disclosure is advantageous in that 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 reduces processing time for mass cell production as the bioreactor system uses perfusion culture. Advantageously, a switch from cell proliferation (also known as expansion) media to cell differentiation media can be made and the ability to support cell proliferation and differentiation within the same system is leveraged using a porous scaffold (e.g., a polymeric scaffold). Cell proliferation is associated with the phase of cell growth and cell division. Cell differentiation is associated with the phase of the cell becoming a more specific cell type (e.g., mesenchymal stem cells differentiating into muscle cells (muscle cells)). It also addresses the additional problem of non-uniform flow and thereby the problem of unhealthy cell growth and even death due to compaction, packing, or collapse of the porous scaffold as back pressure increases as cells continue to grow within the mechanically fragile scaffold structure when there is no support structure to hold the scaffold in place as perfusion occurs.

[0040] 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 who practice the embodiments as described herein, or will be learned by practice of the embodiments as described herein, as will be realized by one skilled in the art upon reading the detailed description which follows in conjunction with the drawings where provided.

[0041] C. Support Structure

[0042] As disclosed herein, a support structure for a polymeric scaffold that facilitates large scale and commercial scale mass cell production has been developed. As Figures 1 to 4 As shown, the bioreactor system 100 includes a support structure 110. The support structure 110 includes an outer wall 125, an inner wall 160, and a plurality of channels 135 defined by the inner wall 160. In some embodiments, the outer wall is solid. A solid outer wall means that it is impermeable to liquids. The support structure (including its outer wall) can be cell-adherent or cell-non-adherent. In a preferred embodiment, the inner wall 160 around each channel is not cell-adherent, preventing cell attachment so that cells only grow within the polymeric scaffold.

[0043] According to one embodiment, the outer wall of the support structure has an outer edge and an inner edge. Figure 2 A non-limiting example of this embodiment is shown. In some embodiments, the distance between the outer edge and the inner edge of the outer wall of the support structure remains approximately constant around the outer wall. Figure 3A A non-limiting example of this embodiment is shown. In some embodiments, the distance between the outer edge and the inner edge of the outer wall of the support structure can vary around the outer wall to accommodate the shape and placement of individual channels of the plurality of channels in the support structure. Figure 3B A non-limiting example of this embodiment is shown. In one embodiment, the cross-sectional shape of the outer wall 125 is approximately circular, asFigure 2 and 3B As shown. The cross-sectional shape of the outer wall can be any desired shape, such as approximately circular, approximately rectangular, approximately square, approximately elliptical, or approximately polygonal.

[0044] The support structure includes an inner wall. The inner wall can be very thin, such that it occupies a small volume within the entire support structure, leaving a large void volume (void space) to accommodate the polymer scaffold. In one embodiment, the outer wall of the support structure is thicker than the inner wall. The thickness of the inner wall can be about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 0.5 mm, about 0.2 mm to about 2 mm, about 0.2 mm to about 1 mm, or about 0.2 mm to about 0.5 mm. In one embodiment, the support structure is a modular structure, such as... Figure 4 As illustrated in the example, the thickness of the outer wall 125 of the support structure 110 is approximately 0.5 mm to approximately 5 mm, approximately 0.5 mm to approximately 4 mm, approximately 0.5 mm to approximately 3 mm, approximately 0.2 mm to approximately 2 mm, approximately 1 mm to approximately 5 mm, approximately 1 mm to approximately 4 mm, approximately 1 mm to approximately 3 mm, or approximately 1 mm to approximately 2 mm. In another embodiment, the support structure is a freestanding structure, such as... Figure 1 As illustrated in the examples, the outer wall 125 of the support structure 110 can be thicker than when the support structure is a modular structure to meet the mechanical stability and integrity requirements of the bioreactor and production operations. In such freestanding structures, the thickness of the outer wall 125 of the support structure 110 is approximately 2 mm to approximately 20 mm, approximately 2 mm to approximately 10 mm, approximately 5 mm to approximately 20 mm, or approximately 10 mm to approximately 20 mm. The thickness of the outer wall 125 of the support structure 110 depends on the size of the reactor. The larger the reactor, the greater the thickness of the outer wall.

