Method for producing gene product from gene introduced into transient gene expression vector

Abstract

To provide a method for producing a gene product from a gene introduced into a transient gene expression vector.SOLUTION: The present invention provides a method for producing a gene product from a gene introduced into a transient gene expression vector, the method comprising the steps of: (1) culturing cells settled on a polymer porous membrane while shaking during and / or after transfection with a transient gene expression vector; and (2) collecting the cells obtained in step (1) or a gene product produced from the cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a gene product from a gene introduced into a transient gene expression vector. [Background technology]

[0002] In recent years, the pharmaceutical market has been shifting away from traditionally used small molecular compounds to biopharmaceuticals, with biopharmaceuticals expected to account for approximately 40% of global pharmaceutical sales as of 2023. Biopharmaceuticals are pharmaceuticals created using biotechnology such as genetic recombination and cell culture techniques, and examples include vaccines, enzymes, hormones, antibodies, cytokines, and vectors.

[0003] Generally, biopharmaceuticals are produced by introducing an expression vector that expresses a desired gene product into cells, culturing the cells, and then recovering and purifying the gene product produced. For example, a viral vector as a gene product can be obtained by introducing one or more expression vectors incorporating genes necessary for the production of the viral vector into producer cells by a gene transfer technique such as transfection, followed by culturing the cells, and recovering and purifying the viral vector produced from the cells. Various attempts have been made to improve the production efficiency of biopharmaceuticals such as viral vectors, and development is ongoing (e.g., Patent Documents 1 to 4).

[0004] Cell culture and gene transfer technologies are particularly important elemental technologies in the production of biopharmaceuticals. When culturing cells in an artificial environment, the main methods used are plate culture, in which cells are cultured two-dimensionally by adhering them in a monolayer on the culture surface of a culture vessel, or suspension culture, in which cells are cultured suspended in a liquid culture medium. In plate culture, the areas where cells can adhere are limited to the surface of the culture vessel, so the surface area of ​​the culture vessel must be increased to culture large numbers of cells. Furthermore, when two-dimensional cells grow and adhere to each other, contact inhibition occurs, which can cause problems such as a decrease in the amount of proteins produced by the cells and a significant decrease in gene transfer efficiency.

[0005] Suspension culture can be applied to cells that can naturally be cultured in a suspended state (e.g., hematopoietic cells), cells that have been adapted to suspension culture from adherent cells (e.g., CHO cells), and cells that can form spheroids and be cultured in suspension (e.g., iPS cells and ES cells), but it is not suitable for all adherent cells, and the cell types that can be used are limited. Furthermore, while suspension culture can culture more cells than plate culture, many cells are fragile to shear forces, and it is necessary to optimize the culture and agitation conditions according to the cell type.

[0006] Microcarriers and polyimide porous membranes are known as cell culture substrates that enable the cultivation of adherent cells and are used for culturing them in suspension. Microcarriers are small particles to which cells can attach and be cultured, and are also used in the production of vaccines and proteins (Patent Document 5). Polyimide porous membranes are membranes that have excellent permeability to substances such as gases, high porosity, and excellent surface smoothness. They are also relatively strong substrates with numerous macrovoids that, despite their high porosity, have excellent resistance to compressive stress in the membrane thickness direction (Patent Documents 6 and 7). Adherent cells adhere to the surface of the polyimide porous membrane and within the internal macrovoid structure, allowing for the cultivation of large numbers of cells (Patent Document 7). It is also known that cells cultured on such polyimide porous membranes promote the production of biological substances such as proteins (Patent Document 6). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 6165752 [Patent Document 2] Special Publication No. 2023-518919 [Patent Document 3] Special Publication No. 2022-527407 [Patent Document 4] Special Publication No. 2021-519108 [Patent Document 5] International Publication No. 2003 / 054174 [Patent Document 6] International Publication No. 2016 / 121768 [Patent Document 7] International Publication No. 2016 / 121773 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for producing a gene product from a gene introduced into a transient gene expression vector. [Means for solving the problem]

[0009] As a result of intensive research into solving the above problems, the inventors discovered that using a porous polymer with a special structure to culture a transient gene expression vector with shaking during and / or after transfection resulted in the transient gene expression vector being effectively taken up by cells, improving the production of gene products, and thus arrived at the present invention.

[0010] The present invention includes, but is not limited to, the following aspects.

[0011] [1] A method for producing a gene product from a gene introduced into a transient gene expression vector, comprising: (1) Cultivating cells engrafted on a polymer porous membrane with shaking during and / or after transfection with a transient gene expression vector; and (2) recovering the cells obtained in step (1) or the gene product produced from the cells; Including, wherein the polymer porous membrane is a three-layer structure polymer porous membrane having surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the pores in the surface layer A is smaller than the average pore size of the pores in the surface layer B, and the macrovoid layer has partition walls bonded to the surface layers A and B and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, and the pores in the surface layers A and B are in communication with the macrovoids. The method. [2] The method according to Item 1, wherein the transient gene expression vector is a vector for producing a viral vector, and the gene product is a viral vector. [3] The method according to Item 2, wherein the viral vector is one or more selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, and a lentiviral vector. [4] The method according to any one of items 1 to 3, wherein the transfection is one or more selected from the group consisting of electroporation, lipofection, infection with a viral vector, magnetofection, and microinjection. [5] The method according to any one of items 1 to 4, wherein the transfection is carried out using a cationic polymer, a cationic lipid, or DEAE-dextran. [6] The method according to any one of items 1 to 5, wherein the transfection is carried out using a polyethyleneimine-based transfection reagent. [7] The method according to any one of items 1 to 6, wherein the shaking is carried out at 10 to 300 rpm. [8] The method according to any one of items 1 to 7, wherein the cells are animal cells. [9] The method according to any one of items 1 to 8, wherein the porous polymer membrane has a plurality of pores with an average pore size of 0.01 to 100 μm.

[10] The method according to any one of items 1 to 9, wherein the surface layer A has an average pore size of 0.01 to 50 μm.

[11] The method according to any one of items 1 to 10, wherein the surface layer B has an average pore size of 20 to 100 μm.

[12] The method according to any one of items 1 to 11, wherein the total thickness of the porous polymer membrane is 5 to 500 μm.

[13] The method according to any one of items 1 to 12, wherein the polymer porous membrane is a polyimide porous membrane.

[14] The method according to Item 13, wherein the polyimide porous film is a polyimide porous film containing a polyimide obtained from a tetracarboxylic dianhydride and a diamine.

[15] The method according to Item 13 or 14, wherein the polyimide porous film is a colored polyimide porous film obtained by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic dianhydride and a diamine and a colored precursor, followed by heat treatment at 250°C or higher.

[16] A method for introducing a transient gene expression vector into cells, comprising a step of culturing cells attached to a polymer porous membrane by shaking at 10 to 300 rpm during and / or after transfection with the transient gene expression vector, wherein the polymer porous membrane is a three-layer structure having surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between surface layers A and B, wherein the average pore size of the pores in surface layer A is smaller than the average pore size of the pores in surface layer B, and the macrovoid layer has partitions attached to surface layers A and B and a plurality of macrovoids surrounded by the partitions and surface layers A and B, and the pores in surface layers A and B are connected to the macrovoids.

[17] A method for introducing a transient gene expression vector into cells, comprising a step of culturing cells engrafted on a polymer porous membrane by shaking at 10 to 300 rpm during transfection with the transient gene expression vector, wherein the polymer porous membrane has a three-layer structure having surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between surface layers A and B, wherein the average pore size of the pores in surface layer A is smaller than the average pore size of the pores in surface layer B, and the macrovoid layer has partitions bonded to surface layers A and B and a plurality of macrovoids surrounded by the partitions and surface layers A and B, and the pores in surface layers A and B are in communication with the macrovoids.

[18] The method according to Item 16 or 17, wherein the transient gene expression vector is a vector for producing a viral vector, and the gene product is a viral vector.

[19] The method according to Item 18, wherein the viral vector is one or more selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, and a lentiviral vector.

[20] The method according to any one of items 16 to 19, wherein the transfection is one or more selected from the group consisting of electroporation, lipofection, infection with a viral vector, magnetofection, and microinjection.

[21] The method according to any one of items 16 to 20, wherein the transfection is carried out using a cationic polymer, a cationic lipid, or DEAE-dextran.

[22] The method according to any one of items 16 to 21, wherein the transfection is carried out using a polyethyleneimine-based transfection reagent.

