TW200306349A - The cell culture method using a porous membrane - Google Patents

Abstract

To provide a cell culture method that allows a high-density culture and so on. There is provided a cell culture method that uses a porous membrane 1 such as a membrane filter dipped in culture medium as a foothold for the growth of cells 2. The method allows the formation of an environment closely analogous to the inside of the living body where cells can grow spatially. Furthermore, waste products 3 are removed by suction under vacuum from the cell-growing surface 101 of the porous membrane 1 to the opposite surface 102 thereof or by applying pressure on the cell-growing surface 101 to allow a continuous culture of high-density cells 2 (21).

Description

200306349 V. Description of the invention (1) 1 1. [Technical field to which the invention belongs] The present invention relates to cell culture technology. Specifically, it is particularly related to the cell culture technology, which is useful in the fields of regenerative medicine and the like, when using porous membranes as a foothold in cell proliferation. _ ^ 2. [Previous technology] Cell culture technology is to use the w-μ 被 which is transplanted to culture crying when the goods are delivered ... < The cells of the silkworm premises are 'slowly slowed down according to the chemotaxis of the cells' A technique that moves to the area around i 'to perform proliferation. 〃 ^

Cell proliferation depends on the mobility of the cells. Therefore, if the cells are initially moved in, they cannot be cleverly fixed in the culture place. They cannot be clever: the mobility of the cells cannot be used for vigorous cell proliferation. Therefore, the scaffold of 1 cell is an important guarantee for cell culture technology. Bad 1 Here, Patent Document 1 suggests that a milk supply device is needed instead of the West, and that the supply of cell metabolism is often and adequately necessary for the cell metabolism. Dyeing, easy transportation, and at least the container _ # Αώ, # γ ω '... ,, field cells / knockable knives are made of air-permeable plastic materials, which are previously cultured in cells, and are usually performed by instruments, and then developed by plastics. Hydrophilic treatment, or covering treatment with collagen or polyamic acid, etc., cell culture efficiency, and increase the culture. In the previous cell culture, the cells were moved to the surface of the culture place. Based on this idea, various glass special culture systems were developed. Dish, dish, Erlenmeyer flask culture equipment, and then the surface of the equipment or intercellular substance such as fibronectin makes cells more easily attached, and enhances the cell type. Technology, the main points of its improvement focus on improving adhesion to ensure its foothold, and using appliances.

200306349 V. Description of the invention (2) Container for cell culture. Patent Document 1 Japanese Patent Laid-Open Publication No. Hei 08-083. 3. [Summary of the Invention] However, the cells in the living body are surrounded by cells and interstitial cells. Usually, the entire surface of the cells is supplemented with nutrients, discharged from old wastes, and transmitted signals. ° That is, in the living body environment, there is almost no physical obstacle to the direction of cell proliferation, but the previous cell culture performed outside the living body, the cell culture is performed on the hard surface of glass or plastic culture equipment that cannot move material at all Therefore, there are technical issues that necessarily limit the direction of cell proliferation or the form of cell proliferation. Therefore, the present invention uses a "porous membrane" that can function as a foothold during cell proliferation and can move materials, and provides a cell culture method capable of easily realizing high-density cell culture. In order to solve the above technical problems, first, the present invention provides a cell culture method that seeks to perform cell culture with a porous membrane immersed in a culture medium. The porous membrane that may be used in the present invention includes a wide range of porous membranes with uniform and fine pores. Membrane body for substance passing function (filter function), not

:, Meaning explained. A representative example of the "porous membrane" is a polymer synthetic membrane generally called a membrane: an ane filter. Because in the porous membrane shape Sheng. Compared with the above-mentioned cell culture method in which cell proliferation is based on the use of porous membranes in glass, it has a strong foothold, so it promotes cell mobility and proliferates cell culture on the surface of culture equipment made of plastic or plastic. Restriction eases and cells easily overlap. that is,