[0045] 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 about 20 cm to about 500 cm, about 20 cm to about 400 cm, about 20 cm to about 300 cm, about 20 cm to about 200 cm, about 30 cm to about 300 cm, or about 30 cm to about 200 cm. In one embodiment, the diameter of each of the plurality of channels in the support structure is about 0.5 cm to about 10 cm, about 0.5 cm to about 8 cm, about 0.5 cm to about 5 cm, about 1 cm to about 10 cm, about 1 cm to about 8 cm, about 1 cm to about 5 cm, about 2 cm to about 10 cm, about 2 cm to about 8 cm, about 2 cm to about 5 cm, about 5 cm to about 50 cm, or about 5 cm to about 40 cm. Because the scaffold structure (such as a polymer scaffold) itself is highly porous (due to the presence of an interconnected network of pores and because they are made of soft polymer or biopolymer materials), it can generally withstand well any dimensional variations (e.g., thickness and diameter) of the internal channels of the support structure.

[0046] When scaling up production (e.g., from 1 kg to 10 kg of cultured cells), it is preferable to keep the height of the support structure and the size of each of the multiple channels approximately the same, but to increase the diameter of the support structure proportionally, whether as a modular structure or as a standalone structure. In this way, both the operating parameters for cell attachment to the polymer scaffold and the total surface area can be linearly scaled up.

[0047] The support structure can be made of plastic, ceramic, glass, or other materials such as stainless steel. In one embodiment, the support structure is plastic or ceramic. In some embodiments, the support structure can be manufactured using a single process or technology (e.g., injection molding, extrusion, or 3D printing). In some embodiments, the outer wall of the support structure can be made of a different material than the inner wall of the support structure. For example, the outer wall can be made of a plastic material, and the inner wall can be made of a ceramic material. In such examples, the outer wall and the inner structure can be manufactured separately.

[0048] The support structure provides separation for the polymer (including biopolymer) scaffold. The outer walls of the support structure create internal spaces, which are spaces surrounded by the inner edges of the outer walls. In other words, the internal spaces have outer edges defined by the inner edges of the outer walls. The internal spaces have multiple inner walls. The spaces between the inner walls define individual channels among multiple channels. In some embodiments, the inner walls may be solid. In some embodiments, the inner walls may be porous. In some embodiments, the porosity of the inner walls is less than about 30%, about 20%, about 10%, about 5%, about 2%, or about 1%. In other embodiments, the inner walls (whole walls or alternative segments 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 segment of the inner wall may be porous, and another segment of the same inner wall may be non-porous.

[0049] Individual channels among the multiple channels can be of 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 of the multiple channels is open at each end of the channel, so that all cells can be collected after culture. In some embodiments, the inner wall is porous, and a certain percentage of the channels have a closed end at the end closest to the fluid inflow into the bioreactor system. In such embodiments, the percentage of channels with closed ends can be less than about 50%, about 40%, about 30%, about 20%, about 10%, or about 5%. In such embodiments, the polymer scaffold is placed within channels with two open ends, and once the culture medium flows into the channels with two open ends, the culture medium can diffuse into nearby channels with only one end open through the porous inner wall, which helps to minimize or eliminate back pressure during high-density cell culture. In these embodiments, cells are preferably introduced into the bioreactor system through a port located at the outlet of the bioreactor system, such that the cells are distributed only within a channel with two open ends. Once the cells have adhered to the polymer scaffold and begun to grow, culture medium can be introduced from the open end of the support structure (i.e., through the port located at the inlet of the bioreactor system).

[0050] exist Figures 3A-3B In the middle, the opening of the channel of the support structure 110 is shown to be hexagonal in shape. Figure 3A ) or approximately hexagonal shape ( Figure 3B ).like Figure 3AAs shown, the opening of the channel in the support structure 110 has a diameter D. In some embodiments of this disclosure, the diameter of the channel opening is from about 0.5 mm to about 50 cm, depending on the desired size of the bioreactor. In other embodiments, the diameter of the channel opening 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. The preferred diameter of the channel in the support structure depends on the type and mechanical properties of the scaffold structure (e.g., a polymer scaffold). Generally, the lower the stiffness of the scaffold structure, the smaller the diameter of the internal channel of the support structure can be. In some embodiments, the diameter along the length of the channel is approximately the same as the diameter of the channel opening.

