Carrier for cell biomass production and cell culture device including same

Abstract

The present invention claims and discloses a three-dimensional porous growth surface (carrier) made by multiple layers of net or mesh, specifically capable of forming a column-type fixed bed by rolling the layers, or other shapes of fixed bed by stacking or randomly arranging and packing the carriers, which is disclosed to enhance the cell culture surface and form a packed bed for the layers of cell culture, facilitating cell recovery after cell growth, and minimizing the possibility of particle release during culture and cell recovery around the multi-layer net / mesh structure, thus increasing cell production.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 953,575, filed December 25, 2019, entitled "Cell Culture Matrix for Biomass Production," the disclosure of which is incorporated herein by reference. The present invention relates to the field of cell culture supports, in particular to the field of cell culture supports for the production of eukaryotic biomass. [Background technology]

[0002] With the rapid advancement of biotechnology, any cell culture technique for prokaryotic or eukaryotic cells is becoming more and more important. In general, eukaryotic cells grow slowly and are susceptible to damage from shear stress and contamination. The majority of eukaryotic cells are anchorage-dependent and require a growth surface to attach and grow. To accommodate this type of eukaryotic cell culture, various supports with growth surfaces have been developed. Currently, the most available supports are smooth surface supports made from dextran-based materials, porous matrices made from polyurethane or polyethylene terephthalate, and semi-permeable membranes such as hollow fibers made from polysulfone or cellulose acetate. However, cell harvesting from those supports is time-consuming, susceptible to contamination, and often close to impossible, especially for supports with porous structures. Therefore, scale-up for anchorage-dependent cells has been a slow, labor-intensive, and expensive process. Due to this, there is a strong need to develop a culture support that can solve this cell harvesting problem.

[0003] There are two main types of carriers for anchorage-dependent cells, including particulate smooth surface carriers (non-porous or non-porous) and porous carriers. Smooth surfaces do not provide a large growth surface area by themselves, and therefore the number and growth of attached cells is limited. On the other hand, porous carriers provide at least one three-dimensional cavity for accommodating cells. The porosity of the carrier also creates additional surface area for cell scaffolding, which protects cells from indirect contact with shear stress caused by aeration, agitation, and feeding. However, the task of collecting cells from porous carriers is often very difficult. The first porous carrier capable of cell collection is BioNOC® II carrier (CESCO® Bioengineering Co., Ltd.), which is made of polyester nonwoven fabric. The ability of cell recovery from porous BioNOC® II carriers is due to the pores formed by stacking the fibers horizontally on a flat surface and heat pressing them to form a multi-layer structure where the morphology of the openings between the fibers all face the same direction (which makes cell release easier to achieve than other porous materials where the pores are shaped as channels in a matrix with random exit directions). However, there are some drawbacks to the design of BioNOC® II carriers, especially for cell recovery. 1. The pores formed by randomly stacking the fibers result in a multi-layer structure of 20 or more with irregular pore sizes ranging from a few microns to hundreds of microns. Due to the physical obstacles of having more than 20 layers of fibers, cell release is not as easy as with flat surfaces. 2. The fibers are easily released during the cell recovery process and are contaminated with the collected cells. 3. These fibers have similar dimensions to the cells, making it more difficult to separate them from the cells. The cost of isolation and the loss of cells during the process hinder the application of BioNOC® II carriers in the cell production field. The above drawbacks limit certain applications or increase production costs, especially when the cells themselves are the subject of biological products.

[0004] Revolutionary developments in biotechnology and genetic engineering have created a large market demand for pharmaceutical proteins, cytokines, monoclonal antibodies, viral products, vaccines, nucleic acids, enzymes, and cellular products such as cells and / or tissues. The demand for these products therefore creates an ever-increasing need for efficient and economical production methods.

[0005] Eukaryotic cells such as mammalian cells are the most popular for providing high quality and quantity of effective protein or cell products. Mammalian cell culture has long been used to produce vaccines, genetically modified proteins, pharmaceuticals, and other cell products. In general, eukaryotic cells can be anchorage-dependent, anchorage-independent, or both. Anchorage-dependent cells require a growth surface to anchor, mature, and produce the desired cell product. Examples of anchorage-dependent cells are eukaryotic stem cells, mesenchymal stem cells, fibroblasts, epithelial cells, and endothelial cells. Eukaryotic cells such as lymphocytes, some recombinant cells, and some cancer cells are "anchorage-independent" cells and can grow in suspension. Regardless of their type, most eukaryotic cells in culture have the following characteristics in common, which have important roles in the design of efficient growth surfaces and culture devices.

[0006] The attachment of anchorage-dependent cells to a growth surface is important for cell survival and is fundamental to all types of culture techniques, including but not limited to traditional monolayer culture or culture using carriers and / or microcarrier systems. Since the growth of anchorage-dependent cells can only occur after attachment to a suitable growth surface, it is important to use surfaces and culture procedures that promote cell attachment. Cell attachment involves the adsorption of attachment factors such as proteins to the culture surface, contacting the cells to the culture growth surface, attaching the cells to a treated surface suitable for cell attachment, and spreading and replicating the attached or adhered cells across the growth surface until they contact another surface-grown cell (i.e., "contact inhibition").

[0007] For the cultivation of viable anchorage-dependent cells, the culture requires a suitable culture growth surface or support, a mechanism for circulating culture medium specific to the type of cell being cultured, and proper aeration with a sufficient supply of gas to support and maintain cell growth. There are several different means for culturing cells: batch systems, where oxygen is added as needed but nutrients are not replenished during cultivation, fed-batch systems, where nutrients and oxygen are monitored and replenished as needed, and perfusion systems, where nutrients and waste products are monitored and controlled by continuous replenishment of fresh medium.

[0008] There are several types of culture supports currently known in the art. For example, dextran-based (e.g., Cytodex I, DEAE dextran, and Cytodex III, porcine collagen-coated dextran; Cytiva, USA, formerly GE Healthcare Life Sciences) or coated polystyrene-based (e.g., SoloHill, USA) microcarriers. Microcarriers are typically very small, with a diameter of about 50-250 micrometers, although larger or smaller sized microcarriers have been used (U.S. Patent No. 5,114,855 issued to Hu et al. on May 19, 1992). The second type of cell culture support includes porous matrix materials made from ceramics, polyurethane foams, or fabrics made from polyethylene terephthalate (PET), or biodegradable materials from PLGA, collagen, and chitosan.