[23] The method according to any one of items 16 to 22, wherein the shaking is carried out at 10 to 300 rpm.

[24] The method according to any one of items 16 to 23, wherein the cells are animal cells.

[25] The method according to any one of items 16 to 24, wherein the porous polymer membrane has a plurality of pores with an average pore size of 0.01 to 100 μm.

[26] The method according to any one of items 16 to 25, wherein the surface layer A has an average pore size of 0.01 to 50 μm.

[27] The method according to any one of items 16 to 26, wherein the surface layer B has an average pore size of 20 to 100 μm.

[28] The method according to any one of items 16 to 27, wherein the total thickness of the porous polymer membrane is 5 to 500 μm.

[29] The method according to any one of items 16 to 28, wherein the polymer porous membrane is a polyimide porous membrane.

[30] The method according to Item 29, wherein the polyimide porous film is a polyimide porous film containing a polyimide obtained from a tetracarboxylic dianhydride and a diamine.

[31] The method according to Item 29 or 30, wherein the polyimide porous film is a colored polyimide porous film obtained by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic dianhydride and a diamine and a colored precursor, followed by heat treatment at 250°C or higher. [Effects of the Invention]

[0012] Conventionally, to increase gene transfer efficiency, it was necessary to transfer genes to cells in the logarithmic growth phase. Therefore, it was necessary to adjust the cell density to be seeded before gene transfer or the timing of gene transfer. The present invention can promote the production of gene products produced from transient gene expression vectors even when transfection with the transient gene expression vector is performed without particularly adjusting the cell seeding density or the timing of gene transfer. [Brief explanation of the drawings]

[0013] [Figure 1]Figure 1 shows the amount of AAV vector genome produced in the culture supernatant after AAVpro293T cells seeded on a polyimide porous membrane (PPI membrane) or a dish were transfected with a transient gene expression vector that produces an adeno-associated virus (AAV) vector, shaken for 1 day, and then left to stand and cultured for an additional 1 day (day 2 after transfection) or 4 days (day 5 after transfection). [Figure 2] Figure 2 shows the amount of AAV vector genome produced in the culture supernatant after transfection of AAVpro293T cells seeded on a polyimide porous membrane (PPI membrane) or a dish with a transient gene expression vector that produces an adeno-associated virus (AAV) vector and culturing with shaking for 2 days (day 2 after transfection) or after subsequent static culturing for an additional 3 days (day 5 after transfection). [Figure 3] Figure 3 shows the amount of AAV vector genome produced in the culture supernatant after AAVpro293T cells seeded on a polyimide porous membrane (PPI membrane) or a dish were transfected with a transient gene expression vector that produces an adeno-associated virus (AAV) vector and cultured with shaking for 5 days (day 5 after transfection). Note that the results for the PPI membrane and 30 rpm experiment (*) were not measured. [Figure 4] Figure 4 shows the ZsGreen fluorescence intensity per cell (ZsGreen1 / cell) when AAVpro® 293T cells seeded on a porous polyimide membrane (PPI) or dish were transfected with a transient gene expression vector producing an adeno-associated virus (AAV) vector and cultured with shaking for 1 day. [Figure 5] Figure 5 shows the ZsGreen fluorescence intensity (ZsGreen1) per cell when AAVpro® 293T cells seeded on a porous polyimide membrane (PPI) or dish were transfected with a transient gene expression vector producing an adeno-associated virus (AAV) vector and cultured with shaking for 2 days. [Figure 6]Figure 6 shows the ZsGreen fluorescence intensity (ZsGreen1) per cell when AAVpro® 293T cells seeded on a porous polyimide membrane (PPI) or dish were transfected with a transient gene expression vector producing an adeno-associated virus (AAV) vector and cultured with shaking for 5 days. [Figure 7] Figure 7 shows the relative fluorescence intensity (ZsGreen1 fluorescence intensity / Hoechst fluorescence intensity) (ZsGreen1 / Hoechst) when AAVpro® 293T cells seeded on a porous polyimide membrane (PPI) or a dish were transfected with a transient gene expression vector that produces an adeno-associated virus (AAV) vector and cultured with shaking for one day. [Figure 8] Figure 8 shows the relative fluorescence intensity (ZsGreen1 fluorescence intensity / Hoechst fluorescence intensity) (ZsGreen1 / Hoechst) when AAVpro® 293T cells seeded on a porous polyimide membrane (PPI) or a dish were transfected with a transient gene expression vector that produces an adeno-associated virus (AAV) vector and cultured with shaking for two days. [Figure 9] Figure 9 shows the relative fluorescence intensity (ZsGreen1 fluorescence intensity / Hoechst fluorescence intensity) (ZsGreen1 / Hoechst) when AAVpro® 293T cells seeded on a porous polyimide membrane (PPI) or a dish were transfected with a transient gene expression vector that produces an adeno-associated virus (AAV) vector and cultured with shaking for 5 days. [Figure 10] FIG. 10 shows the results of regression analysis using a second-order nonlinear regression model with "ZsGreen1 fluorescence intensity per cell" as the response variable and "rotation speed" as the explanatory variable. DETAILED DESCRIPTION OF THE INVENTION

[0014] Hereinafter, embodiments for carrying out the present invention will be described, but the technical scope of the present invention is not limited to the following embodiments. Note that the prior art documents cited in this specification are incorporated herein by reference.

[0015] In one embodiment, the present invention provides a method for producing a gene product from a gene introduced into a transient gene expression vector, comprising the steps of: (1) Cultivating cells engrafted on a polymer porous membrane with shaking during and / or after transfection with a transient gene expression vector; and (2) recovering the cells obtained in step (1) or the gene product produced from the cells; Including, wherein the polymer porous membrane is a three-layer structure polymer porous membrane having surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the pores in the surface layer A is smaller than the average pore size of the pores in the surface layer B, and the macrovoid layer has partition walls bonded to the surface layers A and B and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, and the pores in the surface layers A and B are in communication with the macrovoids. The method relates to the above.

[0016] Conventionally, to increase gene transfer efficiency, it was necessary to transfer genes into cells in the logarithmic growth phase, and it was necessary to adjust the density of cells seeded before gene transfer or the timing of gene transfer. However, the present invention makes it possible to promote the production of gene products produced from transient gene expression vectors even when transfection with a transient gene expression vector is performed without particularly adjusting the cell seeding density or the timing of gene transfer. Therefore, the present invention makes it possible, for example, to increase the amount of gene product produced per cell, thereby reducing the cost of producing a target substance (e.g., a protein, a virus or virus-like particle (e.g., a viral vector), or cells that produce them).

[0017] In step (1) of the method of the present invention, the method for obtaining cells engrafted on the porous polymer membrane is not particularly limited, but may include, for example, the following aspects. (i) applying a medium containing suspended cells to a polymeric porous membrane; (ii) maintaining the polymer porous membrane at a temperature suitable for cell culture and adsorbing the cells to the polymer porous membrane; and (iii) a step of floating the porous polymer membrane to which the cells have been adsorbed in a medium in a state where the porous polymer membrane is not fixed in a cell culture vessel for suspension culture, and culturing the porous polymer membrane while shaking and / or stirring it in a state where the porous polymer membrane is subjected to a rotational motion, a reciprocating motion, an up-and-down motion, a wave-type rocking motion, a rotational motion, or a combination of these motions; Includes:

[0018] As used herein, the term "culture medium" refers to a cell culture medium for culturing cells, particularly animal cells. The term "culture medium" is used synonymously with "cell culture solution." Therefore, the medium used in the present invention refers to a liquid medium. The type of medium may be any commonly used medium, and is determined appropriately depending on the type of cells to be cultured. For example, D-MEM, E-MEM, IMDM, Ham's F-12, etc. may be used, but is not limited to these.

[0019] The temperature at which cells can be cultured in step (ii) may be any temperature at which cells can be adsorbed to the porous polymer membrane, and is 10° C. to 45° C., preferably 15° C. to 42° C., more preferably 20° C. to 40° C., and even more preferably 25° C. to 39° C. The time for adsorbing cells in step (ii) is, for example, 5 minutes to 24 hours, preferably 10 minutes to 12 hours, and more preferably 15 minutes to 500 minutes.

