JP2016146777A - Bag for cell cultivation and cell cultivation method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a bag for cell cultivation capable of preparing cell containing liquid in which floating cell, multipotent stem cell etc. are contained at a high concentration and living cells are included at a high ratio.SOLUTION: A bag for cell cultivation comprises a bag shape container made of tetrafluoroethylene (TFE)-hexafluoropropylene (HFP) copolymer, in which calculated average roughness (Ra) in surface roughness of inner surface of the bag shape container is 3.5 to 6.5nm, root-mean-square roughness (RMS) in surface roughness of the inner surface of the bag shape container is 4.5 to 8.0nm, and surface free energy in inner surface of the bag shape container is 16.5 to 18.5 (mJ/m). When human marrow mesenchymal stem cells (hMSC cells) are cultured in the bag shape container, multipotent stem cells are efficiently induced due to improvement in floating condition, thus preparation of suspension of multipotent stem cell may be carried out at high concentration and at high ratio in living cell, enabling providing cell suspension for transplantation in regenerative therapy of high quality.SELECTED DRAWING: None

Description

The present invention includes a bag-like container made of a tetrafluoroethylene (TFE) -hexafluoropropylene (HFP) copolymer, and the arithmetic average roughness (Ra) of the surface roughness of the inner surface of the bag-like container is 3.5. -6.5 nm, the root mean square roughness (RMS) of the surface roughness of the inner surface of the bag-shaped container is 4.5-8.0 nm, and the surface free energy of the inner surface of the bag-shaped container is 16.5- The present invention relates to a cell culture bag of 18.5 (mJ / m ² ) and a cell culture method for culturing cells in suspension in the bag-like container of the cell culture bag.

  In the field of basic research and regenerative medicine, it is a common practice to subculture and propagate mammalian cells in vitro. In particular, pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are expected to be put to practical use in regenerative medicine and drug discovery research. It is necessary to stably mass-culture cells. However, the conventional adhesion culture method using a glass or synthetic resin flask or petri dish is not suitable for mass production, so a bag capable of culturing pluripotent stem cells or a culture method using the bag Development is underway.

  Bags for floating culture consisting of a poly (ethylene butylene) polystyrene block copolymer and a polymer alloy in which an ethylene acrylate copolymer is mixed with a polymer mixture of polypropylene are excellent in transparency and gas permeability. (Patent Document 1). Commercially available bags for suspension culture with excellent transparency and gas permeability are also known. For example, CultiLife [registered trademark] Spin bag (made by TAKARA) made of ethylene vinyl acetate, TFE-HFP VueLife FEP Bag 32-C (manufactured by American Fluoroseal Corporation), culture bag A-1000NL (manufactured by Nipro Corporation), and the like, which are made of a copolymer (FEP), are commercially available.

  Recently, it has been reported that cells can be efficiently proliferated by suspension culture of ES cells and iPS cells using a culture solution containing methylcellulose and gellan gum and a gas permeable culture bag developed by Nipro (non-patent document). 1).

JP-A-3-65177

Otsuji, T.G., et al. Stem Cell Reports. 2: 734-745 (2014).

  An object of the present invention is to provide a cell culture bag capable of preparing a cell-containing solution having a high concentration and a high proportion of living cells such as suspension cells and pluripotent stem cells.

The present inventors are diligently studying to solve the above problems, and using a TFE-HFP copolymer film as a material, the surface roughness Ra is 3.5 to 6.5 nm, and the surface roughness is A cell culture bag having an inner surface of a bag-like container having an RMS of 4.5 to 8.0 nm and a surface free energy of 16.5 to 18.5 (mJ / m ² ) is manufactured, and the bag is used. When human peripheral blood-derived mononuclear cells (hPBMC) were cultured, it was found that the decrease in cell viability was effectively suppressed. It was also confirmed that when human bone marrow-derived mesenchymal stem cells (hMSC cells) were cultured using the bag, pluripotent stem cells were efficiently induced by improving the floating state. The present invention has been completed based on these findings.