[0009] Cell culture carriers can also be classified according to the porosity of the surface. For example, there are non-porous or non-porous carriers, and porous carriers. Porous carriers are generally more advantageous than non-porous carriers, since they provide a larger surface-to-volume ratio and protection for isolated cells. Due to their porous nature, these carriers form multiple three-dimensional cavities in the growth surface, thus maximizing cell attachment and protecting cells from being dislodged and / or damaged by shear stress resulting from aeration, agitation, and impact during the feeding and / or harvesting process.

[0010] Many cell culture systems currently available in the art employ microcarriers that are porous and / or non-porous or non-porous. These microcarriers, such as currently available microcarrier beads, are used in anchorage-dependent cell production systems. These microcarriers need to be used in conjunction with stirring equipment and / or aeration capabilities. However, a common problem with microcarrier systems is that the stirring action required to maintain cell culture can damage or even kill cells, thereby reducing the efficiency of the culture system and the production of desired cell products.

[0011] Microcarrier systems can also be fabricated in small spheres made of ion exchange gel, dextran, polystyrene, polyacrylamide, or collagen-based materials. These materials were selected for their compatibility with cells, their resilience to agitation, and their specific gravity that can keep the microcarriers suspended in growth medium. The microcarriers are generally kept in suspension in growth medium with gentle agitation in a container to ensure equal distribution of nutrients and air to all cells. Microcarrier systems are currently considered the most suitable system for large-scale cell culture because they have the highest surface-to-volume ratio, allowing for equal distribution of nutrients to the cells.

[0012] Nonetheless, current microcarrier culture systems have serious drawbacks. These drawbacks include high cost and high cell mortality due to exposure to high levels of shear forces generated by agitation and aeration during culture. The most commonly used microcarriers utilize porous, non-rigid dextran as a support matrix. The compressible matrix is ​​thought to reduce potential damage to the microcarriers and attached cells when the microcarriers collide in stirred reactors (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, resulting in adsorption of growth factors and other components from the medium (Butler, M., "Growth Limitations in Microcarrier Cultures", Adv. Biochem. Eng. / Biotech. 4:57-84 (1987)).

[0013] US Patent No. 5,015,576, issued May 14, 1991 to Nilsson et al., relates to making particles that enclose cavities by adding a water-insoluble solid, liquid, or gas cavity-generating compound to an aqueous solution of a matrix material. After forming the particles by dispersion in a non-aqueous dispersion medium, the matrix becomes insoluble in water by cooling, covalent cross-linking, or polymerization. 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, optionally after derivatization of the particles. The particles can also be used as microcarriers for culturing anchorage-dependent cells.

[0014] US Patent No. 5,385,836, issued to Kimura et al. on January 31, 1995, relates to a carrier for animal cell attachment in cell culture or for immobilization of animal cells. The carrier is produced by coating a porous substrate with a cell adhesive material in the form of a mixture including chitosan. The porous substrate is a nonwoven fabric prepared by impregnating the meshes of the nonwoven fabric with a binder resin including silk fibroin, gelatin, and chitosan. The coating is carried out by contacting the nonwoven fabric with a solution prepared by adding silk fibroin and gelatin to an acidic aqueous solution of chitosan to coat the nonwoven fabric, drying the coated nonwoven fabric, and treating the dried nonwoven fabric with alkali to render the chitosan insoluble.

[0015] U.S. Patent No. 5,565,361, issued to Mutsakis et al. on October 15, 1996, relates to a bioreactor with static mixing elements that uses a cell attachment method for enhancing cell culture and growth in the bioreactor. The bioreactor has a housing and static mixing elements, and allows the attached cells to grow and divide by attaching the cells to the mixing elements and a nutrient composition. The static mixing elements and bioreactor have a porous fibrous sheet material, such as a wrinkled or woven wire mesh material, such as stainless steel or titanium, with predetermined dimensions of fiber height and diameter to provide maximum surface area for attachment of the cells to be cultured.

[0016] U.S. Patent No. 5,739,021, issued to Katinger et al. on April 14, 1998, relates to a porous support for biocatalysis using a water-insoluble inorganic excipient and a polyolefin binder selected from polyethylene and polypropylene, with open pores that allow cells to pass through and grow within the pores. The density is 1 g / cm. 3 That's all.

[0017] U.S. Patent No. 6,214,618, issued to Hillegas et al. on April 10, 2001, relates to a method of making microcarrier beads by forming beads made of a lightly crosslinked styrene copolymer core with functional groups on the surface of the bead and washing the microcarrier beads with base and acid solutions to make the beads suitable for cell culture. The microcarrier beads can also be made of a styrene copolymer core with a trimethylamine exterior that has been washed in base and acid solutions to make the beads suitable for cell culture.

[0018] Although various carriers for cell culture are taught in the above art, none of the carriers can cover the requirements of high cell density in 3D culture, easy cell recovery, and no release of any particles along with the harvested cells. Summary of the Invention

[0019] To overcome the shortcomings of these carriers, our aqueous solution firstly uses net or mesh instead of fiber as the base of the carrier, secondly uses sheets of net / mesh to stack the net / mesh to mimic the structure of non-woven fabric carrier, i.e. BioNOC® II carrier, thirdly seals the edges surrounding the multi-layer net / mesh structure by heat, ultrasound or adhesive to avoid any release of the fabric, and fourthly rolls or stacks or packs (randomly arranged) multiple layers of net / mesh into a column or other defined shape or structure to act as a fixed bed in fixed bed culture systems, especially BelloCell® and TideCell® systems with TideMotion® culture mechanism, which definition refers to a culture method by intermittently immersing and floating a fixed bed with a porous matrix to deliver nutrients and oxygen to nurture the cells growing on / in the porous matrix, and to remove waste and excess carbon dioxide from the porous matrix. This design offers several advantages over nonwoven fabric carriers: 1. The pore (opening) shape and pore (opening) size in each layer are consistent. 2. Sheets made of multiple layers of net or mesh can be stacked or rolled or packed in random arrangement or other formats to build a matrix bed in a packed bed bioreactor to enable high cell density cell culture and cell harvesting. 3. The number of layers can be reduced over nonwoven fabric carriers which can facilitate cell release from the carrier. 4. The net or mesh is sturdy and less prone to release particles or fibers than nonwovens, especially after the perimeter of the net structure is sealed. 5. The yarn diameter of the net / mesh is much larger than the cells and any particles generated due to release from the net / mesh can be easily separated from the cells through filtration.6. Matrix beds by packing in rolls or stacks or random arrangements with multiple layers of net / mesh structures can maximize the surface area for cell growth, especially when applied to TideCell® or BelloCell® systems. As a result, high cell density culture and high yield cell production can be achieved with less chance of particle release through the novel design of the carrier.