[0020] In step (ii), the cells may be adsorbed onto the porous polymer membrane while shaking and / or stirring, or may be allowed to stand to allow the cells to be adsorbed onto the porous polymer membrane. The shaking method is not particularly limited, and for example, a culture vessel containing the modularized polyimide porous membrane of the present invention and the cells may be placed on a commercially available shaking device and shaken. Shaking may be performed continuously or intermittently, for example, by alternating between shaking and standing, and the shaking rate may be adjusted as appropriate. The stirring method is not particularly limited, and for example, the porous polymer membrane of the present invention and the cells may be placed in a commercially available spinner flask and stirred by rotating a stirrer. Stirring may be performed continuously or intermittently, for example, by alternating between stirring and standing, and the shaking rate may be adjusted as appropriate.

[0021] As used herein, "transient expression" refers to the introduction of an exogenous gene into a cell or microorganism to temporarily express a protein. This term is distinct from stable expression, in which a gene introduced by transformation such as transfection is incorporated into the host genome and maintained in progeny (cells). While selecting cells that stably express a desired protein requires significant effort and cost, transient expression is advantageous in that the desired protein can be expressed simply by introducing a vector that expresses the desired protein into cells. Conventionally, however, to increase gene transfer efficiency, gene transfer must be performed on cells in the logarithmic growth phase. However, according to the present invention, the production of a gene product produced by a transient gene expression vector can be promoted even when the transient gene expression vector is transfected without particularly controlling the cell seeding density or the timing of gene transfer.

[0022] As used herein, the term "transient gene expression vector" refers to any vector that allows for the transient expression of a desired gene product incorporated therein, and includes, but is not limited to, for example, a plasmid vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, a viral vector, etc. The transient gene expression vector that can be used in the present invention may be any vector that can incorporate a desired gene product and express the gene product without being incorporated into the genome of the cell when introduced into a cell.

[0023] In one embodiment of the present invention, the transient gene expression vector may be a vector for producing a viral vector, and in this case, the gene product expressed from the transient gene expression vector is a viral vector. The viral vector produced may be a commonly used viral vector, for example, one or more selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors.

[0024] In one embodiment, the transfection performed in step (1) of the present invention may be performed using a known method used in gene transfer manipulation, such as one or more methods selected from the group consisting of electroporation, lipofection, infection with a viral vector, magnetofection, and microinjection. In one embodiment, the transfection performed in step (1) of the present invention may be performed using a cationic polymer (e.g., cationic peptides and their derivatives (e.g., polylysine, polyornithine, etc.), linear or branched synthetic polymers (e.g., polybrene, polyethyleneimine, etc.), polysaccharide-based transfection molecules (e.g., cyclodextrin, chitosan, etc.), natural polymers (e.g., histone, collagen, etc.), and activated and inactivated dendrimers, etc.), cationic lipids (e.g., Lipofectamine (registered trademark), etc.), or DEAE-dextran. In one embodiment, the transfection performed in step (1) of the present invention may be performed using a polyethyleneimine-based transfection reagent (e.g., PEIpro (registered trademark), etc.).

[0025] In one embodiment, the shaking during and / or after transfection in step (1) of the present invention may be performed, for example, by a swirling motion, a reciprocating motion, an up-and-down motion, a wave-type rocking motion, a rotational motion, or a combination of these motions.

[0026] As used herein, "orbital movement" refers to a movement in which a culture vessel is rotated in a plane parallel to the horizontal plane to agitate the liquid in the culture vessel. When orbital movement is applied to the present invention, a commercially available culture shaker, such as a rotary shaking shaker (e.g., rotation mode: orbital, etc.), may be used. The orbital movement applied to the present invention may be performed at, for example, 10 rpm to 300 rpm, preferably 10 rpm to 200 rpm, more preferably 20 rpm to 180 rpm, and even more preferably 30 rpm to 170 rpm. The orbital movement applied to the present invention may be performed with a diameter of 5 mm to 100 mm, 10 mm to 50 mm, or 15 mm to 25 mm, for example.

[0027] As used herein, "reciprocating motion" refers to a motion in which a culture vessel is moved left and right or back and forth to agitate the liquid in the culture vessel. When reciprocating motion is applied to the present invention, a commercially available culture shaker, for example, a reciprocating shaking shaker, may be used. The reciprocating motion applied in the present invention may be performed, for example, at a speed of 10 to 300 reciprocating motions / minute, preferably 10 to 200 reciprocating motions / minute, more preferably 20 to 180 reciprocating motions / minute, and even more preferably 30 to 170 reciprocating motions / minute. The travel distance of the reciprocating motion applied in the present invention may be, for example, 5 to 100 mm, 10 to 50 mm, or 15 to 25 mm.

[0028] As used herein, "up-and-down movement" refers to movement in which a culture vessel is moved up and down in the vertical direction to agitate the liquid in the culture vessel. When up-and-down movement is used, a commercially available culture shaker, such as an up-and-down shaking shaker, may be used. The up-and-down movement used in the present invention may be performed, for example, at a rate of 10 to 300 reciprocations / minute, preferably 10 to 200 reciprocations / minute, more preferably 20 to 180 reciprocations / minute, and even more preferably 30 to 170 reciprocations / minute. The travel distance of the reciprocating movement used in the present invention may be, for example, 5 to 100 mm, 10 to 50 mm, or 15 to 25 mm.

[0029] As used herein, "wave-type rocking motion" refers to a movement that generates a wave in the liquid in a culture vessel by a see-saw movement back and forth and / or left and right, thereby stirring the liquid. A commercially available culture shaker, such as a WAVE reactor, may be used to perform the wave-type rocking motion. The wave-type rocking motion used in the present invention may be, for example, rocking with the bottom surface of the cell culture vessel tilted at an angle of 4 to 15 degrees. Furthermore, the wave-type rocking motion used in the present invention may be performed at, for example, 10 to 300 rpm, preferably 10 to 200 rpm, more preferably 20 to 180 rpm, and even more preferably 30 to 170 rpm.

[0030] As used herein, "rotational movement" refers to a movement in which the liquid in the culture vessel is shaken by rotating a physical stirring means. Rotational movement can be performed using, for example, a stirred reactor, such as a spinner flask or a stationary stirred reactor with stirring blades. The rotational movement used in the present invention may be performed at, for example, 10 rpm to 300 rpm, preferably 10 rpm to 200 rpm, more preferably 20 rpm to 180 rpm, and even more preferably 30 rpm to 170 rpm.

[0031] In one embodiment, the shaking in step (1) may be a rotational movement, a reciprocating movement, an up-and-down movement, a wave-like rocking movement, a rotational movement, or a combination of these movements. The shaking period can be appropriately selected, and may be, for example, 0.5 to 240 hours, 6 to 168 hours, 6 to 144 hours, 12 to 120 hours, or 24 to 36 hours after the start of transfection.

[0032] In one embodiment, the culture conditions in step (1) may be in accordance with known conditions for culturing cells, and may be, for example, a temperature of 30 to 40°C, preferably 35 to 39°C, more preferably 36 to 38°C (e.g., about 37°C), in an atmosphere of 1 to 20% (V / V) CO2, preferably 3 to 10% (V / V) CO2 (e.g., about 5% (V / V) CO2), and saturated water vapor.

[0033] In one embodiment, the step (2) of recovering the gene product produced from the cells can be carried out by a known method depending on the type and properties of the gene product. When the produced gene product is a substance secreted by the cells, the substance can be recovered from the cell culture medium. When the produced gene product is a substance that remains intracellularly, the substance can be released from the cells and recovered by disrupting the cells using known methods, such as chemical treatment with a cell lysing agent or physical treatment using a homogenizer or disposable disruption tube. Those skilled in the art can apply appropriate cell disruption methods depending on the type of cell, substance, etc. Furthermore, when the produced gene product is a virus or virus-like particle (e.g., a viral vector), the gene product can be recovered and concentrated by ultracentrifugation of the cell culture medium. Alternatively, the desired gene product may be recovered using any column capable of specifically adsorbing and / or separating the gene product. Furthermore, in one embodiment, step (2) may be a step of recovering the cells themselves that produce the desired gene product expressed from a transient gene expression vector. The method for recovering the cells may be according to a known method, and is not particularly limited, for example, a method in which the cells are detached by enzymatic treatment (such as trypsin) or physical treatment (such as a scraper or pipetting) and then recovered by centrifugation, or a method in which a filter for recovering the cells is used.