[0051] The support structure disclosed herein provides additional strength in bioreactor systems for large-scale or commercial-scale cell production, and for high-density perfusion cell culture in large-scale or commercial-scale production. Polymer (including biopolymer) scaffolds are typically soft, highly porous, and frequently deform under mild pressure. As cells grow to high densities within a bioreactor, the culture medium flow rate needs to increase over time to meet the increasing pressure on the soft scaffold during perfusion due to the cellular metabolic demands for nutrients and oxygen, which in turn causes compaction, clumping, or collapse of the polymer scaffold. For high-density cell cultures using polymer scaffolds, the back pressure caused by the culture medium flow can be as high as 10 Pa to 1000 Pa, which can cause deformation of the polymer scaffold. The support structure described herein offers a solution to these problems by providing support for the polymer scaffold. Using the support structure in a bioreactor system allows cells to grow in a smaller and more uniform space, enabling cells to be more evenly distributed within the polymer scaffold across multiple channels without the problems just described. Furthermore, the inner wall can guide fluid flow and helps maintain the structural integrity of the polymer scaffold during perfusion cell culture. When the inner wall is porous, this can further guide fluid movement and reduce back pressure during high-density cell culture.

[0052] In some embodiments, the support structure will withstand a pressure of at least about 67,000 Pa (about 10 psi). The pressure can be monitored or tested using state-of-the-art sensors, such as PendoTECH disposable pressure sensors. As used herein, a support structure subjected to pressure means that it will not permanently collapse or deform. 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 of about 10 Pa to about 60,000 Pa, about 10 Pa to about 30,000 Pa, about 10 Pa to about 10,000 Pa, about 10 Pa to about 5,000 Pa, or about 10 Pa to about 1,000 Pa.

[0053] D. Polymer scaffold

[0054] The bioreactor system disclosed herein comprises a polymer (including biopolymer) scaffold. The polymer scaffold is a substrate that supports cell attachment, expansion, or differentiation during large-scale or commercial-scale mass cell production. In embodiments where the polymer scaffold is made of edible biomaterial, the entire final cell culture product can be ejected from the bioreactor system using high pressure or mechanical force and then used for downstream food processing. In embodiments where the polymer scaffold is made of a biocuttable material for cultured meat production, cells can be collected and harvested either by directly dissolving the scaffold within the bioreactor via an enzymatic reaction or by first ejecting the scaffold structure from the bioreactor and then dissolving the scaffold via an enzymatic reaction. The cells are collected and then used for downstream food processing. For stem cell culture, a scaffold made of a biocuttable material is preferred because it is necessary to collect cells with high viability (at least 90%). Automated in situ cell collection can be performed using the reactor of the present invention. Specifically, after cell culture is complete, the cells are washed with a buffered medium, followed by enzymatic dissolution of the scaffold, and then cell collection. All of these steps can be performed using the bioreactor system. The collected cells can then undergo downstream washing and concentration steps. Stem cells can grow into 3D structures (e.g., spheres, aggregates, organoids) within porous polymer scaffolds, depending on the type of stem cells and culture conditions.

[0055] In one embodiment, the polymer scaffold is made of a biocutterable material. A biocutterable material is one that can be degraded or broken down by enzymes. Suitable biocutterable biomaterials for preparing soluble scaffolds include, but are not limited to, animal-derived collagen, recombinant collagen, gelatin, polygalacturonic acid (PGA), pectin, dextran or other peptides, and cellulose. Polymer scaffolds formed from collagen can be degraded by collagenases, while polymer scaffolds formed from PGA or pectin can be rapidly degraded by combining EDTA (ethylenediaminetetraacetic acid) with pectinase. Scaffolds formed from gelatin or other peptides can be rapidly degraded by streptoprotein proteases. Proteases are mixtures of several nonspecific endopeptidases and exopeptidases that digest proteins into individual amino acids. Polymer scaffolds formed from dextran can be degraded by dextranases, while polymer scaffolds formed from cellulose can be rapidly degraded by cellulases. The amount and concentration of enzymes used to degrade the polymer scaffold depend on several factors, including, but not limited to, the size and structure of the scaffold, cell density, protocol (e.g., temperature, solution composition), and the time required to complete the degradation of the scaffold structure. The required amount and concentration of enzymes can be optimized experimentally. Scaffold cell product complexes are typically washed with a buffer solution first, followed by an enzymatic reaction. Higher concentrations of enzyme solutions can be used for faster degradation.