[0020] The present invention generally teaches cell culture growth surfaces and structures made from sheets or strips, as well as multiple layers of nets or meshes, which significantly improve the efficiency of culturing anchorage-dependent cells and recovering cells from the growth surface. More specifically, the present invention teaches novel growth surfaces and structures, as well as methods for producing the same. The present invention claims and discloses novel growth surfaces and structures for culturing cells that maximize cell attachment and cell growth, increase cell density, enhance cell recovery, and avoid particulate release from the growth surface. Thus, the unique cell culture growth surfaces, structures, and methods of application according to the present invention remedy the shortcomings and deficiencies of existing technologies and make cell / tissue culture feasible for cell production applications.

[0021] The present invention teaches a novel carrier structure formed by multiple layers of net or mesh with consistent pore structure and the dimensions of each layer. The layers of net or mesh can have large dimensions, whereby they are rolled or stacked to form a single fixed bed unit. Or, the layers of net or mesh can have small dimensions, whereby small pieces of the carrier are placed in a defined narrow space to form the fixed bed. To prevent or reduce fibers or particles from being released from the net mesh, the periphery of the multi-layer net / mesh structure is sealed by heat pressing, ultrasonic pressing, or adhesive. The dimensions of the sheets made from the net / mesh fabric can be 1 cm x 20 cm to 10 m to 100 m, depending on the size of the fixed bed. More precisely, the dimensions of the sheets made from the net / mesh fabric can be 3 cm x 50 cm to 2 m x 20 m. The matrix bed can also be formed by combining multiple layers of cell culture net / mesh with layers of spacer net / mesh, randomly arranged and packed with small pieces of carrier with the same structure. When having small dimensions for placement on a fixed bed space, the dimensions of the sheet made by the net / mesh fabric can be 0.3cm-1cm x 0.5cm-10cm. More precisely, the dimensions of the sheet made by the net / mesh fabric can be 0.5cm-1cm x 1cm-5cm. The definition of net or mesh is as follows: Mesh (also called net fabric) is often defined as a fabric with a number of spaced apart holes, characterized by the open appearance of the net and the spacing between the yarns. It can be knitted, woven, extruded, or knotted (net) construction, and is available in a variety of configurations including knit, lace, or crochet fabric.

[0022] Besides woven fabrics, the definition of a nonwoven fabric is a fabric-like material made from staple fibers (short) and long fibers (continuously long) bonded together by chemical, mechanical, thermal, or solvent processes.

[0023] The net / mesh is surface treated to have biocompatibility and hydrophilicity to allow cell attachment and proliferation. The outer surface of the net / mesh has a layer of net / mesh with large pore size, large wall thickness, and with or without surface treatment requirements. It mainly functions as a spacer to allow medium or air to flow freely between the layers of treated net / mesh. Multi-layer net / mesh structure can form a large matrix bed by rolling with a sheet made by stacking multiple layers of cell culture net / mesh and a layer of spacer net / mesh. The dimensions of the sheet before rolling or stacking can be 1cm x 20cm to 10m x 100m depending on the size of the fixed bed. More precisely, the dimensions of the sheet made by net / mesh fabric before rolling or stacking can be 3cm x 50cm to 2m x 20m. The matrix bed can also be formed by combining multiple layers of cell culture net / mesh with a layer of spacer net / mesh, randomly arranged and packed with small pieces of carriers with the same structure. The sheet of net / mesh may have a small size of 0.3cm-1cm x 0.5cm-10cm, more preferably 0.5cm-1cm x 1cm-5cm. The yarn as a line of longitude or latitude for the sheet may have a width of 50μm-1000μm, preferably 50μm-500μm. The yarn is easily separated from the cultured cells by a filter because its width is much larger than the dimensions of the cells (about 5μm-20μm). From the cross-sectional view, the shape of one yarn as a line of longitude or latitude for the sheet may be a circle, an ellipse or a rectangle, but is not limited thereto. Therefore, the size of the yarn may be the dimension or width shown in the present invention. After multiple layers of the net / mesh structure are rolled, stacked or randomly arranged and packed to form a fixed bed for a fixed-bed bioreactor, the structure is best applied in TideMotion® systems such as BelloCell® or TideCell®.The mechanism of cell culture is performed by intermittently immersing the fixed bed in the culture medium and lifting the fixed bed out of the culture medium. Due to the TideMotion® culture mechanism, the culture medium can be removed from the fixed bed and fresh air can be allowed to enter the fixed bed. Thus, maximum surface area for cell growth can be realized.

[0024] In view of both the great importance of cell culture processes and the deficiencies of carrier systems currently known in the art, the present invention teaches and claims a culture carrier system that can enable cell culture in three-dimensional high cell density, which can be allocated to promote the free flow of culture medium in the culture vessel or bioreactor. The unique properties of the carrier system can facilitate the harvesting of cells or tissues after cell culture with minimal possibility of any particles being released from non-cellular sources. Any particles generated from the net or mesh due to the wall diameter larger than the cells can also be easily removed through simple filtration. Thus, the cell culture growth surface and structure according to the present invention cures the shortcomings and deficiencies of the existing technology.

[0025] The present invention provides efficient and novel growth surfaces and structures suitable for culturing cells and amenable to large scale cell or tissue harvesting, and methods for their manufacture. Objectives of the present invention include, but are not limited to, providing novel cell culture growth surfaces capable of supporting cell attachment and growth for more consistent performance consistent with aperture dimensions, providing cell culture growth surfaces that can maximize cell culture surface area by integrating with packed bed systems such as TideMotion® cell culture systems such as BelloCell® and TideCell®, providing cell culture growth surfaces that can facilitate harvesting of tissue or cell populations after culture is complete, and providing cell culture growth surfaces that minimize the probability of releasing non-cell associated particles during cell culture and cell harvesting.

[0026] The present invention teaches a three-dimensional porous growth surface made from a net or mesh with natural or artificial polymeric materials, particularly polyethylene terephthalate (PET), which is disclosed to reinforce the cell culture surface, promote cell immobilization, enhance cell growth, maintain the integrity of the surface structure, and allow cell recovery without releasing any artificial particles, thus increasing cell production.