[0034] Any cells can be used in the methods of the present invention. For example, cells that can be used may be selected from the group consisting of animal cells, insect cells, plant cells, yeast cells, and bacteria, and are preferably animal cells or insect cells. Animal cells are broadly divided into cells derived from animals belonging to the phylum Vertebrates and cells derived from invertebrates (animals other than those belonging to the phylum Vertebrates). The origin of animal cells in this specification is not particularly limited. Preferably, it refers to cells derived from animals belonging to the phylum Vertebrates. The phylum Vertebrates includes the classes Agnathostomata and Gnathostomata, which include the classes Mammalia, Aves, Amphibia, and Reptilia. Preferably, the cells are derived from animals belonging to the class Mammalia, commonly known as mammals. Mammals are not particularly limited, but preferably include mice, rats, humans, monkeys, pigs, dogs, sheep, goats, and the like.

[0035] The animal cells used may be, but are not limited to, for example, Chinese hamster ovary tissue-derived cells (CHO cells), African green monkey kidney-derived cell lines (Vero cells), human hepatoma-derived cells (HepG2 cells), canine kidney tubular epithelial cell-derived cell lines (MDCK cells), human hepatoma tissue-derived established cell lines (huGK-14), normal human fibroblast-like synoviocytes (HFLS cells), rheumatoid arthritis patient-derived fibroblast-like synoviocytes (HFLS-RA cells), human embryonic kidney cell-derived cells (HEK293 cells), or further modified cell lines.

[0036] In one embodiment, cells that can be used in the methods of the present invention may be pluripotent stem cells. Pluripotent stem cells are intended to collectively refer to stem cells that have the ability to differentiate into cells of any tissue (pluripotency). Pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), germline stem cells (GS cells), and the like. ES cells or iPS cells are preferred. iPS cells are particularly preferred for reasons such as the absence of ethical issues. Any known pluripotent stem cells can be used, but for example, the pluripotent stem cells described in International Publication WO2009 / 123349 (PCT / JP2009 / 057041) can be used.

[0037] In one embodiment, tissue stem cells may be used as cells that can be used in the methods of the present invention. Tissue stem cells refer to stem cells whose differentiable cell lineages are limited to specific tissues but have the ability to differentiate into a variety of cell types (pluripotency). For example, hematopoietic stem cells in bone marrow become the source of blood cells, and neural stem cells differentiate into nerve cells. There are various other types of stem cells, such as liver stem cells that form the liver and skin stem cells that form skin tissue. Tissue stem cells may be, for example, mesenchymal stem cells, liver stem cells, pancreatic stem cells, neural stem cells, skin stem cells, or hematopoietic stem cells.

[0038] In one embodiment, the cells that can be used in the methods of the present invention may be somatic cells. Somatic cells refer to cells that constitute multicellular organisms other than germ cells. They are not passed on to the next generation in sexual reproduction. Somatic cells that can be used in the present invention may be selected from hepatocytes, pancreatic cells, muscle cells, bone cells, osteoblasts, osteoclasts, chondrocytes, adipocytes, skin cells, fibroblasts, pancreatic cells, kidney cells, lung cells, cardiac muscle cells, synovial cells, or blood cells such as lymphocytes, erythrocytes, leukocytes, monocytes, macrophages, or megakaryocytes, or diseased cells thereof, such as human osteoblasts, human chondrocytes, human cardiac muscle cells, human fibroblasts, human synovial cells, human hepatocytes, and diseased cells thereof.

[0039] In one embodiment, the cells that can be used in the method of the present invention may be germ cells. Germ cells refer to cells that play a role in transmitting genetic information to the next generation during reproduction. For example, germ cells include gametes for sexual reproduction, i.e., eggs, egg cells, sperm, and sperm cells, and spores for asexual reproduction.

[0040] Cells that can be used in the methods of the present invention may be selected from the group consisting of sarcoma cells, established cell lines, and transformed cells. "Sarcoma" refers to cancers that develop in connective tissue cells derived from non-epithelial cells, such as bone, cartilage, fat, muscle, and blood, and includes soft tissue sarcoma and malignant bone tumors. Sarcoma cells are cells derived from sarcoma. "Established cell lines" refer to cultured cells that have been maintained in vitro for a long period of time, have acquired certain stable properties, and can be subcultured semi-permanently. Cell lines derived from various tissues of various organisms, including humans, exist, including PC12 cells (derived from rat adrenal medulla), CHO cells (derived from Chinese hamster ovary), HEK293 cells (derived from human fetal kidney), HL-60 cells (derived from human leukocytes), HeLa cells (derived from human cervical carcinoma), Vero cells (derived from African green monkey kidney epithelial cells), MDCK cells (derived from canine kidney tubular epithelial cells), and HepG2 cells (derived from human hepatoma). "Transformed cells" refer to cells whose genetic properties have been changed by introducing nucleic acid (such as DNA) from outside the cell.

[0041] In one embodiment, cells applicable to the present invention may be suspension cells or adherent cells, preferably adherent cells. As used herein, "suspension cells" refers to cells that can grow in a suspended state in a medium, including cells that can be cultured in both adherent and suspension culture, and also includes cells that have changed from adherent cells to suspension cells through acclimatization. Examples of such cells include, but are not limited to, CHO cells, HEK293 cells, and hybridoma cells.

[0042] In one embodiment, the area of ​​the porous polymer membrane used in step (1) of the present invention may be 80% to 100% of the bottom area of ​​the culture vessel (e.g., culture dish) to which it is applied. In this case, the shape of the porous polymer membrane may be the same as or similar to the bottom of the culture vessel (e.g., culture dish) to which it is applied, but may be 0.1% to 20% smaller than that shape. This allows the porous polymer membrane to be shaken by the fluid without causing continuous undulating motion. The shape of the porous polymer membrane may be, for example, triangular, rectangular (e.g., rectangular, square), polygonal (e.g., pentagonal, hexagonal, heptagonal, octagonal, nonagonal, decagonal, or n-gonal (n = 10 or more)), or approximately circular. Preferably, the shape is similar to the shape of the bottom of the culture vessel to which it is applied, and more preferably, approximately circular.

[0043] In one embodiment, the porous polymer membrane used in step (1) of the present invention may be used in a single layer, or in two or more layers (two sheets) (for example, two, three, four, five, six, seven, eight, nine, ten, or more sheets).

[0044] <Porous polymer membrane> The average pore size of the pores present in surface layer A (hereinafter also referred to as "side A" or "mesh side") in the polymer porous membrane used in the present invention is not particularly limited, and is, for example, 0.01 μm or more and less than 200 μm, 0.01 to 150 μm, 0.01 to 100 μm, 0.01 to 50 μm, 0.01 to 40 μm, 0.01 to 30 μm, 0.01 to 20 μm, or 0.01 to 15 μm, and preferably 0.01 to 15 μm.

[0045] The average pore size of the pores present in surface layer B (hereinafter also referred to as "side B" or "large hole side") in the polymer porous membrane used in the present invention is not particularly limited as long as it is larger than the average pore size of the pores present in surface layer A, but is, for example, more than 5 μm and not more than 200 μm, 20 μm to 100 μm, 30 μm to 100 μm, 40 μm to 100 μm, 50 μm to 100 μm, or 60 μm to 100 μm, and preferably 20 μm to 100 μm.

[0046] The average pore size on the surface of a polymer porous membrane can be determined by measuring the pore area of ​​200 or more open pores in a scanning electron microscope photograph of the porous membrane surface, and calculating the average diameter from the average pore area according to the following formula (1), assuming that the pores are perfectly circular.

number

[0047] The thickness of the surface layers A and B is not particularly limited, but is, for example, 0.01 to 50 μm, and preferably 0.01 to 20 μm.

[0048] The average pore size of the macrovoids in the macrovoid layer in the plane direction of the membrane in the porous polymer membrane is not particularly limited, but is, for example, 10 to 500 μm, preferably 10 to 100 μm, and more preferably 10 to 80 μm. The thickness of the partition walls in the macrovoid layer is not particularly limited, but is, for example, 0.01 to 50 μm, and preferably 0.01 to 20 μm. In one embodiment, at least one partition wall in the macrovoid layer has one or more pores that connect adjacent macrovoids and have an average pore size of 0.01 to 100 μm, preferably 0.01 to 50 μm. In another embodiment, the partition walls in the macrovoid layer have no pores.

[0049] The total thickness of the surface of the porous polymer membrane used in the present invention is not particularly limited, but may be 5 μm or more, 10 μm or more, 20 μm or more, or 25 μm or more, or may be 500 μm or less, 300 μm or less, 100 μm or less, 75 μm or less, or 50 μm or less, preferably 5 to 500 μm, more preferably 25 to 75 μm.

[0050] The thickness of the porous polymer membrane used in the present invention can be measured using a contact type thickness meter.

[0051] The porosity of the porous polymer membrane used in the present invention is not particularly limited, but is, for example, 40% or more and less than 95%.