That is, the present invention comprises (1) a bag-like container made of a tetrafluoroethylene-hexafluoropropylene copolymer, and the arithmetic average roughness (Ra) of the surface roughness of the inner surface of the bag-like container is 3.5-6. 0.5 nm, the root mean square roughness (RMS) of the inner surface of the bag-shaped container is 4.5 to 8.0 nm, and the surface free energy of the inner surface of the bag-shaped container is 16.5 to 18. 5 (mJ / m ² ), or (2) The cell culture bag according to (1) above, wherein the bag-like container has a thickness of 10 to 100 μm And (3) the cell culture bag according to (1) or (2) above, wherein the cells are mesenchymal stem cells or peripheral blood-derived mononuclear cells.

The present invention also includes (4) a bag-like container made of a tetrafluoroethylene-hexafluoropropylene copolymer, and the arithmetic average roughness (Ra) of the surface roughness of the inner surface of the bag-like container is 3.5-6. 0.5 nm, the root mean square roughness (RMS) of the inner surface of the bag-shaped container is 4.5 to 8.0 nm, and the surface free energy of the inner surface of the bag-shaped container is 16.5 to 18. 5 (mJ / m ² ) cell culturing method characterized in that cells are suspended in the bag-like container, or (5) the bag-like container has a thickness of 10 to 100 μm. The culture method according to (4) above, or (6) the cells according to (4) or (5) above, wherein the cells are mesenchymal stem cells or peripheral blood-derived mononuclear cells. It relates to the culture method of.

  When cells are cultured using the cell culture bag of the present invention, a decrease in cell viability can be effectively suppressed, and pluripotent stem cells can be efficiently induced by improving the floating state. In addition, a cell suspension having a high ratio of living cells can be prepared, and a high-quality cell suspension for transplantation in regenerative medicine can be provided.

FIG. 1A is a diagram showing a microscopic image of the surface of a cell culture bag A (commercial product; comparative sample 1) when measured in a measuring range of 3 μm square. FIG. 1B is a diagram showing a microscopic image of the surface of cell culture bag B (Example sample) when measured in a measurement range of 3 μm square. Six types of cell culture bags (cell culture bag A, cell culture bag B, cell culture bags C to E [Comparative Example Samples 2 to 4], and cell culture bag F [commercial product; comparative sample 5 ]) Is a diagram showing a microscopic image of the cells when culturing hMSC cells for 1 day. It is a figure which shows the microscope image of a cell when hPBMC cell is cultured for 7 days using said 6 types of cell culture bags AF.

The cell culture bag of the present invention is a bag for use in cell culture, comprising a bag-like container made of a TFE-HFP copolymer, and the arithmetic average roughness of the surface roughness of the inner surface of the bag-like container ( Ra) is 3.5 to 6.5 nm, the root mean square roughness (RMS) of the surface roughness of the inner surface of the bag-shaped container is 4.5 to 8.0 nm, and the surface freeness of the inner surface of the bag-shaped container is It is characterized in that the energy is 16.5 to 18.5 (mJ / m ² ). When cells are cultured in a bag-like container having such characteristics, cell adhesion and cell viability decrease are effectively suppressed. Therefore, the cell culture bag of the present invention provided with such a bag-like container In addition, it can be suitably used for suspension culture of adherent cells. When used for suspension culture, the above-mentioned bag-like container is matrigel, entactin, fibronectin, temperature-responsive polymer (PIPAAm), polycation (polylysine, etc.), gelatin, lectin, polysaccharide (hyaluronic acid, etc.), polylactic acid, polyglycol A cell adhesive substance such as acid, ε-aminocaprolactone, type I collagen, type IV collagen, chitosan, laminin, etc., whose inner surface is not coated (or arranged) is preferred.

  The thickness of the bag-like container is preferably in the range of 10 to 100 μm, more preferably in the range of 15 to 95 μm, further preferably in the range of 20 to 92 μm, from the viewpoint of durability and cost effectiveness. Even more preferably within the range, particularly preferably within the range of 30 to 86 μm, particularly preferably within the range of 35 to 83 μm, most preferably within the range of 40 to 80 μm.