[0027] Thus, the carrier for cell culture comprises a net / mesh structural unit, which comprises at least a first net / mesh film having a plurality of first meshes formed by intersecting a plurality of first longitude lines and a plurality of first latitude lines (the first net / mesh film is biocompatible and hydrophilic), and at least a second net / mesh having a plurality of second meshes formed by intersecting a plurality of second longitude lines and a plurality of second latitude lines (the second net / mesh film is parallel to the first net / mesh film and is disposed below or above the first net / mesh film).

[0028] Preferably, the net / mesh structural unit includes two layers of a first net / mesh film and one layer of a second net / mesh film, and the second net / mesh film is disposed between the two layers of the first net / mesh film, or on top of the two layers of the first net / mesh film, or under the two layers of the first net / mesh film.

[0029] Preferably, the net / mesh structural unit includes at least three layers of a first net / mesh film and one layer of a second net / mesh film, with the second net / mesh film being disposed between any two layers of the first net / mesh film, or on top of three layers of the first net / mesh film, or underneath three layers of the first net / mesh film.

[0030] Preferably, the material of the first net / mesh film includes polyethylene terephthalate (PET), and the material of the second net / mesh film includes polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE), polytetrafluoroethylene (PTFE) or nylon.

[0031] Preferably, any one opening (hole size) of the first mesh is 30 μm to 800 μm, and any one opening (hole size) of the second mesh is 1 mm to 5 mm.

[0032] Preferably, the first net / mesh film is a net / mesh material having a hydrophilic surface treatment, and the second net / mesh film is a supporting net / mesh material.

[0033] Preferably, the type of carrier is one or more columns formed by curling a net / mesh structural unit, one or more columns formed by laminating a plurality of net / mesh structural units in sequence, or a stack body formed by randomly arranging and packing the net / mesh structural units.

[0034] Preferably, the peripheral edges of the first net / mesh film and the second net / mesh film are sealed together by heat pressing, ultrasonic or adhesive treatment.

[0035] Preferably, the openings (hole size) of the first mesh of the first net / mesh film are completely or partially identical.

[0036] Preferably, the openings (hole size) of the second mesh of the second net / mesh film are completely or partially identical.

[0037] Preferably, any one of the first longitude lines and any one of the first latitude lines has a width of 50 μm to 500 μm, and any one of the second longitude lines and any one of the second latitude lines has a width of 100 μm to 1000 μm.

[0038] Preferably, any one of the first longitude lines and any one of the first latitude lines has a width of 50 μm to 250 μm, and any one of the second longitude lines and any one of the second latitude lines has a width of 250 μm to 500 μm.

[0039] Thus, the cell culture device includes a carrier for cell culture and a culture chamber in which the carrier for cell culture is disposed. The carrier for cell culture includes at least a first net / mesh film having a plurality of first meshes formed by intersecting a plurality of first longitude lines and a plurality of first latitude lines (the first net / mesh film is biocompatible and hydrophilic), and at least a second net / mesh having a plurality of second meshes formed by intersecting a plurality of second longitude lines and a plurality of second latitude lines (the second net / mesh film is parallel to the first net / mesh film and is disposed below or above the first net / mesh film).

[0040] More preferably, the novel growth surface according to the present invention is made of multiple layers of net or mesh, including at least one layer of surface-treated PET net / mesh with 30 μm-800 μm openings for cell attachment and growth, and a layer of treated or untreated net / mesh with 1 mm-5 mm openings, preferably polypropylene or nylon, which is a spacer that promotes nutrient and air exchange between the layers of carriers, to be rolled, stacked or packed together to form a matrix bed in a packed bed bioreactor, especially in BelloCell® and / or TideCell® bioreactors. The multiple layers of surface-treated PET net / mesh can be from at least one layer up to 10 or more layers, depending on the thickness of the net / mesh and / or the ease of cell culture and cell recovery. When the carriers of the present invention are in the form of rolls, stacks or packs to form a packed bed and when they are applied in BelloCell® or TideCell® cell culture systems, they contribute to the maximum results of biomass production. This is because the TideCell® cell culture system utilizes a culture mechanism called TideMotion® to intermittently submerge and float the culture carriers with culture medium. Under the TideMotion® cell culture mechanism, even dense packaging of packed beds such as rolls, stacks, or packs allows oxygen to enter the matrix bed and does not drown the cells. Other cell culture bioreactors do not have the ability to culture cells under such high density packaging conditions. Therefore, the current invention may have maximum results in terms of performance when combined with the BelloCell® or TideCell® systems. These and other embodiments are disclosed or will be apparent from or encompassed by the following detailed description. [Brief description of the drawings]

[0041] The following figures are given as examples and are not intended to limit the invention to any particular embodiment described. The description may be understood in conjunction with the accompanying figures, which are incorporated herein by reference. [Figure 1] FIG. 2 is an exploded schematic diagram showing the novel carrier structure of the present invention made by two layers of net / mesh in the present invention. [Diagram 2] FIG. 2 is an exploded schematic diagram showing the novel carrier structure of the present invention made by three layers of net / mesh in the present invention. [Diagram 3] FIG. 2 is an exploded schematic diagram showing the novel carrier structure of the present invention made by four layers of net / mesh in the present invention. [Figure 4] FIG. 2 is a top schematic diagram showing the novel carrier structure of the present invention, sealed around the perimeter by heat pressing, sonic pressing, or adhesive. [Diagram 5] FIG. 2 is a schematic side view showing a single roll carrier structure of the present invention. [Figure 6] FIG. 2 is a schematic side view of a multiple roll carrier structure of the present invention; [Figure 7] 1 is a schematic side view of a multiple stack carrier structure of the present invention; [Figure 8] FIG. 2 is an enlarged cross-sectional schematic diagram showing a novel carrier of the present invention having three bi-layer sheets of net / mesh according to the present invention. [Figure 9] FIG. 2 is an enlarged cross-sectional schematic diagram showing a novel carrier of the present invention having three triple layer sheets of net / mesh according to the present invention. [Figure 10] FIG. 2 is an enlarged cross-sectional schematic diagram showing a novel carrier of the present invention having three four-ply sheets of net / mesh in accordance with the present invention. [Figure 11] FIG. 2 is a partial three-dimensional view showing a cultivation carrier system having a roll carrier structure of the present invention. [Figure 12] FIG. 1 is a partial three-dimensional schematic diagram showing a cultivation support system having randomly arranged support structures of the present invention. [Figure 13]FIG. 2 is a partially microscopically enlarged top view schematic diagram showing mesenchymal stem cell proliferation in a carrier forming a dense three-dimensional structure in accordance with the present invention. [Figure 14] 1 shows mesenchymal stem cell proliferation in a carrier forming a dense three-dimensional structure in accordance with the present invention. [Figure 15] In the present invention, the carrier is shown by peeling off one layer to observe the cells remaining after cell collection. [Figure 16] In the present invention, the carrier is shown by peeling off one layer to observe the cells remaining after cell collection. [Figure 17] 1 is a table listing cell counts and recovery rates from carriers in BelloCell® containers after cell harvesting in the present invention. [Figure 18] 1 is a table listing the results of particle counts from harvest broth containing cells in the present invention. These and other embodiments are described in and / or are apparent from the detailed description that follows. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The present invention teaches a three-dimensional porous growth surface made from multiple layers of net / mesh to enhance cell culture surfaces, promote cell immobilization, enable efficient cell recovery, reduce particle release, and thereby increase cell or biological production. The present invention further teaches a method of modifying the growth surface for eukaryotic cells, which includes increasing the surface area by creating porosity and three-dimensional structure, treating the surface to promote cell attachment, promoting cell growth and proliferation, and placing the growth surface on any conventional cell culture device. The growth surface allows cell growth and releases the cell / tissue population by adding trypsin / EDTA, protease, collagenase and / or DNAse to obtain single cells after complete culture.

