Carriers for cell culture and harvesting and cell culture and harvesting devices comprising the same

Abstract

The present invention provides a 3D porous growth surface comprising a fabric with multiple mesh layers, which can be formed into a cylindrical fixed bed by rolling the mesh layers, or other shapes of fixed bed by stacking or filling the multi-layer mesh structure. The present invention also provides a method for enhancing cell growth by maintaining the pore size and structure of the mesh layers. The present invention further provides a method for enhancing cell recovery rate by using the mesh layers described above, which has less obstacles than the carriers made of non-woven fabric or other porous materials, thereby enhancing cell recovery rate. The present invention also provides a method for reducing particle generation during cell collection by sealing the edges of the multi-layer mesh structure to block the release of fibers or particles, and using the yarn diameter of the mesh layers to be larger than the cell wall size for easy filtration separation. The growth surface provided by the present invention can be easily formed into a fixed bed by simply rolling multiple mesh layers, which not only reduces manufacturing costs, but also facilitates mass production of carriers for fixed bed bioreactors.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 953,575, filed on December 25, 2019, entitled “A Cell Culture Substrate for Mass Production of Biological Quality”, the disclosure of which is incorporated herein by reference. Technical Field

[0002] This invention relates to the field of cell culture substrates, and in particular to a cell culture substrate for producing eukaryotic cell biomass. Background Technology

[0003] With the rapid development of biotechnology, cell culture techniques for prokaryotic or eukaryotic cells have become increasingly important. Generally, eukaryotic cells grow slowly and are easily damaged by shear stress and contamination. Most eukaryotic cells are anchorage-dependent, requiring attachment to a growth surface to grow. To culture these anchorage-dependent eukaryotic cells, various culture carriers with growth surfaces have been developed. Commonly used culture carriers include those with smooth surfaces made of dextran-based materials, porous matrices made of polyurethane or polyethylene terephthalate, and semi-permeable membranes with hollow fibers made of polysulfone or cellulose acetate. However, cell collection from these culture carriers is easily contaminated by debris particles released due to shear stress, especially with porous carriers, making cell recovery from the carrier pores particularly difficult. Therefore, scaling up adherent cell culture processes is slow, time-consuming, labor-intensive, and expensive. In light of this, it is necessary to develop suitable cell culture vectors to solve the aforementioned cell collection problems.

[0004] Currently, the main culture carriers used for adherent cells include two types: particulate smooth surface carriers (non-porous or with few pores) and porous carriers. Smooth surface carriers, limited by their own growth surface area, restrict the number of cells that can adhere and grow on them. On the other hand, porous carriers provide a three-dimensional cavity to accommodate cells, and their porosity provides additional growth surface area for cell adhesion, protecting cells from the indirect effects of shear stress caused during cell culture processes such as aeration, stirring, and feeding. However, collecting cells from porous carriers is not easy. The first-generation porous carrier used for cell recovery was the BioNOC II (registered trademark) carrier (CESCO Bioengineering Co., Ltd.), which is made of polyester non-woven fabrics. Cells are collected from porous BioNOC II carriers through a multi-layered structure formed by hot-pressing the pores created by horizontally stacked fibers. In BioNOC II, the openings between fibers are arranged in the same direction, making it easier to release cells compared to other porous materials (such as channel pore structures distributed randomly within a matrix). However, collecting cells using BioNOC II carriers has the following drawbacks: 1. The randomly stacked pores within the fibers form a multilayered structure with more than twenty layers, and the pores have diameters ranging from a few micrometers to several hundred micrometers. Due to the physical obstacle of these more than twenty fiber layers, cell release cannot be as direct as on a flat surface; 2. During cell collection, fibers are easily released simultaneously, causing cell contamination; 3. The released fibers have a similar size to the cells, making them difficult to separate from the collected cells. These processes of separating fibers from cells not only increase costs but also cause cell loss, thus hindering the application of BioNOC II carriers in cellular production. These drawbacks limit applications or increase production costs, especially for production targets that use cells as biological products.

[0005] Revolutionary advancements in biotechnology and genetic engineering have created high demand in the cellular products market, including protein drugs, cytokines, monoclonal antibodies, virus-related products, vaccines, nucleic acids, enzymes, and cells and / or tissues. To meet this demand, there is a growing need for efficient and economical production methods.

[0006] Eukaryotic cells, such as mammalian cells, have become a sought-after target for providing high-quality and high-yield proteins or cell products. Cultured mammalian cells have long been used for the production of vaccines, genetically engineered proteins, drugs, and other cell products. Generally, eukaryotic cells can be adherent, non-adherent, or a combination of both. Adherent cells require a growth surface to anchor, grow, and produce the desired cell products. For example, adherent cells can be embryonic stem cells, mesenchymal stem cells, fibroblasts, epithelial cells, and endothelial cells. Eukaryotic cells, such as lymphocytes, certain metastatic cells, and cancer cells, can be non-adherent and can grow in suspension. However, regardless of the type, most eukaryotic cells share common characteristics during culture (described in detail below), which play a crucial role in designing effective growth surfaces and culture apparatus.

[0007] The dependence of adherent cells on growth surfaces is crucial to cell viability and forms the basis of culture techniques for all cell types, including but not limited to traditional monolayer culture or culture using carrier and / or microcarrier systems. Since adherent cells can only proliferate after attaching to a suitable growth surface, the growth surface and culture procedures used to promote cell attachment are extremely important. Cell attachment involves adsorbing attachment factors (e.g., proteins) onto the culture surface, bringing cells into contact with the culture surface, attaching cells to a treated surface suitable for cell attachment, dispersing and replicating the attached cells throughout the growth surface until these cells come into contact with cells growing on another surface (i.e., contact inhibition).

[0008] For viable adherent cell culture to be successful, the culture apparatus needs to be equipped with a suitable culture surface or carrier, a mechanism for circulating specific cell culture media, and adequate aeration to provide sufficient dissolved oxygen to sustain cell growth. Several different cell culture methods exist, including: batch systems, which require added oxygen but do not provide additional nutrients during culture; fed-batch systems, which require monitoring and replenishment of nutrients and oxygen; and perfusion systems, which monitor and control nutrients and waste products by continuously replenishing fresh culture medium.