[0056] In one embodiment, the polymer scaffold can be made of edible biomaterials. Common edible materials suitable for preparing polymer scaffolds include, but are not limited to, natural or synthetic biomaterials such as polysaccharides (e.g., starch, alginate, carrageenan, chitosan, agarose, cellulose, nanocellulose fibers, carboxymethyl cellulose, soy protein, corn protein, pectin), peptides (e.g., collagen, gelatin, gluten), lipids (e.g., paraffin, shellac), and composites / 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).

[0057] In one embodiment, the polymer scaffold may be made of inedible biological materials. Common inedible materials suitable for preparing polymer 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 polymer not intended for animal food consumption.

[0058] In some embodiments, the polymer scaffold is made from a highly porous and edible decellularized natural material. In one embodiment, a decellularized spinach polymer scaffold made from decellularized spinach leaves can be formed using solvent extraction and buffer washing of the spinach leaves, as described elsewhere, e.g., 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 mechanically shaped (e.g., cut) and used to fill multiple channels within the support structure.

[0059] In one embodiment, the polymer scaffold is porous. The porous polymer scaffold may be commercially available (e.g., textured soy protein is widely available) or prefabricated in batches, and in some embodiments, the porous polymer scaffold is then mechanically shaped (e.g., cut) to fill multiple channels of the support structure.

[0060] In another embodiment, the porous polymer scaffold can be formed directly within multiple channels of the support structure. Common techniques for directly forming porous polymer scaffolds within multiple channels include, but are not limited to, particle leaching, melt molding, freeze drying, and gas foaming. For example, a high-porosity scaffold can be prepared by first providing a solution of gelatin, and hyaluronic acid can be poured in to fill the channels of the support structure. Subsequently, the filled support structure is rapidly frozen to form ice crystals within the simultaneously formed polymer scaffold structure, followed by thawing of the frozen support structure. By using different concentrations of gelatin and hyaluronic acid, and by controlling the crosslinking kinetics, the pore size and porosity of the porous polymer scaffold can be finely tuned. Detailed schemes for freeze-drying methods can be found, for example, Brougham, CM et al., Freeze-drying as a novel biofabrication method for achieving a controlled microarchitecture within large, complex natural biomaterial scaffolds. *Advanced Healthc. Mater.*, Nov 2017; 6(21). doi:10.1002 / adhm.201700598, which is incorporated herein by reference. The exact conditions of the freeze-drying process depend on the desired structure and porosity of the scaffold and the material used to prepare the scaffold. Typically, for high-volume cell production applications, polymer scaffolds with high porosity (i.e., >90%, >80%, >70%, >60%, or >50%) and large-sized pores (e.g., >500 μm, >250 μm, >100 μm, >50 μm) are required.

[0061] In some embodiments, the polymer scaffold is modified to contain cell attachment cofactors, which can be incorporated into the scaffold for cell culture. The cell attachment cofactors can be introduced using conventional methods (e.g., physical absorption or covalent coupling). In one embodiment, the cell attachment cofactor may comprise extracellular matrix proteins (e.g., fibronectin, collagen, fibroinogen, laminin), or peptides containing at least one cell attachment peptide motif, such as RGD or YIGSR (SEQ ID NO: 3). Such cell attachment cofactors are commercially available, such as those from Corning. ® Synthemax ® , used in polymer scaffolds.