200306349 V. Description of the invention (3) The internal environment can be increased three-dimensionally, which can increase the permeability of the gel of cells. It also has a second material. In the present invention, it is managed to overlap and use a majority of porous membranes to form a gap state. It can ensure that cell culture conditions (nutrient, old waste) in multi-layered cell culture conditions become suitable for multi-layered energy based on this porous membrane. That is, it can provide the advantage of using cell membranes, and possibly the quality of production. In addition, the culture method of culture medium sampling (Hollow fiber) culture method is to bundle hollow fibers on the fiber surface or enter the continuation # feature. Secondly, in the present invention, from the opposite side, the negative ink port and the base stream are used to 'try' while the old waste generated above is carried out and the cells formed there are ~ 1.0 / zm. Try to layer the pore membrane and then try to use the various porous free synthesizing methods for cultivation. The control device may be used to make interstitial proliferation fruits, and each protein is suitable for thin stacks. Especially with the multi-membrane culture and high density, the cell biological densitometer may obtain the current advantages. Columns, while the culture unit per unit of production is as high as the above, and the high-density reactor is cultivated with a fine gas cultured in a porous membrane layer. It can be observed that most of the so-called cell density of culture medium and f. Porous "except costly culture. Kong Yan. That is, a suitable method is easy to supply or exchange in a multi-layered (multi-layered) cell garden. The culture becomes a hollow fiber-type hollow fiber type culture cell that can be calculated on the basis of the prediction of the unit porosity of the protein, and the cells continue to flow back to allow them to attach cells to one of the above porous membranes or add from the cell proliferation surface The cell culture method for removing the above-mentioned cell cell culture by pressing the cell proliferation surface of the culture porous membrane. For the culture method, the diameter of the micropores of the porous membrane is not suitable. The reason is that when it is less than 〇 ##, 〇

Page 7 200306349 V. Description of the invention (4)

Without mentioning the pressure during suction or pressurization with negative pressure, it is more difficult to form a proper medium flow through the membrane, so a certain high pressure condition is required, but it is not suitable because of the damage caused by the application of ancient pressure to the cultured cells. In aspects, such as large =, the produced protein is removed along with the old waste. The protein produced by the cell is bound to the peptides and proteoglycans produced by the same, and the amount of surface molecules becomes large. Therefore, the pore diameter below 1 · / z m is blocked and difficult to pass. For this cell culture, the porous membrane has the uric acid and lipid peroxides opposite to each other, and the fine old waste is introduced. As a result, the pore membrane and pore control are related to the present invention. For cell proliferation, there is a technique, which is to combine the regeneration inside and outside the cell (reconstruction). 4. [Embodiment] For the test and experiment 1 ~ 5 of this issue. < Experiment 1 > Method, the passage of old waste wells such as the filtration function, and cell culture with extended cell life removal are used for extensive, cell standing tissue or dirty culture. The best method of biochemical biochemical foot and organ technology is based on multiple running to produce a continuous membrane of the cell membrane. It can be used in the field of cell proliferation, with low-molecular urea, and the cell proliferation surface is removed from the 4 proliferation surface to remove the soil culture. Also choose ^ high-density and continuous seven, especially the regenerative medicine collateral factor, has technical significance in bone formation.

Clarify the effects of the relevant cell culture methods,

Experiments were performed on the comparison of the fertilization point L of the lake fertilization, which caused differences in egg soil quality. For the protein quantification of Test 1, add the same amount of culture supernatant to the wells of the microtiter plate and place the Coomassie protein detection reagent S.

200306349 V. Description of the invention (5) (Coomassie Protein Assay Reagent Kit, PIERCE product), carried out by Bradford method. As a control, BSA (Bovine Serum Albumin, bovine serum albumin) was used. The obtained protein was qualitatively analyzed by SDS-PAGE (sodium lauryl sulfate-polyacrylamide gel electrophoresis) and immunooblot ting method using an anti-collagen antibody, and it was confirmed that most of the protein was collagen. (Comparative Example 1) Inoculation of human primary fibroblasts in a φ 60-band plastic petri dish (containing 10% bovine fetal serum (hereinafter abbreviated as rFBS) in DMEM, Japan Pharmaceutical Co., Ltd.) at 3 7 ° C incubator. That is, cell culture is performed without using a porous membrane. When reaching the (cell) confluent (conn f 1 u e n t) state, the culture supernatant was removed by suction, and the cell culture medium was replaced with a non-blood culture medium (nonFBS-DMEM). The culture supernatant as described above was recovered every week for protein quantification. At this time, the cell culture medium was replaced with a clear medium every time, and antibiotics were added. Also "DEME" #Dulbe (; ^ 〇 ,;