[0009] Several types of culture carriers are known in this art. For example, dextran-based microcarriers (e.g., Cytodex I, DEAE-dextran, and Cytodex III, porcine collagen-coated dextran; Cytiva, US, (formally GE Healthcare LifeSciences)) or polystyrene-based microcarriers (e.g., SoloHill, US) are also known. Microcarriers are typically very small, with diameters ranging from approximately 50 to 250 micrometers; however, larger or smaller microcarriers have also been used (US Pat. No. 5,114,855 issued May 19, 1992 to Hu et al.). The second type of cell culture carrier includes a porous matrix material made of ceramics or polyurethane foam; a fabric made of polyethylene terephthalate (PET); or a biodegradable material made of poly(lactic-co-glycolic acid) copolymer (PLGA), collagen, or chitosan.

[0010] Cell culture carriers can also be classified according to their surface characteristics. For example, there are generally non-porous / poreless carriers and porous carriers. Porous carriers usually have more advantages than non-porous carriers because they offer a larger surface area to volume ratio and provide insulated cells. Due to their porous nature, these carriers form multiple three-dimensional cavities on the growth surface, thereby maximizing cell adhesion and protecting cells from the indirect effects of shear stress caused during cell culture processes such as aeration, agitation, and feeding.

[0011] Many currently available cell culture systems employ porous and / or non-porous microcarriers. These microcarriers, such as the commonly used microcarrier beads, are frequently used in adherent cell production systems. These microcarriers must be used in conjunction with agitation and / or aeration. However, a common problem with microcarrier systems is that the agitation required to maintain cell culture can damage or even kill cells, thereby reducing the efficiency of the cell culture system and the required cell yield.

[0012] Microcarrier systems can also utilize tiny spheres made from ion exchange gels, dextran, polystyrene, polyacrylamide, or collagen-based materials. The choice of these materials depends on their compatibility with cells, their resistance to agitation, and the specific gravity required to keep the microcarriers continuously suspended in the culture medium. Microcarriers are typically kept suspended in the culture medium with slow agitation to ensure even distribution of nutrients and air to all cells. Currently, microcarrier systems are considered the most suitable for large-scale cell culture due to their highest surface area-to-volume ratio and ability to distribute nutrients evenly to cells.

[0013] However, current microcarrier systems have serious drawbacks, including high cost and high cell mortality, caused by high shear stress due to agitation and aeration during culture. Secondly, the most commonly used microcarriers utilize porous non-rigid dextran as the carrier matrix. This compressible matrix is ​​thought to reduce potential damage to the microcarriers and attached cells when collisions occur in the stirred reactor (Microcarrier Cell Culture: Principles and Methods, Pharmacia Fine Chemicals, Uppsala, Sweden, pages 5-33 (1981)). However, these porous microcarriers also have serious drawbacks in retaining cell products, leading to the adsorption of growth factors and other components from the culture medium (Butler, M., "Growth Limitations in Microcarder Cultures", Adv. Biochem. Eng. / Biotech. 4:57-84 (1987)).

[0014] U.S. Patent No. 5,015,576, issued by Nilsson et al. on May 14, 1991, relates to a method for generating cavitary particles by adding a water-insoluble solid, liquid, or gaseous cavity-generating compound to an aqueous solution of a matrix material. These particles are then dispersed in a water-insoluble dispersion medium, followed by cooling, covalent cross-linking, or polymerization to render the matrix insoluble in water. Optionally, after derivatizing the particles, the cavity-generating compound is washed away, and the particles can then be used as ion exchangers in gel filtration processes, hydrophobic chromatography, or affinity chromatography. These particles can also be used as microcarriers for culturing adherent cells.

[0015] U.S. Patent No. 5,385,836, issued by Kimura et al. on January 31, 1995, relates to a carrier for attaching animal cells during cell culture or for immobilizing animal cells. This carrier is produced by coating a cell attachment material containing a chitosan mixture onto a porous substrate. The porous substrate is a nonwoven fabric obtained by impregnating a nonwoven fabric web with a binder resin containing silk fibroin, gelatin, and chitosan. The coating process is completed by contacting the nonwoven fabric with an acidic aqueous solution of chitosan containing added protein and gelatin, drying the coated nonwoven fabric, and treating it with an alkali to make the chitosan less soluble.

[0016] U.S. Patent No. 5,565,361, issued by Mutsakis et al. on October 15, 1996, relates to a bioreactor with a motionless mixing element for immobilizing attached cells, which can be used to enhance cell culture and proliferation in a bioreactor. The bioreactor has a shell and a motionless mixing element to which cells can attach for growth and proliferation. Both the motionless mixing element and the bioreactor are made of porous fibrous sheet material, such as corrugated or braided wires, like stainless steel or titanium. The height and diameter of the fibers are predetermined to provide maximum surface area for attachment of the cells to be cultured.

[0017] U.S. Patent No. 5,739,021, issued by Katinger et al. on April 14, 1998, relates to a porous support for biocatalysts, comprising a water-insoluble inorganic filler and a polyolefin binder selected from polyethylene and polypropylene, and having open pores to allow cells to penetrate and grow within the pores. The density of these pores is approximately greater than 1 g / cm³. 3 .

[0018] Hillegas et al., in their US patent No. 6,214,618 published on April 10, 2001, disclose a method for preparing microbead carriers. These microbead carriers are prepared by forming functional groups with a lightly crosslinked styrene copolymer core on the surface of the beads, followed by washing the microbead carriers with alkaline and acidic solutions to make them suitable for cell culture. Alternatively, the microbead carriers can be made from a styrene copolymer core externally containing trimethylamine, which has been washed with alkaline and acidic solutions before use in cell culture.

[0019] Although various cell culture vectors have been disclosed in the aforementioned prior patent cases, none of these vectors can meet the requirements of high cell density, easy cell recovery, and no additional particle release during cell collection in 3D culture. Summary of the Invention