[0052] The porosity of the porous polymer membrane used in the present invention can be calculated from the basis weight by measuring the thickness and mass of a porous film cut to a predetermined size and then using the following formula (2).

number

[0053] The porous polymer membrane used in the present invention is preferably a three-layer structure porous polymer membrane having surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between surface layers A and B, wherein the average pore size of the pores in surface layer A is 0.01 μm to 15 μm, and the average pore size of the pores in surface layer B is 20 μm to 100 μm, the macrovoid layer has partition walls bonded to surface layers A and B, and a plurality of macrovoids surrounded by the partition walls and surface layers A and B, the partition walls of the macrovoid layer and surface layers A and B having thicknesses of 0.01 to 20 μm, the pores in surface layers A and B communicating with the macrovoids, the total thickness of the porous polymer membrane being 5 to 500 μm, and the porosity being 40% or more but less than 95%. In one embodiment, at least one partition wall in the macrovoid layer has one or more pores that connect adjacent macrovoids and have an average pore size of 0.01 to 100 μm, preferably 0.01 to 50 μm. In another embodiment, the partition wall does not have such pores.

[0054] The porous polymer membrane used in the present invention is preferably sterilized. Sterilization methods include, but are not limited to, dry heat sterilization, steam sterilization, sterilization with a disinfectant such as ethanol, and electromagnetic sterilization using ultraviolet rays or gamma rays.

[0055] The polymer porous membrane used in the present invention is not particularly limited as long as it has the above-mentioned structural characteristics, but is preferably a polyimide porous membrane or a polyethersulfone (PES) porous membrane. The polymer porous membrane used in the present invention may also be a nonwoven fabric.

[0056] <Porous polyimide film> Polyimide is a general term for polymers containing imide bonds in their repeating units, and usually refers to aromatic polyimides, in which aromatic compounds are directly linked by imide bonds. Aromatic polyimides have a rigid and strong molecular structure because aromatics form a conjugated structure via imide bonds, and the strong intermolecular forces of the imide bonds give them very high levels of thermal, mechanical, and chemical properties.

[0057] The polyimide porous film that can be used in the present invention is preferably a polyimide porous film containing (as a main component) a polyimide obtained from a tetracarboxylic dianhydride and a diamine, more preferably a polyimide porous film consisting of a polyimide obtained from a tetracarboxylic dianhydride and a diamine. "Containing as a main component" means that the polyimide porous film does not essentially contain any components other than the polyimide obtained from a tetracarboxylic dianhydride and a diamine as constituent components, or may contain additional components that do not affect the properties of the polyimide obtained from a tetracarboxylic dianhydride and a diamine.

[0058] In one embodiment, the polyimide porous film that can be used in the present invention also includes a colored polyimide porous film obtained by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component and a colored precursor, followed by heat treatment at 250°C or higher.

[0059] Polyamic acid is obtained by polymerizing a tetracarboxylic acid component and a diamine component. Polyamic acid is a polyimide precursor that can be converted to a polyimide by thermal or chemical imidization to close the ring.

[0060] The polyamic acid may be partially imidized as long as the imidization does not affect the present invention. That is, the polyamic acid may be partially thermally or chemically imidized.

[0061] When thermally imidizing polyamic acid, an imidization catalyst, an organic phosphorus-containing compound, fine particles such as inorganic fine particles or organic fine particles, etc. can be added to the polyamic acid solution as needed. When chemically imidizing polyamic acid, a chemical imidizing agent, a dehydrating agent, fine particles such as inorganic fine particles or organic fine particles, etc. can be added to the polyamic acid solution as needed. It is preferable to carry out the process under conditions in which the color precursor does not precipitate even when the above components are blended into the polyamic acid solution.

[0062] In this specification, the term "colored precursor" refers to a precursor that is partially or completely carbonized by heat treatment at 250°C or higher to produce a colored product.

[0063] The color precursor that can be used in producing the polyimide porous film is preferably one that is uniformly dissolved or dispersed in a polyamic acid solution or a polyimide solution, and is thermally decomposed by heat treatment at 250°C or higher, preferably 260°C or higher, more preferably 280°C or higher, and more preferably 300°C or higher, preferably in the presence of oxygen such as air, at 250°C or higher, preferably 260°C or higher, more preferably 280°C or higher, and more preferably 300°C or higher, and carbonized to produce a colored product, more preferably one that produces a black colored product, and more preferably a carbon-based color precursor.

[0064] When heated, the colored precursors become what at first glance appear to be carbonized products, but structurally they contain elements other than carbon, and include layered structures, aromatic cross-linked structures, and disordered structures including tetrahedral carbon.

[0065] The carbonaceous color precursor is not particularly limited, and examples thereof include tar or pitch such as petroleum tar, petroleum pitch, coal tar, and coal pitch, coke, a polymer obtained from a monomer containing acrylonitrile, a ferrocene compound (ferrocene and ferrocene derivatives), etc. Among these, a polymer and / or a ferrocene compound obtained from a monomer containing acrylonitrile is preferred, and polyacrylonitrile is preferred as a polymer obtained from a monomer containing acrylonitrile.

[0066] In another embodiment, the polyimide porous film that can be used in the present invention also includes a polyimide porous film that can be obtained by molding a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component, followed by heat treatment, without using the above-mentioned colored precursor.

[0067] A polyimide porous film produced without using a coloring precursor may be produced, for example, by casting a polyamic acid solution containing 3 to 60% by weight of a polyamic acid having an intrinsic viscosity of 1.0 to 3.0 and 40 to 97% by weight of an organic polar solvent into a film, immersing or contacting the film with a coagulation solvent containing water as an essential component to produce a porous polyamic acid film, and then heat-treating the polyamic acid porous film to imidize it. In this method, the coagulation solvent containing water as an essential component may be water or a mixture of 5% to less than 100% by weight of water and more than 0% to 95% by weight of an organic polar solvent. After the imidization, at least one surface of the resulting porous polyimide film may be subjected to a plasma treatment.

[0068] The tetracarboxylic dianhydride that can be used in the production of the polyimide porous film may be any tetracarboxylic dianhydride, and can be appropriately selected depending on the desired properties, etc. Specific examples of tetracarboxylic dianhydrides include pyromellitic dianhydride, biphenyltetracarboxylic dianhydrides such as 3,3',4,4'-biphenyltetracarboxylic dianhydride (s-BPDA) and 2,3,3',4'-biphenyltetracarboxylic dianhydride (a-BPDA), oxydiphthalic dianhydride, diphenylsulfone-3,4,3',4'-tetracarboxylic dianhydride, bis(3,4-dicarboxyphenyl)sulfide dianhydride, 2,2-bis(3,4-dicarboxyphenyl)-1,1,1,3,3,3-hexafluoropropane dianhydride, 2,3,3',4'-benzophenonetetracarboxylic dianhydride, 3,3',4,4'-benzophenonetetracarboxylic dianhydride, bis(3,4-dicarboxyphenyl)methane dianhydride, and 2,2-bis(3,4-dicarboxyphenyl)propane. dianhydride, p-phenylene bis(trimellitic acid monoester acid anhydride), p-biphenylene bis(trimellitic acid monoester acid anhydride), m-terphenyl-3,4,3',4'-tetracarboxylic acid dianhydride, p-terphenyl-3,4,3',4'-tetracarboxylic acid dianhydride, 1,3-bis(3,4-dicarboxyphenoxy)benzene dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)benzene dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)biphenyl dianhydride, 2,2-bis[(3,4-dicarboxyphenoxy)phenyl]propane dianhydride, 2,3,6,7-naphthalenetetracarboxylic acid dianhydride, 1,4,5,8-naphthalenetetracarboxylic acid dianhydride, 4,4'-(2,2-hexafluoroisopropylidene)diphthalic acid dianhydride, and the like. It is also preferable to use aromatic tetracarboxylic acids such as 2,3,3',4'-diphenylsulfonetetracarboxylic acid, etc. These may be used alone or in combination of two or more.

[0069] Among these, at least one aromatic tetracarboxylic dianhydride selected from the group consisting of biphenyltetracarboxylic dianhydride and pyromellitic dianhydride is particularly preferred. As the biphenyltetracarboxylic dianhydride, 3,3',4,4'-biphenyltetracarboxylic dianhydride can be suitably used.