[0062] In some embodiments, the polymer scaffold may comprise a covalently linked cofactor, the cofactor comprising a cell attachment peptide motif. In one embodiment, the cell attachment cofactor may be a peptide comprising at least one of the following: the amino acid sequence Arg-Gly-Asp (referred to as “RGD”), the amino acid sequence Pro-His-Ser-Arg-Asn (referred to as “PHSRN”) (SEQ ID NO: 1), the amino acid sequence Arg-Glu-Asp-Val (referred to as “REDV”) (SEQ ID NO: 2), the amino acid sequence Tyr-Ile-Gly-Ser-Arg (referred to as “YIGSR”) (SEQ ID NO: 3), the amino acid sequence Ile-Lys-Val-Ala-Val (referred to as “IKVAV”) (SEQ ID NO: 4), or the amino acid sequence Asp-Gly-Glu-Ala (referred to as “DGEA”) (SEQ ID NO: 5). 5) The amino acid sequence Gly-x-Hyp-Gly-Glu-Arg (referred to as "GxOGER", where "O" amino acid is hydroxyproline and "x" is a hydrophobic amino acid, such as 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 (referred to as "GxPGER", where "x" is a hydrophobic amino acid, such as 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 (referred to as "RRETAWA") (SEQ ID NO: 8).

[0063] In one embodiment, the polymer scaffold can be crosslinked with a drug that is generally considered safe (GRAS) by the U.S. Food and Drug Administration (FDA). In one specific embodiment, the natural product genipin can be used to crosslink the amine-containing polymer scaffold. In another embodiment, the amine-containing polymer scaffold crosslinked with genipin includes corn protein, soy protein, collagen, gelatin, chitosan, gluten, or other proteins. In another embodiment, food-safe enzymes, including transglutaminase, can be used to crosslink the polymer scaffold. In another embodiment, photoactivated crosslinking agents (including sodium benzoate) can be used to crosslink the polymer scaffold. In another embodiment, physical crosslinking methods can also be used to crosslink the polymer scaffold, including heat treatment of soy protein and corn protein. Common crosslinking agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) and glutaraldehyde do not have FDA generally considered safe (GRAS) status and cannot be used in food production and should be avoided.

[0064] When the polymer scaffold is crosslinked, the stiffness of the polymer scaffold increases. In one embodiment, when the polymer scaffold is crosslinked, the stiffness increases by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 30%. The stiffness can be determined by using Young's modulus, such as that obtained by tensile testing.

[0065] E. Bioreactor System

[0066] The bioreactor system disclosed herein includes at least one support structure and a polymer support. The bioreactor system may further include at least one fluid handling unit. The fluid handling unit allows fluid to enter and exit the bioreactor system more uniformly. The fluid handling unit includes at least one port to allow fluid passage and may have a fluid flow guide plate (also referred to as a fluid flow distributor plate) to assist flow within the bioreactor system. The fluid flow guide plate may be made of any material that allows fluid to flow on the plate and distributes the fluid more uniformly across all channels of the support structure. It may have any shape that helps to distribute the fluid more uniformly across all channels of the support structure compared to a bioreactor system without a fluid flow guide plate.

[0067] In some embodiments, the bioreactor system may be a fixed-bed (also known as 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.

[0068] Figure 1 A mid-plan side view schematic diagram of a stand-alone bioreactor system with a fixed bed for mass cell production is shown. The bioreactor system 100 includes a first end portion 120 and a second end portion 130. A support structure 110 has a plurality of channels 135 and polymer supports 140 within the channels. The first end portion 120 may include at least one inlet port 112, and the second end portion 130 may include at least two outlet ports 115. In this non-limiting embodiment, fluid enters the bioreactor system 100 from the bottom of the system and exits from the top. It should be understood that the inlet and outlet ports can be reversed; that is, fluid can enter from the top and exit from the bottom.

[0069] In a stand-alone bioreactor system, the support structure 110 can be assembled into a bioreactor system 100 by attaching a first fluid flow treatment unit to a first end portion 120 of the support structure 110 and a second fluid flow treatment unit to a second end portion 130 of the support structure 110. The first fluid flow treatment unit includes an inlet port 112 and a fluid flow guide plate, the latter optionally contacting the first end portion 120 of the support structure 110. The fluid flow guide plate of the first fluid flow treatment unit is used to uniformly distribute fluid or culture medium over a region within the support structure 110. The second fluid flow treatment unit includes an outlet port 115 and a second fluid flow guide plate, the latter optionally contacting the second end portion 130 of the support structure 110. Waste fluid can exit the support structure 110 through the outlet port 115 and the second fluid flow guide plate. When both the fluid flow guide plate and the first and second fluid flow treatment units are present, the two fluid flow guide plates can have the same design, or each fluid flow guide plate can have a different design. In a stand-alone bioreactor system, the outer wall 125 of the support structure 110 serves as the outer shell container of a polymer scaffold 140, which is housed within a plurality of channels 135 within the support structure 110. When the culture medium flows into the fluid flow guide plate through the inlet port 112, the medium is more evenly distributed into the plurality of channels 135 of the support structure 110, passes through the porous scaffold 140 within the channels 135, and ultimately reaches the second fluid flow guide plate and flows out of the bioreactor through the outlet port 115. Similarly, it should be understood that the inlet and outlet ports can be reversed; that is, fluid can enter from the top and exit from the bottom.