[0020] To address the aforementioned drawbacks, this invention provides a cell culture substrate: 1. A substrate using a mesh or mesh structure as a substitute for fibers; 2. A thin mesh structure is used, and this thin mesh structure is layered in a manner similar to a nonwoven fabric carrier (e.g., BioNOC II carrier); 3. The layers are sealed around their perimeter using heat pressing, ultrasonic treatment, or adhesive bonding to prevent the mesh fibers from detaching; 4. The layers are rolled, stacked, or filled into columnar or other shapes / structures to serve as a fixed bed in a fixed-bed culture system, particularly in TideMotion. TM Bellocell (registered trademark) and TideCell (registered trademark) bioreactor systems within the culture system. TideMotion TM A culture system is defined as a fixed bed with a porous matrix that is intermittently submerged and exposed to bring in nutrients and oxygen to allow cells to grow on / inside the porous matrix, while removing waste and excess carbon dioxide from the porous matrix. With this design, compared to nonwoven fabric carriers, the present invention has the following advantages: 1. The shape and size of the pores in each layer remain consistent; 2. The multi-layer thin-sheet mesh structure can be stacked, rolled, or made into other shapes to construct a packed bed for the bioreactor with higher cell culture density and higher cell recovery rate; 3. Compared to the twenty-plus layers of a nonwoven fabric carrier, the number of layers in the thin-sheet mesh structure of the present invention is reduced, which is more conducive to the release of cells from the carrier; 4. The mesh structure is more robust than nonwoven fabric and is less likely to release particles and fibers, especially after sealing the periphery of the multi-layer mesh structure, which can significantly reduce the release of fibers or particles; 5. The diameter of the yarns in the mesh structure is much larger than that of cells, so they can be easily filtered and separated from the cell fluid, reducing the cost and loss of cell purification and recovery; 6. By rolling, stacking, or filling the multi-layer thin-sheet mesh structure, the surface area available for cell growth can be maximized, especially for use in Bellocell and TideCell bioreactor systems. In summary, the carrier structure designed in this invention enables high-density cell culture and high-yield cell production.

[0021] Overall, this invention provides a method for significantly improving the culture efficiency and cell recovery rate of adherent cells by using a thin sheet or strip mesh structure and stacking this mesh structure as a cell growth surface and structure. More specifically, this invention provides a novel cell growth surface and structure, and a method for manufacturing the same. This invention provides a novel growth surface and structure for culturing cells that maximizes cell adhesion and cell viability, increases cell growth density, increases cell recovery rate, and prevents the release of particles from the carrier from the growth surface. Therefore, the unique cell culture growth surface, structure, and manufacturing method provided by this invention overcomes the shortcomings and deficiencies of the prior art and can be applied to cell / tissue culture for cell production applications.

[0022] This invention provides a novel carrier structure, which is a layered mesh structure, with each layer having a consistent pore structure and size. When this carrier structure is large, it can be easily rolled or stacked to form a single fixed bed unit. This carrier structure can also be small, formed by filling multiple carrier structures into a defined space to create a fixed bed. The perimeter of the layered structure formed by the multi-layered mesh stack is sealed by heat pressing, ultrasonic waves, or adhesive bonding, thereby preventing or reducing the release of mesh fibers or particles. The large-size sheets made of the mesh fabric can range from 1 cm × 20 cm to 10 m × 100 m, depending on the size of the fixed bed. More precisely, the sheets made of the mesh fabric can range from 3 cm × 50 cm to 2 m × 20 m. The matrix bed can also be formed by randomly disposing of multiple small carrier blocks with the same structure within a container. For small-sized carrier structures, the dimensions can be (0.3 cm - 1 cm) × (0.5 cm - 10 cm). More precisely, small sheets made of mesh fabric can be (0.5 cm - 1 cm) × (1 cm - 5 cm). A mesh structure is defined as a mesh (also called mesh fabric), typically defined as a fabric with numerous interlocking openings characterized by their open appearance and the spacing between the yarns. This mesh structure can be constructed by knitting, woven, extruded, or knotted methods and can be applied to various structures, including knitted fabrics, laces, or crocheted fabrics.

[0023] Nonwoven fabric is defined as a fabric-like material made of man-made staple fibers (short) and long fibers (continuous long fibers) bonded together by chemical, mechanical, thermal or solvent treatment.

[0024] The surface of the mesh structure can be treated to be biocompatible and hydrophilic, allowing cells to attach and grow on it. The outer surface of the mesh structure has another mesh layer with larger pores and coarser yarns. This mesh layer, which may or may not be surface-treated, primarily serves as a spacer layer, allowing culture medium or air to freely circulate between the multiple surface-treated mesh layers. A large matrix bed can be formed by curling together the stacked multi-layered mesh structures used for cell attachment and growth, as well as the mesh layers serving as spacers. The size of this large sheet made of mesh fabric can range from 1 cm × 20 cm to 10 m × 100 m, depending on the size of the fixed bed. More precisely, before curling and stacking, the size of the mesh structure can range from 3 cm × 50 cm to 2 m × 20 m. The matrix bed can also be formed by randomly disposing of multiple small carrier blocks with the same structure within a container. For small-sized carrier structures, the dimensions can be (0.3 cm - 1 cm) × (0.5 cm - 10 cm). More precisely, small sheets made of mesh fabric can be (0.5 cm - 1 cm) × (1 cm - 5 cm). The width of the warp and weft yarns within the mesh fabric can be from 50 μm to 1000 μm, more precisely, from 50 μm to 500 μm. This width is much larger than the cell diameter (approximately 5-20 μm), thus facilitating easy separation from cells even after detachment from the mesh fabric. It should be noted that the cross-section of the aforementioned warp and weft yarns can be cylindrical, elliptical, or rectangular, depending on the actual situation. Therefore, in this specification, the width or diameter of the yarn can be used to describe the size of the yarn. The aforementioned small carrier comprises a multi-layered mesh structure for cell attachment and growth, as well as mesh layers serving as spacers. This multi-layered mesh structure is best suited for TideMotion after being rolled or stacked to form a fixed bed for a bioreactor. TM Systems (such as Bellocell or TideCell). In these systems, cell culture is performed by intermittently submerging and exposing the fixed bed to the culture medium. Based on the TideMotion described above... TMThe system's cell culture mechanism allows the culture medium to leave the fixed bed while allowing fresh air to enter, thus maximizing the surface area for cell growth.

[0025] Considering the importance of cell culture and the shortcomings of known carrier systems in the art, this invention provides a cell culture carrier system that allows cells to be cultured at high density in a 3D structure and can be advantageously applied in culture containers or bioreactors with free-flowing culture media. The carrier system of this invention is unique in that it facilitates the recovery of cells or tissues after cell culture and minimizes the release of additional particles of non-cellular origin. Although particle release is still possible in the mesh structure, it can be easily removed by filtration because the diameter of the mesh lines (yarn) of the carrier is much larger than the cell size. Therefore, the surface and structure for cell culture growth provided by this invention overcome the shortcomings and deficiencies of the prior art.

[0026] This invention provides a novel and efficient growth surface and structure suitable for cell culture, enabling large-scale cell or tissue harvesting. The invention also provides a method for manufacturing this growth surface and structure. One object of this invention is, but is not limited to: providing a novel cell culture growth surface containing a consistent pore structure and size, thereby allowing cell attachment and resulting in more consistent cell growth; and providing a cell culture growth surface that can be integrated into TideMotion. TM Culture systems, such as the Bellocell and TideCell bioreactor systems, provide maximum cell growth surface; provide cell culture growth surface that facilitates cell or tissue recovery after culture; and provide cell culture growth surface that significantly reduces the release of additional non-cell-related particles during cell culture and cell recovery.