Modefied Eagle Medium (Examples 1 to 4) The cultivation of f acres of used membranes (hereinafter abbreviated as "MF") was fixed in advance (straight 13_ or 47_) at the bottom of the Mocm petri dish, which is the same as the above comparative example, ΓΛ Connection: V knife-grade primary fibroblasts (containing 1 °% bovine fetal blood = mouth mi I, _, Yueben Pharmaceutical Co., Ltd. II I wide stand, cultured. The inoculated cells are cultured in MOcm oral culture dishes When the bottom is uniformly attached and proliferated, the confluent state, A can be deduced on the cells: = Bottom ° 为 cells are fused I 邳 fused state. After this culture

200306349

Replace with serum-free medium. And every MF was moved to a new φ10 cm petri dish and the serum-free medium was changed once every 5 days. After the medium-grade primary fibroblasts were cultured to a confluent state in a culture dish for cell culture (culture area: 2.0 cm2), they were replaced with serum-free culturing substrates, and the quality of the protein in the serum-free medium was measured and compared. The diameter of the cells was 47onnMF (culture area: 173cm2) and the protein mass in serum-free culture. Soil At this time, four types of materials A to D are used. Stare at the type to abbreviate ―A, swollen B, MF-C, MF-D. MF-A (Example 1) is a hydrophilic polytetrafluoroethylene (pore diameter ···· 5 // m), and MF-B (Example 2) is a hydrophilic polyvinylidene difluoride (pore diameter 1.45 // m), MF-C (Example 3) is a non-hydrophilic polytetrafluoroethylene (pore size: 0.5 #m), and MF to D (Example 4) is polycarbonate (pore size: 0.4 / zm). The results are shown in Table 1 below. [Table 1] — redundant " cultivation equipment-7? ^ Total protein amount Ug during culture> protein mass / area / say ^ (Mg / cm2 / say; μ Comparative Example Petri dish P 7 ^ 2L :! 0.15. Implementation Example MF II A »405 ^ 4 7 ^ Example MF MF Bo 5 ^ 285-3 · 3ρ Example 1 MF 205 5, ^ 63, 0.73. Example 'MF-D-5 ^ -60 — 0.69 From Table 1 shown above, it is known that when cell culture is performed on MF, the amount of protein produced increases, and the protein production per unit area also increases, so the cell density per unit area increases. In particular, the use of MF-A and MF-B is When using a porous membrane, compared with the use of a petri dish, the production of protein is significantly increased, and cells can be cultured at a higher density. That is, the hydrophilic polytetrafluoroethylene of the hydrophilic porous membrane,

200306349 V. Description of the invention (7) Hydrophilic polyvinylidene difluoride is particularly suitable as a porous membrane material. < Experiment 2 > Experiment 2 was performed to verify that different cell footholds (comparing plastic culture JDI with filter membranes) caused differences in cell life. U-shaped, primary, and primary fibroblasts are cultured in plastic petri dishes (F a 1 c ο η male 1 ^ b petri dishes) as "Comparative Example 2", and cultured on a filter membrane: & Example 5 ”. Comparing the two, the cells were separated from the bottom to the planktonic position. After that, the cells were kept in a confluent state and cultured in serum-free.

30% of the culture supernatant was replaced with medium. The results of Experiment 2 are shown in Table 2. After culture ia > state p 70 # 70, the lamellar cells were detached from the bottom surface. 142. "Sheet cells have not been peeled off by MF cells. Cell culture using MF L has shown that cell culture using MF is suitable for increasing footholds to ^ ^ ^ °, .r... Its cell life is longer than that of culture (Comparative Example 2), that is, continuous culture of cells.

Here, the tube, A, and π are regarded as abnormal from the perspective of life expectancy of ordinary primary cell culture in January. However, in consideration of the metabolism of the skin fibers in the living body at a period of ~ 6 years, the culture conditions of Example 5 can be regarded as approximate the living body environment. This, again, should be considered that the cells do not stop cell proliferation in a fused state and continue cell proliferation with heavy abundance. When it is connected, the cells are connected into a thin sheet, and the field cells are not stripped by MF. Therefore, although each cell died, it was as if it were living.