[0070] Figure 2A top view of a schematic diagram of a bioreactor system having a fixed bed for mass cell production is shown. The bioreactor system includes a support structure 110 having an outer wall 125 and an inner wall 160, wherein the inner wall 160 is configured to generate a plurality of channels, and wherein the channels are filled with a polymer scaffold 140. The outer wall 125 includes an outer edge 145 and an inner edge 150.

[0071] Figure 3A Cross-sections of multiple channels 135 of the support structure are shown (in Figure 3A In this configuration, the channels are located inside a hexagonal shape. Each channel has a hexagonal shape with a diameter of "D", which is a cross-sectional area defined by an inner wall 160. Figure 3B This is a top view of the support structure 110 of the bioreactor system, wherein the support structure 110 has an outer wall 125 and a plurality of channels separated by an inner wall, and wherein the cross-sectional area of ​​each channel has an approximately hexagonal shape.

[0072] In another embodiment (a non-limiting example thereof) Figure 4(As shown in the diagram), the support structure 110 of the polymer scaffold 140 is a modular structure, meaning it can be placed inside the outer shell container 190 such that the outer wall 125 of the support structure 110 is in close contact with the inner wall of the outer shell container 190, and once assembled into the bioreactor system 100, the culture medium will not flow between the outer shell container 190 and the modular support structure 110. In some embodiments, a sealing structure (e.g., an O-ring) 192 is added to seal any gaps between the inner wall of the outer shell container and the outer wall 125 of the support structure 110. Methods for forming a tight seal between the end of the support structure and the fluid processing unit include O-rings, adhesive-based bonding, ultrasonic welding, and clamping mechanisms. The support structure 110 has the polymer scaffold 140 within a plurality of channels 135. In some embodiments, the bioreactor system 100 also has a first fluid flow processing unit 111 and a second fluid flow processing unit 114, the first fluid flow processing unit having a fluid flow guide plate 113 and the second fluid flow processing unit having a fluid flow guide plate 116. In these embodiments, the support structure 110 is located between a fluid flow guide plate 113 at a first end portion of the bioreactor system 100 and another fluid flow guide plate 116 at a second end portion of the bioreactor system 100. The fluid flow guide plate 113 is placed at the first end portion after the inlet port 112 is placed on the outer casing 190, and the other fluid flow guide plate 116 is placed at the second end portion before the outlet port 115 is placed on the outer casing 190. When the culture medium flows into the fluid flow guide plate 113 through the inlet port 112, the culture medium is evenly distributed to all channels of the support structure 110, passes through the polymer support 140, and eventually reaches the other fluid flow guide plate 116, and flows out of the outer casing 190 through the outlet port 115. It should be understood that the inlet and outlet ports can be reversed; that is, fluid can enter from the top and exit from the bottom.

[0073] According to this disclosure, a single bioreactor system may contain more than one support structure. In one embodiment, the bioreactor system includes at least two support structures. In another embodiment, the bioreactor system includes at least two support structures configured in a stacked arrangement.