[0027] This invention provides a three-dimensional porous growth surface, which is composed of a mesh structure made of natural or artificial polymer materials, particularly polyethylene terephthalate (PET). This growth surface can strengthen the cell growth surface, promote cell fixation and proliferation, maintain the integrity of the surface structure, facilitate cell recovery, and is less likely to cause the release of any artificial particles, thereby increasing cell yield.

[0028] One object of the present invention is to provide a carrier for cell culture, comprising a mesh structure unit, the mesh structure unit comprising at least one first mesh layer having a plurality of first mesh openings formed by the interweaving of first warp and first weft, and the first mesh layer being biocompatible and hydrophilic; and at least one second mesh layer having a plurality of second mesh openings formed by the interweaving of second warp and second weft, the second mesh layer being disposed parallel above or below the first mesh layer.

[0029] Preferably, the mesh structure unit may include two first mesh layers and one second mesh layer, wherein the second mesh layer is disposed between the two first mesh layers, or above the two first mesh layers, or below the two first mesh layers.

[0030] Preferably, the mesh structure unit may include at least three first mesh layers and one second mesh layer, wherein the second mesh layer is disposed between any two of the three first mesh layers, or above the three first mesh layers, or below the three first mesh layers.

[0031] Preferably, the material of the first mesh layer may include polyethylene terephthalate (PET), while the material of the second mesh layer may include polyethylene terephthalate (PET), polypropylene, polyethylene (PE), polytetrafluoroethylene (PTFE), or nylon.

[0032] Preferably, the aperture of the plurality of first meshes can be from 30 μm to 800 μm, while the aperture of the plurality of second meshes can be from 1 mm to 5 mm.

[0033] Preferably, the first mesh layer is a mesh material with a hydrophilic surface treatment, and the second mesh layer is a supportive mesh material.

[0034] Preferably, the carrier can be one or more pillars formed by curling up network structural units, one or more pillars formed by sequentially stacking multiple network structural units, or a stack formed by randomly stacking multiple network structural units.

[0035] Preferably, the periphery of the first mesh layer and the periphery of the second mesh layer are sealed by hot pressing, ultrasonic waves or adhesive bonding.

[0036] Preferably, in the first mesh layer, the apertures of the plurality of first mesh holes may all be the same or partially the same.

[0037] Preferably, in the second mesh layer, the apertures of the multiple second meshes may be all the same or partially the same.

[0038] Preferably, the width of any first meridian or any first parallel can be from 50 μm to 500 μm, while the width of any second meridian or any second parallel can be from 100 μm to 1000 μm.

[0039] Preferably, the width of any first meridian or any first parallel can be from 50 μm to 250 μm, while the width of any second meridian or any second parallel can be from 250 μm to 500 μm.

[0040] According to the above, a cell culture device includes a culture chamber and a carrier for cell culture. The carrier is disposed in the culture chamber and has a mesh structure unit. The mesh structure unit includes at least one first mesh layer having a plurality of first mesh openings formed by the interweaving of first warp and first weft, and the first mesh layer is biocompatible and hydrophilic; and at least one second mesh layer having a plurality of second mesh openings formed by the interweaving of second warp and second weft, the second mesh layer being disposed parallel above or below the first mesh layer.

[0041] More specifically, the novel growth surface provided by this invention comprises at least one layer of PET-treated mesh structures with pores of 30 μm to 800 μm for cell attachment and growth; and a surface-treated or untreated mesh layer with pores of 1 mm to 5 mm, preferably made of polypropylene or nylon, which acts as a spacer to facilitate nutrient and air circulation between the multiple mesh structure layers. By curling, stacking, or filling the stacked multilayer mesh structures for cell attachment and growth with the mesh layers serving as spacers, a matrix bed can be formed for packed-bed bioreactors, particularly Bellocell or TideCell bioreactors. This surface-treated PET multilayer mesh structure can be at least one layer or up to ten or more layers, depending on the thickness of the mesh structure and / or the ease of cell culture and cell recovery. When the carriers of the present invention are formed into carrier beds via coiling, stacking, or filling, and when applied to Bellocell or TideCell cell culture systems, they achieve maximum biomass production results. This is because the TideCell cell culture system uses TideMotion. TMThe cultivation mechanism involves intermittently submerging and exposing the carrier to the culture medium. (TideMotion) TM In this culture mechanism, even when the packed bed is tightly stacked (e.g., rolled, stacked, or filled), oxygen can still enter the substrate bed without causing cell drowning. Other cell culture bioreactors cannot perform cell culture under such high-density packing conditions. Therefore, the present invention, combined with the Bellocell or TideCell cell culture system, achieves maximum performance, as can be seen from the following examples and detailed description. Attached Figure Description

[0042] The figures provided below are merely illustrative and are not intended to limit the invention to any particular embodiment. These descriptions are to be understood as being incorporated herein by reference in conjunction with the accompanying drawings.

[0043] Figure 1 This is an exploded view of a novel carrier structure according to an embodiment of the present invention.

[0044] Figure 2 This is an exploded view of a novel carrier structure according to another embodiment of the present invention.

[0045] Figure 3 This is an exploded view of a novel carrier structure according to another embodiment of the present invention.

[0046] Figure 4 This is a schematic diagram of the peripheral sealing of the first mesh layer and the second mesh layer of the present invention.

[0047] Figure 5 This is a schematic diagram of a novel carrier structure according to another embodiment of the present invention.

[0048] Figure 6 This is a schematic diagram of a novel carrier structure according to another embodiment of the present invention.

[0049] Figure 7 This is a schematic diagram of a novel carrier structure according to another embodiment of the present invention.

[0050] Figure 8 This is a cross-sectional microscopic schematic diagram of a novel carrier structure according to an embodiment of the present invention.

[0051] Figure 9 This is a cross-sectional microscopic schematic diagram of a novel carrier structure according to another embodiment of the present invention.

[0052] Figure 10 This is a cross-sectional microscopic schematic diagram of a novel carrier structure according to another embodiment of the present invention.

[0053] Figure 11 This is a schematic diagram illustrating the use of a novel carrier structure according to an embodiment of the present invention.

[0054] Figure 12 This is a schematic diagram illustrating the use of a novel carrier structure according to an embodiment of the present invention.