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5. Description of the invention (8) In vivo, new cells proliferate one after another to fill the space where cell death occurs. MF is configured in multiple layers, and it is possible to culture cells at high density: a system for high density culture is called a bioreactor (reactor) for animal cell culture. Animal cells are divided into two types due to their proliferation patterns, so the above reactor There are generally two types.

First, the cells have planktonic cells that can proliferate in the culture medium and adherent cells that cannot proliferate without a foothold. The reactor needs to work for the above-mentioned attachment cells, so a horizontal roller type (roller bottle) that increases the attachment area, or a method of suspending the pellets in the reactor after attaching the cells to the surface of a microcar rier (suspension suspension) Method, air lift, cell lift, etc.). In addition, in the high-density culture, the development of the hollow fiber (Hoi low fiber) and ceramic matrix (Ceramic matrix) 〇 In this way, although the cell density has increased dramatically, it also occurs that can not be used to expand cell culture or monitor cell soil environment Problems with sampling. This monitoring is necessary when managing cell production to homogenize the cell environment. < Experiment 3 > "Experiment 3" was performed to verify the morphology of cells cultured on MF. After human primary fibroblasts were cultured on MF for 1 to 2 months, the stained cells were observed with a stereo microscope. As a result, overlapping of cells was observed, and a part of them showed a three-dimensional structure. FIG. 1 shows the above-mentioned microscope photograph. In the photo shown in Fig. 1, the trypan blue (or trypan blue, trypan b 1 ue) stains the darker part, which is the part with higher cell density. Cell culture methods using general plastic culture instruments

Page 12 200306349 V. Description of the invention (9) The surface is hard but not porous', so cells with this three-dimensional structure cannot be formed. Therefore, the proliferation of this three-dimensional cell can be considered as one of the major characteristics when MF is the foothold. As a result of multi-layered MF, the number of cells per unit area is increased, and a high protein production amount can be maintained (verified by Experiment 4 described later). Darker parts of the photo indicate higher cell density. In the above embodiment, the filtration membrane (MF) is used as a porous membrane, but it is not limited to this. In the cell culture method related to the present invention, the membrane body has at least a uniform fine pore membrane, and the porous membrane suitable for cell culture is uniform. Can be used, if it can be multi-layered, it is more suitable for use.

Here, a suitable development form of the cell culture method related to the present invention will be described with reference to FIG. 2. FIG. 2 is a conceptual diagram briefly showing the above development form. In FIG. 2, the cell 2 is attached to one side 10m of the porous membrane 1, and the opposite side 102 of the other side is attracted with negative pressure to form a medium flow F through the porous membrane i. The proliferation surface 101 removes the old waste 3 produced by the cells 2 and performs cell culture. Alternatively, the cell proliferation surface of the porous membrane 1 can be pressurized to make the old waste 3 produced by the cell 2 flow from the cell proliferation surface 101 to the opposite surface 102 with the medium flow f to remove it. Method.

The method of suction or pressurization by negative pressure may be a method of forming the flow of the culture medium 4 (medium flow F) that meets the purpose of the present invention, and the method of removing only the old waste 3 to the opposite side by 102 cells 4 Using these methods is also applicable to MF polyphonic media-Xi a medium flow F. Even layered ¥, in a multi-layered MF shape. Also shown in Figure 2 is the symbol 21 for the pattern.

200306349 V. Description of the Invention (ίο) Three-dimensional proliferation is performed on the pore membrane 1. < Experiment 4> Experiment 4

First, in order to make the MF easier, a frame having a large number of protrusions for holding and fixing a circular film-shaped MF with a diameter of 47 mm was produced. This vertical frame is designed so that when the frame itself is overlapped, the two phases are fixed. As a result of overlapping frames, it is possible to overlap (multi-layer) the evaluations that are fixed by the failure of each frame in order to ensure that the detail is equal to (space). Secondly, prepare a cylindrical container to house the overlapping MF and fix the liver's frame. This cylindrical container is made of a material with high air permeability (polymethylpentene or polycarbonate). In addition, the lid of this cylindrical container is also selected from the above-mentioned materials having air permeability. (Comparative Example 3 and Examples 6 to 9 related to Experiment 4) First, to attach cells to MF, a MF (MF-A or MF-B) with a diameter of 47 bands + fixed with white vaseline at 60 mm in diameter and cultured Dish, inoculated with human primary fibroblasts (DMEM medium containing 10% FBS), and cultured in a 37 ° C incubator. MF made 9 small holes with a diameter of 1 cm in a cross shape in advance,