[0043] The following detailed description is given by way of example and is not intended to limit the invention to any particular embodiment described. The detailed description may be understood in conjunction with the attached figures, which are incorporated herein by reference. Without wishing to unnecessarily limit the above, the present invention shall be disclosed below with reference to certain preferred embodiments. The embodiments according to the present invention are suitable for eukaryotic cell culture, particularly animal and / or mammalian cells. The present invention teaches novel growth surfaces and structures suitable for culturing anchorage-dependent cells, which may be easily harvested after cell growth is complete.

[0044] The novel growth surface according to the present invention is made of a combination of multiple layers of net / mesh, including a layer of net or mesh for support and space, and one or more nets or meshes for cell attachment and cell growth. The layer of net or mesh for cell growth can be at least one layer (Figure 1), two layers (Figure 2), three layers (Figure 3) or more layers depending on the requirements for cell growth.

[0045] FIG. 1 shows the novel carrier structure of the present invention, which is made of two layers of net / mesh. The top layer is made of smaller opening size of 30 μm to 800 μm. The bottom layer is made of larger opening size of 1 mm to 5 mm. The top layer is made of a material that is easy to surface treat, such as polyethylene terephthalate. The bottom layer is made of any material, such as rigid polyethylene terephthalate (PET), polypropylene, polyethylene (PE), polytetrafluoroethylene (PTFE) or nylon, which can act as a spacer and supporter during the construction of the matrix bed. The top layer of net / mesh is surface treated to be biocompatible and hydrophilic, which can promote cell attachment and proliferation. The bottom layer of net / mesh, which acts as a spacer when constructing the matrix bed, does not require any treatment, even if the treatment does not affect its function. Furthermore, the periphery of the entire two-layer net / mesh structure is sealed by heat pressing, ultrasonic pressing, or adhesive to further prevent or reduce the release of net fibers and particles.

[0046] As shown in FIG. 1, the carrier or carrier structure 100 includes a net / mesh structural unit 30. The net / mesh structural unit 30 includes at least a first net / mesh film 10 (the first net / mesh film 10 is biocompatible and hydrophilic) having a plurality of first meshes 11 formed by crossing a plurality of first longitude lines 101 and a plurality of first latitude lines 102, and at least a second net / mesh film 20 (the second net / mesh film 20 is parallel to the first net / mesh film 10 and is disposed below or above the first net / mesh film 10) having a plurality of second meshes 21 formed by crossing a plurality of second longitude lines 201 and a plurality of second latitude lines 202. In one embodiment, the opening (hole size) of the second mesh 21 is larger than the opening (hole size) of the first mesh 11. Furthermore, the multiple first meshes 11 have an opening direction perpendicular to both the first longitude lines 101 and the first latitude lines 102. The multiple second meshes 21 have opening directions perpendicular to both the second longitude line 201 and the second latitude line 202 .

[0047] FIG. 2 shows a novel carrier structure made by three layers of net / mesh, which may include two layers of a first net / mesh film 10 and one layer of a second net / mesh film 20. FIG. 2 shows that the top two layers are made with smaller opening sizes of 30 μm to 800 μm, and the bottom layer is made with larger opening sizes of 1 mm to 5 mm. The top two layers are made of materials that are easy to surface treat, such as polyethylene terephthalate. The bottom layer is made of any material, such as rigid polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE), polytetrafluoroethylene (PTFE) or nylon, which can act as a spacer and supporter during the construction of the matrix bed. The top two layers of net / mesh are surface treated to be biocompatible and hydrophilic, which can promote cell attachment and proliferation. The bottom layer of net / mesh, which acts as a spacer when constructing the matrix bed, does not require any treatment, even if the treatment does not affect its function. Additionally, the perimeter of the entire three layer net / mesh structure is sealed by heat pressing, sonic pressing, or adhesive to further prevent or reduce the release of net fibers and particles.

[0048] 2, the second net / mesh film 20 is disposed below the two layers of the first net / mesh film 10, but this is not limited in the present invention. The second net / mesh film 20 can be disposed between or above the two layers of the first net / mesh film 10.

[0049] FIG. 3 shows a novel carrier structure made by four layers of net / mesh, which may include three layers of a first net / mesh film 10 and one layer of a second net / mesh film 20. The top three layers are made of smaller opening sizes of 30 μm to 800 μm. The bottom layer is made of larger opening sizes of 1 mm to 5 mm. The top three layers are made of materials that are easy to surface treat, such as polyethylene terephthalate. The bottom layer is made of any material, such as rigid polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE), polytetrafluoroethylene (PTFE) or nylon, which may act as a spacer and supporter during the construction of the matrix bed. The top three layers of net / mesh are surface treated to be biocompatible and hydrophilic, which may promote cell attachment and proliferation. The bottom layer of net / mesh, which acts as a spacer when constructing the matrix bed, does not require any treatment, even if the treatment does not affect its function. The perimeter of the four layer construction is sealed by heat pressing, sonic pressing, or adhesive to further prevent or reduce the release of net fibers and particles.

[0050] 3, the second net / mesh film 20 is disposed below three layers of the first net / mesh film 10, but this is not limited in the present invention. The second net / mesh film 20 can be disposed between any two layers of the first net / mesh film 10 or above three layers of the first net / mesh film 10.