[0055] Figure 13 This is a schematic diagram of mesenchymal stem cells growing on the carrier structure of the present invention.

[0056] Figure 14 This is a schematic diagram of mesenchymal stem cells growing on the carrier structure of the present invention.

[0057] Figure 15 This is a schematic diagram of the carrier structure, which consists of one layer of a mesh structure, after cell harvesting.

[0058] Figure 16 This is a schematic diagram of the carrier structure, which consists of one layer of a mesh structure, after cell harvesting.

[0059] Figure 17 The results show the number of cells and recovery rate recovered from the carrier structure in the BelloCell culture system.

[0060] Figure 18 This is the result of counting additional particles in a culture medium containing cells. Detailed Implementation

[0061] This invention provides a three-dimensional porous growth surface composed of a multi-layered mesh structure. It enhances the cell growth surface, promotes cell fixation, facilitates cell resorption, reduces particle release, and thus increases cell yield. This invention also provides a method for improving the growth surface, applicable to eukaryotic cell culture, comprising the following steps: providing a porous and three-dimensional growth surface; treating the growth surface to promote cell attachment; and placing the growth surface in any cell culture apparatus to allow cells to grow and proliferate therein. The aforementioned growth surface enables cell growth, and after culture, adding trypsin / EDTA, protease, collagenase, and / or DNase to the growth surface can release cells / tissues to obtain single cells.

[0062] The following description is merely illustrative and is not intended to limit the invention to any particular embodiment. These descriptions, in conjunction with the accompanying drawings, are provided to give the reader a more complete understanding of the invention. To avoid unnecessarily limiting the foregoing, preferred embodiments of the invention will be disclosed below. The embodiments of the invention are applicable to the culture of eukaryotic cells, and particularly to animal cells and / or mammalian cells. The invention specifically provides novel growth surfaces and structures suitable for culturing adherent cells, facilitating cell collection after growth is complete.

[0063] The novel growth surface of this invention is made of a multilayer mesh structure, comprising mesh layers for support or spacing, and one or more mesh structures for cell attachment and growth, wherein the mesh structure for cell attachment and growth may be at least one layer (e.g., Figure 1 As shown), two layers (as shown) Figure 2 As shown), three layers (as shown) Figure 3 (as shown), or more layers depending on the cells to be cultured.

[0064] Figure 1 This is a schematic diagram of a novel carrier structure according to an embodiment of the present invention, wherein the structure is made of two layers of sheet-like mesh structures. The top layer has a smaller pore size (30 μm to 800 μm), while the bottom layer has a larger pore size (1 mm to 5 mm). The top layer is made of a grid material that is easy to surface treat, such as polyethylene terephthalate (PET), while the bottom layer is made of a supportive grid material, such as polyethylene terephthalate (PET), polypropylene, polyethylene (PE), polytetrafluoroethylene (PTFE), or nylon. This material is rigid and supportive and can serve as a spacer and support during the construction of a matrix bed. The top layer mesh structure is surface-treated to be biocompatible and hydrophilic, thereby allowing cells to adhere to and grow thereon. The bottom mesh layer can serve as a spacer and does not require surface treatment; even if surface treatment is performed, its function will not be affected. The entire two-layer mesh structure is sealed around its perimeter using hot pressing, ultrasonic welding, or adhesive bonding (not shown in the diagram), which further prevents the release of mesh fibers and particles.

[0065] like Figure 1As shown, the carrier structure 100 of the present invention includes a mesh structure unit 30, which includes: at least one first mesh layer 10 having a plurality of first mesh openings 11 formed by interlacing a plurality of first warp lines 101 and a plurality of first weft lines 102, the first mesh layer 10 being biocompatible and hydrophilic; and at least one second mesh layer 20 having a plurality of mesh openings 21 formed by interlacing a plurality of second warp lines 201 and a plurality of second weft lines 202. The second mesh layer 20 is disposed parallel above or below the first mesh layer 10. The aperture of the plurality of second mesh openings 21 is larger than the aperture of the plurality of first mesh openings 11, and the opening direction of the plurality of first mesh openings 11 is perpendicular to the first warp lines 101 and the first weft lines 102, while the opening direction of the plurality of second mesh openings 21 is perpendicular to the second warp lines 201 and the second weft lines 202.

[0066] However, the present invention is not limited thereto; the mesh structure may include two first mesh layers 10 and one second mesh layer 20, such as... Figure 2 As shown. Figure 2 This is a schematic diagram of a novel carrier structure according to another embodiment of the present invention, wherein the structure is made of a three-layer mesh structure. The top two layers have small pore sizes (30 μm to 800 μm), while the bottom layer has larger pore sizes (1 mm to 5 mm). The top two layers are made of a mesh material that is easy to surface treat, such as polyethylene terephthalate (PET), while the bottom layer is made of a supportive mesh material, which can be made of materials such as polyethylene terephthalate (PET), polypropylene, polyethylene (PE), polytetrafluoroethylene (PTFE), or nylon, and has rigidity and support, and can be used as a spacer and support when constructing a matrix bed. The top two mesh structures are surface treated to be biocompatible and hydrophilic, thereby allowing cells to attach and grow thereon. The bottom mesh layer can serve as a spacer and does not require surface treatment; even if surface treatment is performed, its function will not be affected. The entire three-layer mesh structure is sealed around its perimeter using hot pressing, ultrasonic welding, or adhesive bonding to further prevent the release of mesh fibers and particles.

[0067] exist Figure 2 In this invention, the second mesh layer 20 is disposed below the two first mesh layers 10. However, the invention is not limited to this. The second mesh layer 20 may be disposed between the two first mesh layers 10 or on top of the two first mesh layers 10 as needed.

[0068] In another embodiment of the invention, the mesh structure may include three first mesh layers 10 and one second mesh layer 20, such as... Figure 3 As shown. Figure 3 This is a schematic diagram of a novel carrier structure according to another embodiment of the present invention, wherein the structure is made of a four-layer mesh structure. The top three layers have small pore sizes (30 μm to 800 μm), while the bottom layer has larger pore sizes (1 mm to 5 mm). The top three layers are made of a mesh material that is easy to surface treat, such as polyethylene terephthalate (PET), while the bottom layer is made of a supportive mesh material, such as polyethylene terephthalate (PET), polypropylene, polyethylene (PE), polytetrafluoroethylene (PTFE), or nylon, which is rigid and supportive and can be used as a spacer and support when constructing a matrix bed. The top three mesh structures are surface treated to be biocompatible and hydrophilic, thereby allowing cells to attach and grow thereon. The bottom mesh layer can serve as a spacer and does not require surface treatment; even if surface treatment is performed, its function will not be affected. The entire four-layer mesh structure is sealed around its perimeter using hot pressing, ultrasonic welding, or adhesive bonding to further prevent the release of mesh fibers and particles.