The proliferation of field cells to these small holes means that the evaluated cells have reached a confluent state. Evil: The MF of this attached cell was directly fixed to a new 60 mm diameter petri dish with Vaseline as "Comparative Example 3". The MF was fixed to the above frame at a time, and placed in the above-mentioned cylindrical container. One MF is "Example 6", two MFs are "Example 7", 5 MFs are "Example 8", and 6 MFs are "Example 9". Compare

200306349 V. Description of the invention (11) Example 3 and Examples 6-9 were cultured in 10% FBS-containing DMEM medium, washed one week later and replaced with serum-free (DMEM) medium. The amount of protein in the culture supernatant was measured on the 10th day thereafter. The measurement results are shown in "Table 3" below. [表 有 有 FRAME # #number of y protein mass / area (ren g / cm2)> Comparative Example 3 · No ρ 1 (single layer) 5.6 ^ Example & yes-1 (single layer) 19.2 = Example 7 Yes 2 (multi-layer) 19 4. Examples are p 5 (multi-layer> 12.5, examples ~ yes. 6 (multi-layer) 12.6 ·: First, compare the comparative example 3 shown in Table 3 above with the implementation The result of Example 6 clarifies the use of the frame to form a gap (space) between the MF and the bottom surface of the container to improve the productivity of the protein. The reason may be that the above gap is formed, and it is easy to freely move materials (nutrition, old waste) around the cell or Supply or exchange of gas. Next, as shown in Examples 6 to 9, it can be seen that the frame of MF is superimposed and fixed. When multilayered MF is used, the protein is more productive than Comparative Example 3. That is, the structure of multilayered MF is also explained. Valid: When there are MFs (Embodiment 6) and when there are 2 MFs (Embodiment 7) and -------- V month, / when dual MFs (Embodiment 8) or 6 MFs (implementation) Example 9), there is a difference of approximately 35% in protein production. This means that it is possible to estimate and calculate the expanded production or the total protein production. As described above, in the present invention, using MF as the foothold of the cell may allow cell culture to be performed with a high degree of efficiency and good efficiency. Furthermore, a multi-layered structure is formed between the feet and the space, rather than a multi-layered structure that closely contacts MF and MF Improve what was previously considered the most

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200306349

The efficiency of the hollow fiber type MF is easy to predict. It is possible to observe the cells on the MF < Experiment 5 > It can also be counted as the amount of protein produced, or culture medium sampling, that is, simple expansion of production based on adherent cells. Supporting appliances are also easy to design. = The suitable material of the container (cylindrical container) containing the MF during inspection is for the purpose of the following "Experiment 5".

Cylindrical containers for multi-layered MF were prepared in three materials. Cells were cultured in each container, and the protein content in the culture supernatant was measured to investigate the effectiveness of gas exchange (breathability) during protein production. As the material of the container, polypropylene (Example 丨 0), polycarbonate (Example 丨 丨), and polymethylpentene (Example 12) were used. In addition, an embodiment (Example 3) in which the upper part of the cylindrical container with a high protein content in the culture supernatant is provided with a gas exchange property (air permeability) and a filter to further improve the gas exchange property. In addition, the cylindrical trough is highly airtight in the form of a closed lid, and does not leak when the water is inverted. The method of Experiment 5 will be described in detail. To attach cells to MF, fix straight # 4 7mm MF (MF-A or MF-B) with white vaseline at a diameter of 60 °.