[0051] In addition, to meet the design requirements, the openings (hole size) of the first mesh 11 of the first net / mesh film 10 can be fully or partially identical by adjusting the spacing between each of the first longitude lines 101 and each of the first latitude lines 102. As well as the second mesh 21 of the second mesh 20 with the help of adjusting the second longitude lines 201 and the second latitude lines 202. Furthermore, the diameter of the yarn as the longitude line or latitude line in the present invention is much longer than the cell, so that it can be easily filtered to separate from the cell. In one embodiment, the width of the yarn of the net / mesh structure can be 50 μm to 1000 μm, or exactly 100 μm to 500 μm, which is much larger than the cell diameter, which is 5 to 20 μm. Thus, any yarn that even falls off the net / mesh structure can be easily separated from the cell by any suitable filtering means. As shown in Figure 4, the peripheral edges of the first net / mesh film 10 and the second net / mesh film 20 are sealed together by heat pressing, ultrasonic, or adhesive treatment, and the peripheral openings are filled with the molten material resulting from the heat pressing treatment in Figure 4. Such an approach may prevent or reduce unwanted fibers or particles being released from the net / mesh structure during cell collection.

[0052] A rigid support (second net / mesh film 20), e.g., a net or mesh made from polypropylene or nylon, is a porous layer above or below the cell growth net / mesh to support and space (FIGS. 5, 6, 7) the carriers when they are rolled (FIGS. 5 and 6) or stacked (FIG. 7) together to form a packed bed in a bioreactor (e.g., a BelloCell® or TideCell® bioreactor).

[0053] FIG. 5 shows the novel carrier structure of the present invention, where the matrix bed can be constructed by rolling multiple net / mesh structural units 30 into a column to form a matrix bed 200, while the bottom layer net / mesh serves as a spacer between each structure to allow the culture medium or oxygen to flow freely during cell culture. However, such columns are not limited to the present invention. FIG. 6 shows a matrix bed 210 constructed by arranging multiple rolled net / mesh structural units 30. FIG. 7 shows the novel growth surface structure of the present invention, where the matrix bed 300 can be constructed by stacking the carriers of the present invention (net / mesh structural units 30) in a container, where the carriers are cut into any shape that can form the matrix bed 300, or sheets with dimensions preferably ranging from 2 cm×2 cm to 2 m to 2 m in size. However, in another embodiment, the matrix bed can be constructed by randomly arranging and packing many net / mesh structural units 30, such as those shown in FIG. 12.

[0054] Figure 8 is an enlarged cross-sectional schematic diagram showing the novel carrier of the present invention with three two-layer sheets of net / mesh. With reference to Figure 1 and Figure 8, Figure 8 shows a microscopic view of the novel carrier structure after being packed in rolls or stacks or randomly arranged to form a matrix bed in a bioreactor. The carrier is made by one layer of hydrophilic net / mesh (first net / mesh film 10) and one layer of non-treated net / mesh (second net / mesh film 20) as a spacer. The net / mesh for the spacer may have a larger diameter and allow the culture medium, solution, air to pass through freely.

[0055] Figure 9 is an enlarged cross-sectional schematic diagram showing the novel carrier of the present invention with three three-layer sheets of net / mesh. With reference to Figure 2 and Figure 9, Figure 9 shows a microscopic view of the novel carrier structure after being packed in rolls or stacks or randomly arranged to form a matrix bed in a bioreactor. The carrier is made by two layers of hydrophilic net / mesh (first net / mesh film 10) and one layer of non-treated net / mesh (second net / mesh film 20) as a spacer. The net / mesh for the spacer may have a larger diameter to allow the culture medium, solution, air to pass through freely.

[0056] Figure 10 is an enlarged cross-sectional schematic diagram showing the novel carrier of the present invention with three four-layer sheets of net / mesh. With reference to Figure 3 and Figure 10, Figure 10 shows a microscopic view of the novel carrier structure after being packed in rolls or stacks or randomly arranged to form a matrix bed in a bioreactor. The carrier is made by three layers of hydrophilic net / mesh (first net / mesh film 10) and one layer of non-treated net / mesh (second net / mesh film 20) as a spacer. The net / mesh for the spacer may have a larger diameter and allow the culture medium, solution, air to pass through freely.

[0057] The cell culture net or mesh (first net / mesh structure film 10) is made by any natural or artificial polymer, preferably polyethylene terephthalate (PET), which is easy to surface treat. Due to the net / mesh having precise and consistent dimensions of the openings, it can improve the culture with better and consistent performance. Referring again to Figures 1, 2 and 3, the net / mesh 11 of the first net / mesh film 10 is in an opening direction perpendicular to both the first longitude line 101 and the first latitude line 102. Similarly, the net / mesh 21 of the second net / mesh film 20 is open in a direction perpendicular to both the second longitude line 201 and the second latitude line 202. Therefore, it is beneficial for cell culture and consistent preformation that the opening direction of the net / mesh 11 of the first net / mesh film 10 is consistent with the net / mesh 12 of the second net / mesh film 20. Cell release and recovery from the growth surface may be facilitated due to the openings on multiple layers of the net or mesh facing in the same direction.Multi-layer net / mesh structures combining both cell growth layers and non-cell growth support layers are optionally sealed around the perimeter of the structure by heat pressing, ultrasonic, or adhesive treatment to prevent or reduce fiber and particle release.

[0058] The openings (pore size) in the porous structure for cell growth (first net / mesh film 10) may range from 30 μm to 800 μm. More preferably, the openings (pore size) of the first net / mesh film 10 may range from 50 μm to 200 μm. The openings (pore size) in the support net / mesh (second net / mesh film 20) may range from 1 mm to 10 mm. More preferably, the openings (pore size) of the second net / mesh film 20 may range from 2 mm to 5 mm.

[0059] Carriers according to the present invention provide maximum surface area to facilitate cell attachment, cell adhesion, cell proliferation and ease of cell removal, thereby providing maximum cell density and therefore maximum cell product.