[0069] exist Figure 3 In this invention, the second mesh layer 20 is disposed below the three-layer first mesh layer 10. However, the invention is not limited to this. The second mesh layer 20 may be disposed between any two layers of the three-layer first mesh layer 10 or on the three-layer first mesh layer 10 as needed.

[0070] In the first mesh layer 10, the spacing between each first warp 101 and first weft 102 can be adjusted as needed to change the aperture of the plurality of first mesh openings 11 to be all or partially the same. In another embodiment, the spacing between the second warp 201 and second weft 202 in the second mesh layer 20 can also be adjusted as needed to make the aperture of the plurality of second mesh openings 21 all or partially the same. Furthermore, the diameter of the yarns, which are warp or weft, is much larger than that of cells, allowing them to be easily filtered from the cell fluid and separated from the cells. The width of the yarns within the mesh fabric can be from 50 μm to 1000 μm, more precisely, from 50 μm to 500 μm. Its width is much larger than the cell diameter (approximately 5-20 μm), therefore, even if detached from the mesh fabric, it is easily separated from the cells by filtration. Furthermore, as... Figure 4 As shown, the periphery of the first mesh layer 10 and the periphery of the second mesh layer 20 are sealed by heat pressing, ultrasonic bonding, or adhesive bonding (see...). Figure 4 The mesh material melts and fills the holes around the perimeter due to heat and pressure, thus preventing the formation or generation of additional particles during cell harvesting, such as particles from the carrier being released.

[0071] The second mesh layer 20, serving as a support or spacer, is rigid, for example, made of polypropylene or nylon, and is porous. It can be positioned above or below the mesh structures for cell attachment and growth, or between the growing mesh structures. Figure 5 , Figure 6 and Figure 7 As shown. When the entire culture medium curls up (as shown). Figure 5 and Figure 6 (as shown) or stack (as shown) Figure 7 (As shown) or filling, can be set in a bioreactor (e.g., Bellocell or TideCell bioreactor) to form a packed bed.

[0072] Figure 5 This is a schematic diagram of a novel carrier structure according to another embodiment of the present invention, in which a multi-layered mesh structure unit 30 can be rolled into a columnar shape to construct a carrier 200 as a substrate bed, wherein the bottom mesh layer serves as a spacer between the structures to allow the culture medium or oxygen to flow freely during cell culture. However, the present invention is not limited to a single columnar carrier structure, such as… Figure 6 As shown, the carrier 210 can be constructed by multiple mesh structural units 30 that are rolled into columnar shapes. Figure 7This is a schematic diagram of a novel carrier structure according to another embodiment of the present invention, in which multiple layers of mesh structure units 30 are sequentially stacked in a container to construct a carrier 300 as a matrix bed. The multiple layers of mesh structure units 30 can be cut into thin sheets of arbitrary shapes, preferably with dimensions from 2 cm × 2 cm to 2 m × 2 m, thus forming a matrix bed. However, the present invention is not limited thereto. In another embodiment of the present invention, multiple mesh structure units 30 can be randomly stacked to form a stack body as a matrix bed (see reference). Figure 10 ).

[0073] Figure 8 This is a cross-sectional microscopic schematic diagram of a novel carrier structure according to an embodiment of the present invention. Please also refer to... Figure 1 The carrier structure is coiled, stacked, or filled to form a matrix bed in the bioreactor. In this embodiment, the mesh structure unit consists of a hydrophilic mesh structure (first mesh layer 10) and an untreated mesh layer as a spacer (second mesh layer 20). The spacer mesh layer has a large pore size and provides support, allowing culture medium, solution, and air to permeate freely.

[0074] Figure 9 This is a cross-sectional microscopic schematic diagram of a novel carrier structure according to another embodiment of the present invention. Please also refer to... Figure 2 The carrier structure is coiled, stacked, or filled to form a substrate bed in a bioreactor. In this embodiment, the mesh structure unit consists of two layers of hydrophilic mesh structure (first mesh layer 10) and one untreated mesh layer as a spacer (second mesh layer 20). The spacer mesh layer has a large pore size and allows culture medium, solution, and air to permeate freely.

[0075] Figure 10 This is a cross-sectional microscopic schematic diagram of a novel carrier structure according to an embodiment of the present invention. Please also refer to... Figure 3 The carrier structure is coiled, stacked, or filled to form a substrate bed in a bioreactor. In this embodiment, the mesh structure unit consists of three layers of hydrophilic mesh structure (first mesh layer 10) and one untreated mesh layer as a spacer (second mesh layer 20). The spacer mesh layer has a large pore size and allows culture medium, solution, and air to permeate freely.

[0076] The mesh structure (first mesh layer 10) for cell attachment and growth can be made of any natural or artificial polymer, but is preferably made of polyethylene terephthalate (PET), which is easier to surface treat, and the mesh structure has precise and consistent opening dimensions (see reference). Figures 1 to 3The opening direction of the mesh 11 of the first mesh layer 10 is perpendicular to the first meridian 101 and the first parallel 102, while the opening direction of the mesh 21 of the second mesh layer 20 is perpendicular to the second meridian 201 and the second parallel 202 (the opening directions of the mesh 11 of the first mesh layer 10 and the mesh 21 of the second mesh layer 20 are consistent), thus optimizing cell culture and providing more consistent performance. Since the openings on the multi-layered mesh structure all face the same direction, it facilitates cell release from the growth surface and collection. This multi-layered mesh structure, consisting of one or more mesh structures for cell attachment and growth and mesh layers for support or spacing, can be sealed around its perimeter by heat pressing, ultrasonic treatment, or adhesive bonding, which can prevent or reduce the release of fibers or particles.

[0077] The porous structure of the mesh structure (first mesh layer 10) for cell attachment and growth has a pore size of 30 μm to 800 μm. Preferably, the pore size of the first mesh layer 10 can be in the range of 50 μm to 200 μm. The mesh layer (second mesh layer 20) used for support and spacing has a pore size of 1 mm to 10 mm. More preferably, the pore size of the second mesh layer 20 can be in the range of 2 mm to 5 mm.

[0078] The carrier structure of the present invention can maximize the growth surface area of ​​cells, promote cell adhesion and cell proliferation, and facilitate cell shedding, thus providing maximum cell density and cell products.