[0074] The support structure of the bioreactor system disclosed herein can have any of the configurations described herein. In one embodiment, the support structure of the bioreactor system comprises a honeycomb structure, wherein the internal space of the support structure includes inner walls configured to form channels with cross-sectional regions between the inner walls. The cross-sectional regions can be approximate shapes, such as circular, elliptical, triangular, quadrilateral, pentagonal, hexagonal, or octagonal. More specifically, in one embodiment, the support structure has inner walls configured to form channels with approximately hexagonal cross-sectional regions between the inner walls. Figure 3B The diagram illustrates a non-limiting example of a channel with an approximately hexagonal shape. The hexagon can be a regular hexagon or an irregular hexagon. In another embodiment, the support structure of the bioreactor system has a channel between its inner walls, the channel having a cross-sectional area that is approximately circular or approximately elliptical. In another embodiment, the support structure of the bioreactor system has inner walls configured to form a channel between the inner walls having a cross-sectional area with an approximately triangular shape. The triangular shape includes equilateral triangles, isosceles triangles, right triangles, or scalene triangles. In another embodiment, the support structure of the bioreactor system has inner walls configured to form a channel having a cross-sectional area with an approximately quadrilateral shape. The quadrilateral shape includes squares, rectangles, parallelograms, trapezoids, or rhombuses. Figure 2 The diagram illustrates a non-limiting example of a channel with an approximately square shape. In another embodiment, the support structure of the bioreactor system has an inner wall configured to form a channel with a cross-sectional area of ​​approximately pentagonal shape. The pentagon can be a regular pentagon or an irregular pentagon. In another embodiment, the support structure of the bioreactor system has an inner wall configured to form a channel with a cross-sectional area of ​​approximately heptagonal shape. The heptagon can be a regular heptagon or an irregular heptagon. In another embodiment, the support structure of the bioreactor system has an inner wall configured to form a channel with a cross-sectional area of ​​approximately octagonal shape. The octagon can be a regular octagon or an irregular octagon. In another embodiment, the support structure of the bioreactor system has an inner wall configured to form a channel with a cross-sectional area of ​​approximately nonagonal shape. The nonagon can be a regular nonagon or an irregular nonagon. In another embodiment, the support structure of the bioreactor system has an inner wall configured to form a channel with a cross-sectional area of ​​approximately decagonal shape. The decagon can be a regular decagon or an irregular decagon.

[0075] Liquid can be added to and / or removed from the bioreactor system of this disclosure. Liquid can also flow through the bioreactor system. Fresh liquid can be supplied to the cell cultures in the bioreactor system through at least one port, which allows fluid inflow into the bioreactor system. Waste liquid can be removed from the cell cultures in the bioreactor system through at least one port, which allows fluid outflow from the bioreactor system. In some embodiments, liquid can enter the 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 through the top of the system and exit from the bottom of the system.

[0076] In some embodiments, the liquid is a cell culture expansion medium. In some embodiments, the liquid is a medium that promotes cell attachment. In another embodiment, the liquid is a cell differentiation medium. In some embodiments, the liquid is a cell harvesting medium or a 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. The type of liquid entering the bioreactor system can be switched at any desired point in time during cell culture production. In one embodiment, the cell expansion medium can be switched to a cell differentiation medium. In other embodiments, the cell expansion medium can be switched to a cell harvesting medium. In yet another embodiment, the cell differentiation medium can be switched to a cell harvesting medium. Switching the cell culture medium allows cell attachment, cell expansion, and cell differentiation to occur within a single bioreactor system. In one embodiment, cell expansion and differentiation occur within a single bioreactor system. In another embodiment, cell attachment and cell expansion occur within a single bioreactor system. In yet another embodiment, cell attachment, cell expansion, and cell differentiation occur within a single bioreactor system.

[0077] A bioreactor system comprising at least one support structure and a polymer scaffold increases the density at which cells can be cultured in large-scale cell production. The bioreactor system disclosed herein can culture cells at densities of approximately 2000 cells / cm³. 2 Approximately 30,000 cells / cm 2 Approximately 5000 cells / cm 2 Approximately 25,000 cells / cm² 2 Approximately 5000 cells / cm 2 Approximately 20,000 cells / cm 2 Approximately 10,000 cells / cm³ 2 Approximately 20,000 cells / cm 2 Approximately 5000 cells / cm 2Approximately 15,000 cells / cm² 2 Approximately 5000 cells / cm 2 Approximately 10,000 cells / cm 2 Approximately 2,500 cells / cm² 2 Approximately 7,500 cells / cm 2 Or less than approximately 5,000 cells / cm 2 Cells are cultured at a specific seeding density. In one specific embodiment, when the support structure of the bioreactor system has an outer wall and multiple porous or non-porous inner walls, the bioreactor system can culture cells at a density of approximately 2000 cells / cm². 2 Approximately 30,000 cells / cm 2 Approximately 5000 cells / cm 2 Approximately 25,000 cells / cm² 2 Approximately 5000 cells / cm 2 Approximately 20,000 cells / cm 2 Approximately 10,000 cells / cm³ 2 Approximately 20,000 cells / cm 2 Approximately 5000 cells / cm 2 Approximately 15,000 cells / cm² 2 or approximately 5000 cells / cm 2 Approximately 10,000 cells / cm 2 Approximately 2,500 cells / cm² 2 Approximately 7,500 cells / cm 2 Or less than about 2,000 cells / cm 2 Cell culture seeding density of cells.