[0060] The novel growth surfaces of the present invention can be of any size, shape, form, structure, or geometric configuration, as long as they are in accordance with the present invention. The growth surfaces of the present invention can be in any suitable form, such as pallets, strips, sheets, or any three-dimensional structure. In one embodiment, the growth surface is in the form of a sheet (net / mesh structural unit 30) that is rolled to form a column (matrix bed 200 shown in FIG. 5) or multiple columns (matrix bed 210 shown in FIG. 6) and placed in a culture tank, culture vessel, or bioreactor. For example, TideCell® or BelloCell® bioreactors. The layer of net or mesh has larger dimensions, whereby instead of placing small pieces of carriers in a restricted and defined area to form a fixed bed, it is rolled to form a single fixed bed unit. The dimensions of the sheet made by the net / mesh fabric before rolling can be from 1 cm (height) x 20 cm (width) to 10 m x 100 m, depending on the size of the fixed bed. More precisely, the dimensions of the sheet made by the net / mesh fabric can be 3cm (height) x 50cm (width) to 2m (height) x 20m (width).

[0061] In another embodiment, the growth surface is in the form of sheets (net / mesh structural units 30) that are stacked to form a column or multiple columns (matrix bed 300 shown in FIG. 7) and placed in a culture tank, culture vessel, or bioreactor. For example, TideCell® or BelloCell® bioreactor. The layers of net or mesh have larger dimensions, whereby instead of placing small pieces of carriers in a limited and defined area to form a fixed bed, they are stacked to form a single fixed bed unit. The dimensions of the sheets made by the net / mesh fabric before stacking can be from 1 cm×1 cm square or 1 cm diameter circle to 2 m×2 m or 2 m diameter circle or any other shape and dimensions, depending on the dimensions and shape of the fixed bed. More preferably, the dimensions of the sheets made by the net / mesh fabric can be from 5 cm×5 cm or 5 cm diameter circle to 0.5 m×0.5 m or 0.5 m diameter circle.

[0062] When the carrier of the present invention is in the form of a roll, and when the carrier is applied to the TideCell® cell culture system, it may contribute to the maximum result of biomass production. This is because the TideCell® cell culture system utilizes a culture mechanism called TideMotion® to intermittently submerge the culture carrier with culture medium and float the culture carrier. Under the TideMotion® cell culture mechanism, even a dense package of packed beds such as rolls, stacks, or packs allows oxygen to enter the matrix bed and does not drown the cells. Other cell culture bioreactors do not have the ability to culture cells under such a highly dense package condition. Therefore, the current invention may have the maximum result in terms of cell culture performance when combined with the BelloCell® or TideCell® system.

[0063] Those skilled in the art will appreciate that certain characteristics of a growth surface may have an impact on its performance. Support or surface characteristics such as surface properties, support density, size, toxicity, and stiffness may affect the performance of the growth surface and therefore the performance of the cell culture, particularly with respect to cell density and overall production of cell products. Specifically, the size of the pores of the growth surface may affect the performance of the cells. Those skilled in the art will appreciate that any known growth surface pore size is suitable, but the pore size is preferably in the range of 30 micrometers to 800 micrometers.

[0064] The cell culture can cover most eukaryotic cells, more specifically animal cells, more specifically mammalian cells, such as fibroblasts, mesenchymal stem cells from bone marrow, mesenchymal stem cells from Wharton's Jelly, adipose-derived mesenchymal stem cells from adipose tissue, etc.

[0065] After cell growth, cells can be easily removed and harvested from the growth surfaces of the invention by conventional cell harvesting procedures, such as through trypsinization or other enzymatic treatment with collagenase, AccuMAX, Accutase, TrypLE, and the like.

[0066] Example 1 Material preparation

[0067] This example describes cell growth on the inventive porous structure. Materials and Methods: A PP net with grid dimensions of 3 mm x 3 mm was prepared. A PET net with grid dimensions of 200 μm x 200 μm was prepared. The size of the net was 43 cm long x 3 cm wide. Two layers of PET net were placed on top of the PP net, and then the sheet was rolled to form a column (matrix bed 200 shown in Figure 5) of approximately 4 cm diameter and 3 cm height. The column was placed in a BelloCell® container (CESCO® Bioengineering Co., Ltd.) and sterilized with gamma radiation at a dose ranging from 25 to 35 kGy (shown in Figure 11). The microscopic structure after rolling into a column is shown in Figure 9. It should be noted that before rolling into the column, the two layers of PET net and one layer of PP net were sealed at the peripheral edges by heat, ultrasound, or adhesive to form an enclosed perimeter.

[0068] Example 2 Cell culture

[0069] Prepare mesenchymal stem cells (MSCs) isolated from human Wharton's Jelly, with a total of 3 × 10 MSCs in 100 ml culture medium. 7 The cells were placed in a BelloCell® container with the carrier of the present invention and seeded for 3 hours. After checking that the seeding efficiency was above 90%, 400 ml of fresh culture medium was added to bring the total volume of culture medium to 500 ml. Then, BioNOC® was installed in a BelloStage® 3000 machine (CESCO® Bioengineering Co., Ltd.) and operated with the following parameters: up rate: 1 mm / sec, T_H: 10 sec, down rate: 1 mm / sec, B_H: 30 min at 37°C and 5% CO2. Cell growth was checked on the 4th and 8th days. After confirming that the cell growth reached culture confluency, cell harvesting was performed on the 8th day.

[0070] The type of carrier in the present invention can be a stack pack, which is constructed by randomly arranging and packing a plurality of net / mesh structural units as shown in Fig. 12. Fig. 12 is a partial three-dimensional schematic diagram showing a culture carrier system having a randomly arranged carrier structure of the present invention. In Fig. 12, the net / mesh structural units 30 can be strips, which are randomly arranged or packed in the culture chamber 400 to form a matrix bed in the BelloCell® container for cell culture.

[0071] FIG. 13 shows mesenchymal stem cells growing in a carrier forming a dense three-dimensional structure. The carrier is constructed by two layers of PET net (mesh) and one layer of PP net (mesh). With reference to FIG. 2 and FIG. 13, an exemplary carrier structure is constructed by two layers of PET net / mesh and one layer of PP net / mesh, and the net / mesh 21 of the second net / mesh film is larger than the net / mesh 11 of the first net / mesh film. In a microscopic view, there may be two net / mesh 11 shown within the net / mesh 21 to provide cell growth. FIG. 14 shows mesenchymal stem cell growth in a carrier forming a dense three-dimensional structure. The carrier is constructed by three layers of PET net (mesh) and one layer of PP net (mesh). With reference to FIG. 3 and FIG. 14, an exemplary carrier structure is constructed by three layers of PET net / mesh and two layers of PP net / mesh, and the three overlapping first net / mesh films appear darker than FIG. 12 under microscopic observation.