[0070] Any diamine can be used in the production of the porous polyimide film. Specific examples of the diamine include the following: 1) Benzenediamines with one benzene nucleus, such as 1,4-diaminobenzene (paraphenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, and 2,6-diaminotoluene; 2) Diaminodiphenyl ethers such as 4,4'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenylmethane, 3,3'-dimethyl-4,4'-diaminobiphenyl, 2,2'-dimethyl-4,4'-diaminobiphenyl, 2,2'-bis(trifluoromethyl)-4,4'-diaminobiphenyl, 3,3'-dimethyl-4,4'-diaminodiphenylmethane, 3,3'-dicarboxy-4,4'-diaminodiphenylmethane, 3 ,3',5,5'-Tetramethyl-4,4'-diaminodiphenylmethane, Bis(4-aminophenyl) sulfide, 4,4'-diaminobenzanilide, 3,3'-Dichlorobenzidine, 3,3'-Dimethylbenzidine, 2,2'-Dimethylbenzidine, 3,3'-Dimethoxybenzidine, 2,2'-Dimethoxybenzidine, 3,3'-Diaminodiphenyl ether, 3,4'-Diaminodiphenyl ether, 4,4'-Diaminodiphenyl ether, 3,3'-Diaminodiphenyl Sulfide, 3,4'-diaminodiphenyl sulfide, 4,4'-diaminodiphenyl sulfide, 3,3'-diaminodiphenyl sulfone, 3,4'-diaminodiphenyl sulfone, 4,4'-diaminodiphenyl sulfone, 3,3'-diaminobenzophenone, 3,3'-diamino-4,4'-dichlorobenzophenone, 3,3'-diamino-4,4'-dimethoxybenzophenone, 3,3'-diaminodiphenylmethane, 3,4'-diaminodiphenylmethane, 4,4'-di Diamines with two benzene nuclei, such as aminodiphenylmethane, 2,2-bis(3-aminophenyl)propane, 2,2-bis(4-aminophenyl)propane, 2,2-bis(3-aminophenyl)-1,1,1,3,3,3-hexafluoropropane, 2,2-bis(4-aminophenyl)-1,1,1,3,3,3-hexafluoropropane, 3,3'-diaminodiphenyl sulfoxide, 3,4'-diaminodiphenyl sulfoxide, and 4,4'-diaminodiphenyl sulfoxide; 3) 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, 1,4-bis(3-aminophenoxy)benzene, 1,4-bis(4-aminophenoxy)benzene, 1,3-bis(3-aminophenoxy)-4-trifluoromethylbenzene, 3,3'-diamino-4-(4-phenyl)phenoxybenzophenone, 3,3'-diamino-4,4'-di(4-phenylphenoxy)benzophenone, 1,3 -Diamines with three benzene nuclei, such as bis(3-aminophenylsulfide)benzene, 1,3-bis(4-aminophenylsulfide)benzene, 1,4-bis(4-aminophenylsulfide)benzene, 1,3-bis(3-aminophenylsulfone)benzene, 1,3-bis(4-aminophenylsulfone)benzene, 1,4-bis(4-aminophenylsulfone)benzene, 1,3-bis[2-(4-aminophenyl)isopropyl]benzene, 1,4-bis[2-(3-aminophenyl)isopropyl]benzene, 1,4-bis[2-(4-aminophenyl)isopropyl]benzene; 4) 3,3'-bis(3-aminophenoxy)biphenyl, 3,3'-bis(4-aminophenoxy)biphenyl, 4,4'-bis(3-aminophenoxy)biphenyl, 4,4'-bis(4-aminophenoxy)biphenyl, bis[3-(3-aminophenoxy)phenyl]ether, bis[3-(4-aminophenoxy)phenyl]ether, bis[4-(3-aminophenoxy)phenyl]ether, bis[4-(4-aminophenoxy)phenyl]ether, bis[3-(3-aminophenoxy)phenyl]ketone, bis[3-(4-aminophenoxy)phenyl]ketone bis[4-(3-aminophenoxy)phenyl]ketone, bis[4-(4-aminophenoxy)phenyl]ketone, bis[4-(4-aminophenoxy)phenyl]ketone, bis[3-(3-aminophenoxy)phenyl]sulfide, bis[3-(4-aminophenoxy)phenyl]sulfide, bis[4-(3-aminophenoxy)phenyl]sulfide, bis[4-(4-aminophenoxy)phenyl]sulfide, bis[3-(3-aminophenoxy)phenyl]sulfone, bis[3-(4-aminophenoxy)phenyl]sulfone, bis[4-(3-aminophenoxy)phenyl]sulfone bis[4-(4-aminophenoxy)phenyl]sulfone, bis[3-(3-aminophenoxy)phenyl]methane, bis[3-(4-aminophenoxy)phenyl]methane, bis[4-(3-aminophenoxy)phenyl]methane, bis[4-(4-aminophenoxy)phenyl]methane, 2,2-bis[3-(3-aminophenoxy)phenyl]propane, 2,2-bis[3-(4-aminophenoxy)phenyl]propane, 2,2-bis[4-(3-aminophenoxy)phenyl]propane, 2,2- Diamines with four benzene nuclei such as bis[4-(4-aminophenoxy)phenyl]propane, 2,2-bis[3-(3-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-bis[3-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-bis[4-(3-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, and 2,2-bis[4-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane.

[0071] These may be used alone or in combination of two or more. The diamine to be used may be appropriately selected depending on the desired properties.

[0072] Among these, aromatic diamine compounds are preferred, and 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether, paraphenylenediamine, 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, and 1,4-bis(3-aminophenoxy)benzene can be suitably used. In particular, at least one diamine selected from the group consisting of benzenediamine, diaminodiphenyl ether, and bis(aminophenoxy)phenyl is preferred.

[0073] From the viewpoints of heat resistance and dimensional stability at high temperatures, the polyimide porous film that can be used in the present invention is preferably formed from a polyimide obtained by combining a tetracarboxylic dianhydride and a diamine, which has a glass transition temperature of 240°C or higher or has no clear transition point at 300°C or higher.

[0074] The polyimide porous film that can be used in the present invention is preferably a polyimide porous film made of the following aromatic polyimides, from the viewpoints of heat resistance and dimensional stability at high temperatures. (i) an aromatic polyimide comprising at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units, and an aromatic diamine unit; (ii) an aromatic polyimide comprising a tetracarboxylic acid unit and at least one aromatic diamine unit selected from the group consisting of a benzenediamine unit, a diaminodiphenyl ether unit, and a bis(aminophenoxy)phenyl unit; and / or (iii) An aromatic polyimide comprising at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units, and at least one aromatic diamine unit selected from the group consisting of benzenediamine units, diaminodiphenyl ether units, and bis(aminophenoxy)phenyl units.

[0075] The polyimide porous membrane used in the present invention is preferably a polyimide porous membrane having a three-layer structure including surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between surface layers A and B, wherein the average pore size of the pores in surface layer A is 0.01 μm to 15 μm, and the average pore size of the pores in surface layer B is 20 μm to 100 μm, the macrovoid layer has partition walls bonded to surface layers A and B, and a plurality of macrovoids surrounded by the partition walls and surface layers A and B, the thicknesses of the partition walls of the macrovoid layer and surface layers A and B are 0.01 to 20 μm, the pores in surface layers A and B are connected to the macrovoids, the total thickness is 5 to 500 μm, and the porosity is 40% or more but less than 95%. At least one partition wall in the macrovoid layer has one or more pores that connect adjacent macrovoids and have an average pore size of 0.01 to 100 μm, preferably 0.01 to 50 μm.

[0076] For example, the polyimide porous films described in WO 2010 / 038873, JP 2011-219585 A, and JP 2011-219586 A can also be used in the present invention.

[0077] <Polyethersulfone (PES) porous membrane> The PES porous membrane that can be used in the present invention contains polyethersulfone, and typically consists essentially of polyethersulfone. The polyethersulfone may be synthesized by a method known to those skilled in the art, for example, by polycondensation reaction of a dihydric phenol, an alkali metal compound, and a dihalogenodiphenyl compound in an organic polar solvent, or by synthesis of an alkali metal disalt of a dihydric phenol and polycondensation reaction of the alkali metal disalt with a dihalogenodiphenyl compound in an organic polar solvent.

[0078] Examples of the alkali metal compound include alkali metal carbonates, alkali metal hydroxides, alkali metal hydrides, alkali metal alkoxides, etc. Sodium carbonate and potassium carbonate are particularly preferred.