[0079] The novel growth surface provided by this invention can have any size, shape, form, structure, or geometry as required, provided it falls within the scope of protection of this invention. The growth surface can be in any suitable form, for example, it can be granular, strip-shaped, sheet-shaped, or any three-dimensional structure. In one embodiment, the mesh structure unit 30 can be sheet-shaped and can be rolled up to form a columnar carrier 200 (e.g., Figure 5 (as shown) or multiple columnar carriers 210 (such as Figure 6 As shown, these mesh structures can be placed within culture tanks, culture containers, or bioreactors (e.g., Bellocell or TideCell bioreactors). These mesh structures have a large size to facilitate curling into a single fixed bed unit, rather than being limited to placing small carriers in a defined area to form a fixed bed. The sheet size made of the mesh fabric can range from 1 cm (height) × 20 cm (width) to 10 m (height) × 100 m (width), depending on the size of the fixed bed. More precisely, the sheet size made of the mesh fabric can range from 3 cm (height) × 50 cm (width) to 2 m (height) × 20 m (width).

[0080] In another embodiment, the mesh structure unit 30 may be in the form of sheets and sequentially stacked to form a columnar carrier 300 or multiple columns, such as Figure 7 As shown. These mesh structures can be placed within culture tanks, culture containers, or bioreactors (e.g., Bellocell or TideCell bioreactors). These mesh structures have a relatively large size to facilitate curling into a single fixed bed unit, rather than being limited to placing small carriers in a defined area to form a fixed bed. The sheet-like mesh structures can be squares ranging from 1 cm × 1 cm to 2 m × 2 m, or circles with a diameter of 1 cm to 2 m. However, they are not limited to these dimensions; the mesh structure can be adjusted to any shape and size depending on the size and shape of the fixed bed. More precisely, the sheet-like mesh fabric can be squares ranging from 5 cm × 5 cm to 0.5 m × 0.5 m, or circles with a diameter of 5 cm to 0.5 m.

[0081] When the carrier structure of this invention is coiled and placed in a TideCell culture system, it provides maximized biomass production. This is because the TideCell culture system uses the TideMotion culture mechanism. TM This method involves intermittently immersing and exposing the culture medium to the substrate. Under this mechanism, even with tightly packed beds (e.g., rolled, stacked, or filled), oxygen can still enter the substrate bed without causing cell drowning. Other cell culture bioreactors cannot perform cell culture under such high-density packing conditions. Therefore, this invention, combined with the Bellocell or TideCell cell culture system, achieves maximum performance.

[0082] For those skilled in the art, it is understandable that certain characteristics of the growth surface can affect its performance. The carrier or growth surface, for example, its surface properties, carrier density, size, toxicity, and rigidity, all affect the performance of the growth surface, thereby affecting cell culture performance (particularly cell density and overall cell yield). Specifically, the pore size on the growth surface affects cell growth performance. The preferred pore size of the first mesh layer 10 is approximately 30 μm to 800 μm.

[0083] Here, the cultured cells comprise mostly eukaryotic cells, more specifically animal cells, and even more specifically mammalian cells. Examples include dermal fibroblasts, mesenchymal stem cells isolated from bone marrow, mesenchymal stem cells purchased from Wharton's Jelly, and adipose-derived mesenchymal stem cells isolated from adipose tissue.

[0084] After cell growth, cells can be collected according to standard cell collection procedures and treated with trypsin or other enzymes, such as collagenase, AccuMAX, Accutase, or TrypLE.

[0085] Example 1: Material Preparation

[0086] This example demonstrates cell growth on the carrier structure provided by this invention.

[0087] Materials and Methods: Polypropylene (PP) mesh layers with a mesh size of 3 mm × 3 mm and polyethylene terephthalate (PET) mesh layers with a mesh size of 200 μm × 200 μm were prepared. Each mesh layer measured 43 cm (length) × 3 cm (width). Two PET mesh layers were placed on a single PP mesh layer, and then the perimeter was heat-sealed by hot pressing. The structure was then subjected to a hydrophilic surface treatment and rolled into a cylindrical carrier 200 with a diameter of approximately 4 cm and a height of 3 cm. This cylindrical carrier 200 was placed in a Bellocell culture flask (culture chamber 400) (CESCO Bioengineering Co., Ltd.) and sterilized using gamma rays at a dose range of 25 kGy to 35 kGy. Figure 11 As shown. The carrier structure is rolled into a columnar microstructure, as shown. Figure 9 As shown. Before being rolled into a column, two layers of PET mesh and one layer of PP mesh are hot-pressed around the perimeter of the mesh layer to press the perimeter of the multi-layer mesh layer into a closed edge.

[0088] Example 2: Cell Culture

[0089] Approximately 3 × 10n mesenchymal stem cells (MSCs) isolated from human Wharton's Jelly were used. 7The cells were seeded in 100 ml of culture medium into BelloCell bottles containing the carrier structure of the present invention for 3 hours. After confirming that the seeding efficiency exceeded 90%, 400 ml of fresh culture medium was added to make a total culture medium of 500 ml. The BelloCell bottles were then installed on a BelloStage-3000 machine (CESCO Bioengineering Co., Ltd.) and cultured for 30 minutes at 37 degrees Celsius and 5% CO2 concentration using the following parameters: ascent speed: 1 mm / s; T_H: 10 seconds; descent speed: 1 mm / s; B_H: 30 seconds. Cell growth was checked on days 4 and 8. After confirming that the cells had reached confluence, the cells were harvested on day 8.

[0090] The carrier protected by this invention can also be a stack formed by randomly stacking multiple mesh structure units, such as... Figure 12 The diagram illustrates the use of a novel carrier structure according to an embodiment of the present invention. The mesh structure units 30 may be strip-shaped and are randomly stacked (or filled) in the culture chamber 400 to form a packed bed in Bellocell (CESCO Bioengineering Co., Ltd) for cell culture.

[0091] Figure 13 This is a partially enlarged schematic diagram showing the growth of mesenchymal stem cells on the carrier structure of this invention, wherein the cells grow in a dense, 3D structural form. Please also refer to... Figure 2 and Figure 13 In this embodiment, the carrier structure is composed of two layers of mesh structure (PET) and one layer of mesh (PP). The mesh openings 21 of the second mesh layer are larger than the mesh openings 11 of the first mesh layer. Therefore, under magnification under a microscope, the two mesh openings 11 can overlap within the range of the mesh openings 21, and cells can grow between them. Figure 14 This is a schematic diagram showing the growth of mesenchymal stem cells in the carrier structure of this invention. Please also refer to... Figure 3 and Figure 14 The cells grow in a dense and 3D structure. In this embodiment, the carrier structure is composed of a three-layer mesh structure (PET) and a one-layer mesh layer (PP). Therefore, the first mesh layer with three overlapping layers appears darker under the magnification of the microscope.