[0072] FIG. 15 shows the carrier by peeling off one layer to observe the remaining cells after cell collection. Here, the carrier is constructed by two layers of PET net / mesh and one layer of PP net / mesh. With reference to FIG. 2 and FIG. 15, no remaining cells are observed on the PET net / mesh after cell collection. FIG. 16 shows the carrier by peeling off one layer to observe the remaining cells after cell collection. Here, the carrier is constructed by three layers of PET net / mesh and one layer of PP net / mesh. With reference to FIG. 3 and FIG. 16, less extracellular matrix and cells were observed in some limited areas after cell collection.

[0073] Example 3: Cell / tissue release

[0074] After cell growth reached confluence, the growth surface was washed three times with D-PBS, and then the growth surface was immersed in trypsin / EDTA for 5 min. The trypsin / EDTA solution was discarded before cell dissociation. Tapping and shaking was performed on the BelloCell® vessel to strike the growth surface against the BelloCell® vessel to forcefully dissociate the cells, and then washed with D-PBS. The tap / shake and wash cycle was repeated three times. All harvests were collected, centrifuged, resuspended, and checked for overall cell count and viability. The results after cell harvest are shown in Figure 17. The data show that the cell density was 9 x 10 per BelloCell® vessel with two-layer (group 1) and three-layer (group 2) PET net structures with a size of 4 cm (diameter) x 3 cm (height). 7 This indicates that more than 8×10 cells can be reached per BelloCell® vessel. The recovery rate was estimated by checking for stained cells on the growth surface. By estimating the overall cell productivity, if the matrix bed is increased to fill the BelloCell® matrix bed space, the cell productivity is estimated to be 8×10 cells per BelloCell® vessel. 8 In contrast to BioNOC II carriers in the BelloCell® vessel, only about 2×10 8Cells can be obtained with a recovery rate of about 70-80% (data not shown). With the carrier of the present invention, a total of 4-fold increase in cell productivity can be reached. Furthermore, no artificial particles were observed after cell harvesting shown in FIG. 18. Through particle release challenges, more than 14,000 fibers were seen in one BelloCell® vessel with BioNOC® II carrier after cell harvesting. Compared to particles generated from BioNOC® II carriers in BelloCell® vessels, with the carrier of the present invention, no extra downstream process for particle removal is required.

[0075] Therefore, the carrier system for cell culture in the present invention has a three-dimensional porous structure composed of multiple net / mesh structures in different nets / meshes. These net / mesh films have openings in a consistent direction, thereby strengthening the surface for cell growth, improving cell scaffolding, favoring cell collection, and improving biomass production. Moreover, compared with previous nonwoven material carriers, the carrier structure in the present invention can reduce the possibility of excess particle production, manufacturing costs, and time waste.

Claims

1. A carrier for cell culture comprising a net / mesh structural unit, the net / mesh structural unit comprising: At least one first net / mesh film having a plurality of first meshes formed by intersecting a plurality of first longitude lines and a plurality of first latitude lines, the first net / mesh film having biocompatibility and hydrophilicity; at least one second net / mesh film having a plurality of second meshes formed by intersecting a plurality of second longitude lines and a plurality of second latitude lines, the second net / mesh film being parallel to the first net / mesh film and disposed below or above the first net / mesh film; Including, The net / mesh structural unit includes two layers of the first net / mesh film and one layer of the second net / mesh film; the second net / mesh film is disposed between the two layers of the first net / mesh film, or on top of the two layers of the first net / mesh film, or under the two layers of the first net / mesh film; the openings of the first meshes of the first net / mesh film are the same size; the second meshes of the second net / mesh film have openings of equal size; The openings of the first mesh are 30 μm to 800 μm, and the openings of the second mesh are 1 mm to 5 mm; A carrier for cell culture, wherein peripheral edges of said first net / mesh film and said second net / mesh film are sealed together by heat pressing, ultrasonic, or adhesive treatment.

2. The net / mesh structural unit further includes another layer of the first net / mesh film and one layer of the second net / mesh film, 2. The carrier for cell culture according to claim 1, wherein the second net / mesh film is disposed between any two layers of the first net / mesh film, or on top of any three layers of the first net / mesh film, or under any three layers of the first net / mesh film.

3. 3. The carrier for cell culture according to claim 1 or 2, wherein the material of the first net / mesh film comprises polyethylene terephthalate (PET), and the material of the second net / mesh film comprises polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE), polytetrafluoroethylene (PTFE), or nylon.

4. The carrier for cell culture according to any one of claims 1 to 3, wherein the first net / mesh film is a hydrophilic surface-treated net / mesh material and the second net / mesh film is a supporting net / mesh material.

5. The carrier for cell culture according to any one of claims 1 to 4, wherein the type of the carrier is one or more columns formed by curling the net / mesh structural unit, one or more columns formed by laminating the plurality of net / mesh structural units in sequence, or a stack body formed by randomly arranging and packing the net / mesh structural units.

6. The carrier for cell culture according to any one of claims 1 to 5, wherein any one of the first longitude lines and any one of the first latitude lines has a width of 50 μm to 500 μm, and any one of the second longitude lines and any one of the second latitude lines has a width of 100 μm to 1000 μm.

7. The carrier for cell culture according to any one of claims 1 to 6, wherein any one of the first longitude lines and any one of the first latitude lines has a width of 50 μm to 250 μm, and any one of the second longitude lines and any one of the second latitude lines has a width of 250 μm to 500 μm.

8. A cell culture device comprising a carrier for cell culture according to any one of claims 1 to 7 and a culture chamber in which said carrier for cell culture is placed.

9. 9. The cell culture device of claim 8, wherein the net / mesh structural unit is curled to form one or more columns that are placed in the culture chamber, or a number of the net / mesh structural units are laminated in sequence to form one or more columns that are placed in the culture chamber, or a number of the net / mesh structural units are randomly arranged or packed to form a stack body that is placed in the culture chamber.

10. The cell culture device of claim 8, wherein the net / mesh structural unit includes two or three layers of the first net / mesh film and one layer of the second net / mesh film, and the second net / mesh film is disposed between any two layers of the first net / mesh film, above all layers of the first net / mesh film, or below all layers of the first net / mesh film.

11. 11. The cell culture device of claim 8, wherein the material of the first net / mesh film comprises polyethylene terephthalate (PET), and the material of the second net / mesh film comprises polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE), polytetrafluoroethylene (PTFE), or nylon.

12. 12. The cell culture device of claim 8, wherein the first net / mesh film is a hydrophilic surface-treated net / mesh material and the second net / mesh film is a supporting net / mesh material.

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