[0079] Examples of dihydric phenol compounds include hydroquinone, catechol, resorcinol, 4,4'-biphenol, bis(hydroxyphenyl)alkanes (e.g., 2,2-bis(hydroxyphenyl)propane and 2,2-bis(hydroxyphenyl)methane), dihydroxydiphenyl sulfones, dihydroxydiphenyl ethers, and compounds in which at least one hydrogen atom on the benzene ring of these compounds has been substituted with a lower alkyl group such as a methyl group, an ethyl group, or a propyl group, or a lower alkoxy group such as a methoxy group or an ethoxy group. Two or more of the above compounds can be mixed and used as the dihydric phenol compound.

[0080] The polyethersulfone may be a commercially available product, examples of which include Sumikaexcel 7600P and Sumikaexcel 5900P (both manufactured by Sumitomo Chemical Co., Ltd.).

[0081] The logarithmic viscosity of the polyethersulfone is preferably 0.5 or more, more preferably 0.55 or more, from the viewpoint of favorable formation of macrovoids in the porous polyethersulfone membrane, and is preferably 1.0 or less, more preferably 0.9 or less, even more preferably 0.8 or less, and particularly preferably 0.75 or less, from the viewpoint of ease of production of the porous polyethersulfone membrane.

[0082] Furthermore, from the viewpoint of heat resistance and dimensional stability at high temperatures, the PES porous membrane or the polyethersulfone used as its raw material preferably has a glass transition temperature of 200°C or higher, or no clear glass transition temperature is observed.

[0083] The method for producing the PES porous membrane that can be used in the present invention is not particularly limited, but may be, for example, A step of casting a polyethersulfone solution containing 0.3% by mass to 60% by mass of polyethersulfone having an inherent viscosity of 0.5 to 1.0 and 40% by mass to 99.7% by mass of an organic polar solvent into a film, and immersing or contacting the film with a coagulation solvent containing a poor solvent or a non-solvent for polyethersulfone as an essential component to produce a coagulated film having pores; A step of heat-treating the solidified membrane having pores obtained in the step above to coarsen the pores and obtain a PES porous membrane. wherein the heat treatment includes raising the temperature of the coagulated film having pores to a temperature equal to or higher than the glass transition temperature of the polyethersulfone, or 240°C or higher.

[0084] The PES porous membrane that can be used in the present invention is preferably a PES porous membrane having a surface layer A, a surface layer B, and a macrovoid layer sandwiched between the surface layer A and the surface layer B, the macrovoid layer has partition walls bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, and having an average pore size of 10 μm to 500 μm in the membrane plane direction; The partition walls of the macrovoid layer have a thickness of 0.1 μm to 50 μm, The surface layers A and B each have a thickness of 0.1 μm to 50 μm, one of the surface layers A and B has a plurality of pores with an average pore size of more than 5 μm and not more than 200 μm, and the other has a plurality of pores with an average pore size of 0.01 μm or more and less than 200 μm; One of the surface layer A and the surface layer B has a surface opening ratio of 15% or more, and the other surface layer has a surface opening ratio of 10% or more, the pores of the surface layer A and the surface layer B communicate with the macrovoids, The PES porous membrane has a total membrane thickness of 5 μm to 500 μm and a porosity of 50% to 95%. It is a PES porous membrane.

[0085] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art can easily make modifications and variations to the present invention based on the description in this specification, and such modifications and variations are within the technical scope of the present invention. [Example]

[0086] The porous polymer membrane used in the following examples was a polyimide porous membrane prepared by molding a polyamic acid solution composition containing a polyamic acid solution obtained from the tetracarboxylic acid component 3,3',4,4'-biphenyltetracarboxylic dianhydride (s-BPDA) and the diamine component 4,4'-diaminodiphenyl ether (ODA), and polyacrylamide, a coloring precursor, followed by heat treatment at 250°C or higher. The resulting porous polyimide membrane had a three-layer structure consisting of surface layers A and B, each having a plurality of pores, and a macrovoid layer sandwiched between surface layers A and B. The pores in surface layer A had an average pore size of 18 μm, the pores in surface layer B had an average pore size of 31 μm, the membrane thickness was 25 μm, and the porosity was 75%.

[0087] <Experiment 1> 1. Experimental Method

[0088] 1-1.Cell seeding Two days before transfection, a polyimide porous membrane (diameter: 19.00 mm) (hereinafter referred to as "PPI membrane") was placed in a 12-well plate (IWAKI, diameter: 22.10 mm, depth: 17.50 mm) and immersed in DMEM (High Glucose, 10% FBS, Wako Pure Chemical Industries, medium volume: 1.4 mL / well) for 30 minutes. AAVpro® 293T (Takara Bio, product number: 632273) was transfected at a density of 50,000 cells / cm. 2 The cells were seeded onto the PPI membrane at a density of 100 μg / cm2 and cultured in D-MEM (high glucose, 10% FBS) for 2 days.

[0089] As a comparative example, AAVpro (registered trademark) 293T (Takara Bio Inc.) was cultured in the above-mentioned 12-well plate (IWAKI, diameter: 22.10 mm, depth: 17.50 mm) at 50,000 cells / cm. 2 The seeds were directly sown at a density of 1000.

[0090] 1-2.Transfection The medium in each well prepared in 1-1 was replaced with fresh medium 4 hours before transfection. Then, the plasmids were mixed at the following mixing ratio to prepare a plasmid mixture. [Table 1]

[0091] 1.386 mL of PEIpro® (Polyplus, France) was diluted with 50 μL of D-MEM. The diluted PEIpro® solution was added to the above plasmid mixture and mixed by vortexing to prepare a PEIpro® + plasmid mixture. After incubating at room temperature for 15 minutes, 100 μL of the PEIpro® + plasmid mixture was added to each well of a 12-well plate. The 12-well plate was then placed in a CO2-resistant shaker (Thermo Scientific™, product number: 88881103) and cultured at 37°C in a 5% CO2 incubator with shaking (rotation mode: orbital, orbital diameter: 19 mm, rotation speed: 30, 60, 100, 150, 170, 190, 210, or 220 rpm). Shaking was performed for 1, 2, or 5 days after transfection.

[0092] Table 2 shows the amount of virus recovered after 1 day of incubation with shaking, 2 days, or 5 days of incubation. [Table 2]

[0093] Table 3 shows the amount of virus recovered after 2 days of incubation with shaking, and after 2 or 5 days of incubation. [Table 3]

[0094] Table 4 shows the amount of virus recovered after 5 days of incubation with shaking. [Table 4]

[0095] 1-3. Medium exchange and supernatant collection Two days after transfection, the culture supernatant was collected from each well, the medium was replaced with fresh D-MEM (high glucose, 2% FBS), and the cells were cultured for an additional 3 days, after which the culture supernatant was collected.

[0096] 1-4.RT-qPCR The amount of viral vector genome (DNA) in the collected culture supernatant was measured. The amount of viral vector genome (DNA) in the collected supernatant was measured using the AAVpro (registered trademark) Titration Kit (for Real Time PCR) Ver. 2 (Takara Bio Inc.).

[0097] 1-5.Hoechst staining Five days after transfection, samples treated under each experimental condition were washed once with DPBS, calcium, and magnesium (Thermo Fisher Scientific). Cellstain® Hoechst 33342 solution (Dojindo Laboratories) was then diluted 250-fold with DPBS, calcium, and magnesium (Thermo Fisher Scientific), and 1 mL was added to each well. The plate was incubated for 10 minutes. To obtain background images, a polyimide porous membrane and a 12-well plate (Corning) without cells were also stained using the same method.

[0098] 1-6. Image acquisition Using a 20x lens on a fluorescence microscope (Zeiss: Axio Observer5), images were taken of the area on side A of each sample where the cell count was >600 cells. Images were taken from three fields of view per sample (a total of nine fields of view, as three samples were observed). Images were also taken of the polyimide porous membrane and 12-well plate without cells seeded thereon using the same method. The excitation light intensity and exposure time conditions for obtaining each fluorescence were set as follows: ZsGreen1: Excitation light intensity 10%, exposure time 10 ms Hoechst: Excitation light intensity 50%, exposure time 800 ms

[0099] 1-7. Image analysis 1 (quantitative comparison of ZsGreen1 fluorescence intensity per cell) Using analysis software (ImageJ), the ZsGreen1 fluorescence intensity of each image was calculated as the "average fluorescence intensity of the sample image" minus the "fluorescence intensity of the background image." The "fluorescence intensity of the background image" was the fluorescence intensity of the image of only the porous polyimide membrane or dish without cells. The total number of cells (Hoechst-positive cells) within the field of view was then counted. Each image was binarized, and the thresholds for the porous polyimide membrane and dish were set to 130 and 80, respectively, and the Hoechst-fluorescent areas were colored black. The total area of ​​the black areas (=Hoechst-fluorescent areas) was then calculated and defined as the total area of ​​cell nuclei. Nine single cell nuclei (those not adjacent to neighboring cells) were randomly selected within the image, and the average area of ​​the nuclei on the porous polyimide membrane and dish was calculated and defined as the unit cell area. The number of cells in each image was then calculated (total area of ​​cell nuclei / unit cell area). From this, the ZsGreen1 fluorescence intensity per cell (ZsGreen1 fluorescence intensity / cell number) was calculated and compared as an index of transfection efficiency.