[0092] Figure 15This is a schematic diagram of a carrier structure used in an example of a carrier consisting of two layers of PET mesh structure and one layer of PP mesh structure. One layer of the PET mesh structure is peeled off for observation after cell harvesting. See also: Figure 2 and Figure 15 It was found that no residual cells were observed after cell harvesting. Figure 16 This is a schematic diagram of a carrier structure used in an example of a carrier consisting of a three-layer PET mesh structure and a single-layer PP mesh structure. One layer of the PET mesh structure is peeled off for observation after cell harvesting. See also: Figure 3 and Figure 16 It can be observed that after cell harvesting, only some extracellular matrixes and a small number of cells can be observed in a limited area.

[0093] Example 3: Cell / Tissue Release

[0094] After cell growth reached confluence, the growth surface was rinsed three times with D-PBS, followed by immersion in trypsin / EDTA for 5 minutes. Before cell separation from the growth surface, the trypsin / EDTA solution was removed. The BelloCell flasks were gently tapped and shaken, with the growth surface impacting the flask walls to force cell separation. The flasks were then rinsed again with D-PBS, and the tapping / shaking and rinsing process was repeated three times. All products were then collected, centrifuged, resuspended, and the cell number and viability were examined. Results are shown in [image / image / etc.]. Figure 17 According to data, the cell density grown in each BelloCell vial using the carrier structure of this invention can reach 9 × 10⁻⁶ cells / vial. 7 Here, carriers with two PET mesh layers and one PP mesh layer (Group 1) and carriers with three PET mesh layers and one PP mesh layer (Group 2) were used, both with a diameter of 4 cm and a height of 3 cm. Cell recovery rate was calculated by examining stained cells on the growth surface. By estimating the overall cell productivity, if the matrix bed were increased to fill the entire matrix bed space of the BelloCell flask, the cell productivity could exceed 8 × 10⁻⁶. 8 Compared to the BioNOC II vector, it can only achieve 2×10⁻⁶. 8 Cells were harvested at a recovery rate of approximately 70-80% (not shown). Using the carrier of this invention can increase cell productivity by four times. Furthermore, no artificial particles, such as…, were observed after cell harvesting. Figure 18 As shown. Regarding the issue of particle release, Bellocell flasks using the BioNOC II carrier may show up to 14,000 fibers after cell harvesting. In contrast, the carrier of this invention does not require additional procedures to remove artificial particles.

[0095] In summary, the cell culture carrier provided by this invention has a three-dimensional porous structure, which is composed of a multi-layered mesh structure with different pore sizes. The openings of these mesh layers are aligned, which strengthens the cell growth surface, promotes cell fixation, facilitates cell recovery, and thus increases cell yield. Furthermore, compared to traditional carriers using non-woven fabrics, the carrier structure provided by this invention reduces the generation of additional particles during cell harvesting, lowering production costs and minimizing wasted time.

Claims

1. A vector for cell culture and harvesting, characterized in that, It comprises a mesh structure unit, which includes: At least one first mesh layer having multiple first mesh openings formed by the interweaving of multiple first meridians and multiple first parallels, and the first mesh layer being biocompatible and hydrophilic; and At least one second mesh layer having a plurality of second mesh openings formed by the interweaving of a plurality of second warp lines and a plurality of second weft lines, the second mesh layer being disposed parallel above or below the first mesh layer; The mesh structure unit comprises two first mesh layers and one second mesh layer, wherein the second mesh layer is disposed between the two first mesh layers, or above the two first mesh layers, or below the two first mesh layers. The width of any first meridian and any first parallel is 50 μm to 250 μm, while the width of any second meridian or any second parallel is 250 μm to 500 μm. The aperture of any of the first meshes is 50 μm to 200 μm, and the aperture of any of the second meshes is 2 mm to 5 mm. The periphery of the first mesh layer and the periphery of the second mesh layer are sealed by hot pressing, ultrasonic waves or adhesive bonding. In the first mesh layer, all of the plurality of first mesh openings have the same aperture. In the second mesh layer, all of the plurality of second mesh openings have the same aperture.

2. The vector for cell culture and harvesting as described in claim 1, characterized in that, The mesh structure unit includes at least three first mesh layers and one second mesh layer, wherein the second mesh layer is disposed between any two of the three first mesh layers, or above the three first mesh layers, or below the three first mesh layers.

3. The carrier for cell culture and harvesting as described in claim 1, characterized in that, The material of the first mesh layer includes polyethylene terephthalate (PET), and the material of the second mesh layer includes polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE), polytetrafluoroethylene (PTFE), or nylon.

4. The carrier for cell culture and harvesting as described in claim 1, characterized in that, The first mesh layer is a single-grid material with a hydrophilic surface treatment, and the second mesh layer is a single-grid material with supporting properties.

5. The carrier for cell culture and harvesting as described in claim 1, characterized in that, The carrier can be one or more pillars formed by curling the mesh structure units, one or more pillars formed by sequentially stacking multiple mesh structure units, or a stack formed by randomly stacking multiple mesh structure units.

6. A cell culture and harvesting apparatus comprising a carrier for cell culture and harvesting as described in claim 1 and a culture chamber for containing the carrier for cell culture and harvesting.

7. The cell culture and harvesting apparatus as described in claim 6, characterized in that, One or more pillars formed by the curling of the mesh structure units are placed in the culture chamber; or one or more pillars formed by the sequential stacking of multiple mesh structure units are placed in the culture chamber; or a stack formed by the random stacking of multiple mesh structure units is placed in the culture chamber.

8. The cell culture and harvesting apparatus as described in claim 6, characterized in that, The mesh structure unit includes two or three first mesh layers and one second mesh layer, wherein the second mesh layer is disposed between any two first mesh layers, or above all first mesh layers, or below all first mesh layers.

9. The cell culture and harvesting apparatus as described in claim 6, characterized in that, The material of the first mesh layer includes polyethylene terephthalate (PET), while the material of the second mesh layer includes polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE), polytetrafluoroethylene (PTFE), or nylon.

10. The cell culture and harvesting apparatus as described in claim 6, characterized in that, The first mesh layer is a single-grid material with a hydrophilic surface treatment, and the second mesh layer is a single-grid material with supporting properties.

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