The dish was inoculated with human primary fibroblasts (containing 10% FBS2DMEM medium) and cultured at 37 C. MF made nine small holes with a diameter of 1 mm in a cross shape with a 1 cm interval in advance. When the cells proliferated to these small holes, it was determined that the cells on the liver had reached a confluent state. The MF of this adherent cell was fixed to a frame, and it was stored in a cylindrical container of the actual yoke example 10 to 12 and then a particulate carrier 1 · 5 g of the adherent cell was placed. After 1 week of culture, the serum-containing medium was sufficiently removed, washed, and replaced with a serum-free medium. The following fourteenth: determination of protein in the culture supernatant

Page 16 200306349 V. Description of the invention (13) Quality. The measurement results are as shown in "Table 4" below. The material of the container θ The amount of medium (πύ > Protein concentration (# g / ml $ Example 10) Polypropylene 4 32.5 ^ ~~ Example 1L · Polycarbonate β 36.5— Implementation Example 12 ^ Polymethylammonium chloride 39.2 ^ 'Example 13 ^ Poly! Based on alkenes (with filter > 38.8,-^… a py 丄The 12-polymethylpentyl cylindrical container is the largest (39.2 // g / mi). The results of the material test conducted separately are that the maximum permeability of polymethylpentene. That is, the protein productivity of cells, and The breathability of the money holder is closely related. ^ Compare Example 12 and Example 13 again, as long as it is a polymethylpentene-based container and add an air permeability filter, the protein concentration does not change much. Therefore 1 It was learned that a cylindrical container made of a material having high air permeability such as polymethylpentene was used to improve the air permeability. 〆 According to the above experiment 5, the multilayered MF is accommodated in a high air permeability. Polyfluorenyl dilute container for cell culture, can carry out high-density cell culture. [Productive effect] [Inventive effect] / According to the cell culture method related to the present invention, cells can form a strong foothold in porous readings, thus promoting cell mobility and vigorously colonizing, slowing cell proliferation. Limitation, and can overlap cells, making high-density culture of cells possible. Also, as a result of high-density culture of cells, it is possible to increase cells

—1 1 丨, _ Page 17 200306349 V. Description of the invention (14) The production of collagen and other proteins is expected to become a regenerative medical technology, especially a cell that uses the basic technology of regenerative medical treatment of autologous tissue obtained from the proliferation of autologous cells Training methods. It is very convenient to use the above-mentioned porous membrane in multiple layers to make higher density cell culture possible. Depending on the number of porous membranes used, it is possible to control the cell density or calculate the amount of protein produced, which is extremely convenient. It is also possible to design culture devices that can observe the sampling of cells or culture media on porous membranes.

Cells are attached to one side of the porous membrane, and the opposite side is attracted by negative pressure or pressurized by the cell proliferation surface to form a medium flow through the porous membrane. If you try to remove the old waste generated by the cells, the cells of the porous membrane may be 捭 = The above efficiency does remove low-molecular urea, urine, and old waste produced by cells. As a result, the cell life is prolonged, and the cell can be surely improved. ,,, Λ. support. Furthermore, by selecting the pore size of the porous membrane, you can control the removal of the material, such as concentrating the cell's autocrine secretion with the porous membrane, that is, the autologous action of the active principle of the Douwangli, and the low-molecular active substance. The waste is removed through the porous membrane, and the raw secretion is replaced by bovine fetal serum (FBS), which can be used for autologous proliferation. θ ,, can be maintained by adding protein-free culture medium ’

Page 18 200306349 Simple illustration of the diagram V. [Simplified illustration of the diagram] FIG. 1 is an enlarged photo of a stereo microscope of the cultured cells of Experiment 3. FIG. Fig. 2 is a conceptual diagram briefly showing a suitable development form of a cell culture method related to the present invention. Explanation of component symbols: 5 porous membrane 6 cells 7 old waste 8 medium F medium flow 2 1 three-dimensional cell proliferation mode 1 0 1 cell proliferation surface of porous membrane 1 102 reverse side of 101 of porous membrane 1

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Claims (1)

200306349 VI. Application Patent Scope 1. A cell culture method, characterized in that the cell culture is performed by a porous membrane immersed in a culture medium. 2. The cell culture method according to item 1 of the application, wherein the porous membrane is multilayered. 3. If the cell culture method according to item 1 or item 2 of the patent application scope, wherein the cells are attached to one side of the porous membrane, the reverse side is attracted by negative pressure or the cell proliferation side is pressurized to form a medium flow, The cell proliferation surface of the porous membrane removes the old waste generated by the above cells while performing cell culture 0 Φ Page 20