[0100] 1-8. Image analysis 2 (quantitative comparison of relative fluorescence intensity (ZsGreen1 fluorescence intensity / Hoechst fluorescence intensity)) Using analysis software (ImageJ), the ZsGreen1 fluorescence intensity of each entire image was calculated as "average fluorescence intensity of sample image" minus "fluorescence intensity of background image." "Fluorescence intensity of background image" is the fluorescence intensity of an image of only the polyimide porous membrane or dish without cells. Furthermore, the Hoechst fluorescence intensity of each entire image was calculated as "average fluorescence intensity of sample image" minus "fluorescence intensity of background image." "Fluorescence intensity of background image" is the fluorescence intensity of an image of only the polyimide porous membrane or dish without cells. From this, the fluorescence intensity ratio (ZsGreen1 fluorescence intensity / Hoechst fluorescence intensity) was calculated and compared as an index of transfection efficiency.

[0101] AAVpro® 293T cells seeded on a polyimide porous membrane or a dish were transfected with a transient gene expression vector that produces an adeno-associated virus (AAV) vector, and the cells were cultured with shaking for 1 day (Figure 4), 2 days (Figure 5), or 5 days (Figure 6). The ZsGreen fluorescence intensity per cell (ZsGreen1 / cell) was compared with the relative fluorescence intensity (ZsGreen1 fluorescence intensity / Hoechst fluorescence intensity) (ZsGreen1 / Hoechst) when the cells were cultured with shaking for 1 day (Figure 7), 2 days (Figure 8), or 5 days (Figure 9).

[0102] Table 5 shows the ZsGreen1 fluorescence intensity per cell after 1, 2, or 5 days of shaking culture. [Table 5]

[0103] 1-9. Regression analysis using nonlinear regression models A regression analysis was performed using a second-order nonlinear regression model with "ZsGreen1 fluorescence intensity per cell" as the dependent variable and "rotation speed" as the explanatory variable, as shown in Figure 10. Regression analysis was performed on the results after one day of shaking, and the following regression equation was obtained. y = 0.02234665 + 0.00056978x - 0.00000168x2 Regression analysis was performed on the results after 2 days of shaking, and the following regression equation was obtained. Y = 0.00801063 + 0.00088452x - 0.00000263x2 Regression analysis was performed on the results after 5 days of shaking, and the following regression equation was obtained. Y = 0.01472702 + 0.00100649x - 0.00000305x2 Table 6 shows the execution environment used for the regression analysis. [Table 6]

[0104] 2.Results AAVpro® 293T cells seeded on a polyimide porous membrane or a dish were transfected with a transient gene expression vector that produces an adeno-associated virus (AAV) vector, and cultured with shaking for 1 day (Fig. 1), 2 days (Fig. 2), or 5 days (Fig. 3). The amount of AAV vector genome produced in the culture supernatant was compared.

[0105] As shown in Figures 1 to 3, when AAVpro (registered trademark) 293T cells seeded on a polyimide porous membrane were transfected with a transient gene expression vector that produces an adeno-associated virus (AAV) vector and then cultured with shaking, the transfection efficiency was improved, suggesting that the amount of AAV vector produced increased.

[0106] As shown in Figures 4 to 9, when AAVpro (registered trademark) 293T cells seeded on a polyimide porous membrane were transfected with a transient gene expression vector that produces an adeno-associated virus (AAV) vector and cultured with shaking, the ZsGreen1 fluorescence intensity and specific fluorescence intensity (ZsGreen1 fluorescence intensity / Hoechst fluorescence intensity) per cell improved, suggesting improved transfection efficiency.

[0107] The results of the regression analysis in Figure 10 predicted that the "ZsGreen1 fluorescence intensity per cell (ZsGreen1 / cell)" at 309 rpm after one day of shaking would be equivalent to the "ZsGreen1 fluorescence intensity per cell (ZsGreen1 / cell)" at 30 rpm.

[0108] Furthermore, for the results after 2 days of shaking, it was predicted that the "ZsGreen1 fluorescence intensity per cell (ZsGreen1 / cell)" at 306 rpm would be equivalent to the "ZsGreen1 fluorescence intensity per cell (ZsGreen1 / cell)" at 30 rpm.

[0109] Similarly, for the results after 5 days of shaking, the "ZsGreen1 fluorescence intensity per cell (ZsGreen1 / cell)" at 300 rpm was predicted to be equivalent to the "ZsGreen1 fluorescence intensity per cell (ZsGreen1 / cell)" at 30 rpm.

Claims

1. 1. A method for producing a gene product from a gene introduced into a transient gene expression vector, comprising: (1) Cultivating cells engrafted on a porous polymer membrane with shaking during and / or after transfection of a transient gene expression vector; and (2) A step of recovering the cells obtained by the step (1) or the gene product produced by the cells. Including, wherein the polymer porous membrane is a three-layer structure polymer porous membrane having surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the pores in the surface layer A is smaller than the average pore size of the pores in the surface layer B, and the macrovoid layer has partition walls bonded to the surface layers A and B and a plurality of macrovoids surrounded by the partition walls and the surface layers A and B, and the pores in the surface layers A and B are in communication with the macrovoids. The method.

2. The method of claim 1, wherein the transient gene expression vector is a vector for producing a viral vector, and the gene product is a viral vector.

3. The method of claim 2, wherein the viral vector is one or more selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, and a lentiviral vector.

4. 2. The method of claim 1, wherein the transfection is one or more selected from the group consisting of electroporation, lipofection, infection with a viral vector, magnetofection, and microinjection.

5. 10. The method of claim 1, wherein the transfection is carried out using a cationic polymer, a cationic lipid, or DEAE-dextran.

6. The method of claim 1, wherein the transfection is carried out using a polyethyleneimine-based transfection reagent.

7. 10. The method of claim 1, wherein the shaking is performed at 10 to 300 rpm.

8. The method of claim 1 , wherein the cell is an animal cell.

9. The method of claim 1, wherein the porous polymer membrane has a plurality of pores with an average pore size of 0.01 to 100 μm.

10. The method according to claim 1, wherein the surface layer A has an average pore size of 0.01 to 50 μm.

11. The method according to claim 1, wherein the surface layer B has an average pore size of 20 to 100 μm.

12. The method according to claim 1, wherein the total thickness of the porous polymer membrane is 5 to 500 μm.

13. The method of claim 1 , wherein the polymer porous membrane is a polyimide porous membrane.

14. The method according to claim 13, wherein the polyimide porous film is a polyimide porous film comprising a polyimide obtained from a tetracarboxylic dianhydride and a diamine.

15. The method according to claim 13, wherein the polyimide porous film is a colored polyimide porous film obtained by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic dianhydride and a diamine and a colored precursor, and then heat-treating the resulting product at 250°C or higher.

16. A method for introducing a transient gene expression vector into cells, comprising a step of culturing cells engrafted on a porous polymer membrane by shaking at 10 to 300 rpm during and / or after transfection of the transient gene expression vector, wherein the porous polymer membrane has a three-layer structure having surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the pores in the surface layer A is smaller than the average pore size of the pores in the surface layer B, and the macrovoid layer has partitions bonded to the surface layers A and B and a plurality of macrovoids surrounded by the partitions and the surface layers A and B, and the pores in the surface layers A and B are in communication with the macrovoids.

17. 17. A method for introducing a transient gene expression vector into cells, comprising the step of culturing cells engrafted on a porous polymer membrane while shaking at 10 to 300 rpm during transfection with the transient gene expression vector, wherein the porous polymer membrane has a three-layer structure having surface layers A and B each having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore size of the pores in the surface layer A is smaller than the average pore size of the pores in the surface layer B, and the macrovoid layer has partitions bonded to the surface layers A and B and a plurality of macrovoids surrounded by the partitions and the surface layers A and B, and the pores in the surface layers A and B are in communication with the macrovoids.

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