[0078] The bioreactor system disclosed herein can culture up to approximately 500,000 cells / cm³. 2 Approximately 1,000,000 cells / cm² 2 The cell density is determined by the culture duration and conditions. When cells are cultured, the final cell density depends on the culture duration and conditions. When the culture is complete (i.e., the cells are ready to be harvested for further downstream processing), the cell density achievable using the bioreactor system of this disclosure can be up to approximately 500,000 cells / cm³. 2 Up to approximately 1,000,000 cells / cm³ 2 Or approximately 500,000 cells / cm 2 Approximately 1,000,000 cells / cm² 2 Any value of .

[0079] While this disclosure includes a limited number of embodiments, those skilled in the art who benefit from this disclosure will understand that other embodiments can be devised without departing from the scope of this disclosure.

Claims

1. A fixed-bed bioreactor system, comprising: First end portion and second end portion; and At least one support structure, the at least one support structure comprising a solid outer wall, multiple inner walls, multiple channels and a polymer support; The channel is a void space defined by the inner wall, and The bioreactor system described herein can culture up to approximately 1,000,000 cells / cm³. 2 . cells.

2. The bioreactor according to claim 1, wherein the bioreactor system culturees approximately 500,000 cells / cm³. 2 Approximately 1,000,000 cells / cm² 2 . cells.

3. The bioreactor system of claim 1, wherein the bioreactor system produces approximately 2,000 cells / cm³. 2 Approximately 30,000 cells / cm 2 Cell culture seeding density of cells.

4. The bioreactor system according to claim 1, wherein the cross-sectional area of ​​each channel is selected 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 wall is porous.

7. The bioreactor system of claim 6, wherein the liquid is capable of flowing across the inner wall.

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

9. The bioreactor system of claim 1, wherein the polymer scaffold comprises a cell attachment aid factor.

10. The bioreactor system of claim 9, wherein the cell attachment cofactor 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 mixtures thereof; Where x is a hydrophobic amino acid and O is hydroxyproline.

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

12. The bioreactor system of claim 11, wherein the first fluid processing unit includes a first port and a fluid flow guide plate, and wherein the second fluid processing unit includes 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 processing unit, the first fluid processing unit comprising a first port and a first fluid flow guide plate; and The second fluid processing unit includes a second port and a second fluid flow guide plate. The internal space has a plurality of inner walls configured to create channels in the space between the inner walls, and a polymer support is accommodated within the channels.

15. The bioreactor support structure according to claim 14, wherein the cross-sectional area of ​​each channel is hexagonal.

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

17. The bioreactor support structure according to claim 14, wherein the inner wall is porous.

18. The bioreactor support structure of claim 17, wherein fluid can pass through the channel via the inner wall.

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

21. The bioreactor support structure according to claim 14, wherein the support structure is an independent unit.

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

23. A bioreactor system comprising: First end portion and second end portion; and At least one support structure, the at least one support structure comprising a solid outer wall, multiple inner walls, multiple channels and a polymer support; The channel is a gap space between the inner walls of the plurality of inner walls, and the polymer support is located within the channel. The support structure described herein can withstand a pressure of at least 1,000 Pa without permanent deformation.

24. The bioreactor system of claim 23, wherein the support structure can withstand a pressure of at least 10,000 Pa without permanent deformation.

25. The bioreactor system of claim 23, wherein the support structure can withstand a pressure of at least 30,000 Pa without permanent deformation.

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