  In the present invention, “TFE-HFP copolymer” means a copolymer containing at least TFE and HFP. That is, the “TFE-HFP copolymer” includes a binary copolymer of TFE and HFP (TFE / HFP copolymer; FEP), and a copolymer of TFE, HFP, and vinyl fluoride (VF). Polymer (TFE / HFP / VF copolymer), TFE / HFP / vinylidene fluoride (VDF) copolymer (TFE / HFP / VDF copolymer), TFE / HFP / perfluoro (alkyl vinyl ether) ( Terpolymers such as copolymers with PAVE) (TFE / HFP / PAVE copolymer), and copolymers of TFE, HFP, VF and VDF (TFE / HFP / VF / VDF copolymer). , TFE / HFP / VF / PAVE copolymer (TFE / HFP / VF / PAVE copolymer), TFE / HFP / VDF / PAVE copolymer (TFE / HFP / VD) Quaternary copolymers such as TFE / HFP / VF / VDF / PAVE copolymer) and quaternary copolymers such as TFE / HFP / VF / VDF / PAVE copolymer. Is also included.

  The TFE-HFP copolymer of the present invention is preferably a TFE / HFP copolymer or a TFE / HFP / PAVE copolymer. 80-97 / 3-20 are preferable and, as for the mass ratio of TFE and HFP in this TFE / HFP copolymer, 84-92 / 8-16 are more preferable. The mass ratio of TFE, HFP, and PAVE in the TFE / HFP / PAVE copolymer is preferably 70 to 97/3 to 20 / 0.1 to 10, more preferably 81 to 92/5 to 16 / 0.3. ~ 5 is more preferred.

  Examples of the suspension cells include suspension cells such as red blood cells and white blood cells (derived from peripheral blood) (neutrophils, mononuclear cells [monocytes, lymphocytes], macrophages, etc.). Nuclear spheres are preferred.

  Examples of the adhesive cells include embryonic stem cells (embryonic stem cells: ES cells), embryonic germ cells (embryonic germ cells: EG cells), germ line stem cells (germline stem cells: GS cells), induced pluripotent stem cells. Pluripotent stem cells (iPS cells; induced pluripotent stem cells), mesenchymal stem cells, hematopoietic stem cells, multipotent stem cells such as neural stem cells, myocardial progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, fat precursors Stem cells such as cells, dermal fibroblasts, skeletal muscle myoblasts, osteoblasts, odontoblasts, etc., cardiomyocytes, vascular endothelial cells, nerve cells, fat cells, skin fibers Mature cells such as cells, skeletal muscle cells, bone cells, hepatocyte (hepatocyte) cells, umbilical vein endothelial cells, cutaneous microlymphatic endothelial cells, epidermal keratinocytes, bronchial epithelial cells, melanocyte cells, smooth muscle cells, ivory cells Raising Mesenchymal stem cells are preferred. In addition to mesenchymal stem cells, pluripotent stem cells are induced by suspension culture of the progenitor cells and the mature cells. Therefore, the cell culture bag of the present invention is advantageous for the preparation of pluripotent stem cells using these cells. Can be used.

In the present invention, “surface roughness Ra” means an average of absolute values of deviations from a reference surface (flat surface which is an average value of heights of designated surfaces) to a designated surface as defined in JIS B0601. Calculated by the following equation.
Ra = 1 / S ₀ ∬ | F (X, Y) −Z ₀ | dXdY
Here, S ₀ represents the area of the reference surface, Z ₀ represents the height of the reference surface, and F (X, Y) represents the height of the designated surface at the coordinates (X, Y).

  Ra of the surface roughness of the inner surface of the bag-like container in the cell culture bag of the present invention may be in the range of 3.5 to 6.5 nm, for example, 3.5 to 6.3 nm, 3.5 to 6 .1 nm, 3.5-5.9 nm, 3.5-5.7 nm, 3.5-5.5 nm, 3.5-5.3 nm, 3.7-6.5 nm, 3.9-6.5 nm 4.1-6.5 nm, 4.3-6.5 nm, 4.5-6.5 nm, 4.7-6.5 nm, 4.9-6.5 nm, 3.7-6.3 nm, 3 0.7-6.1 nm, 3.7-5.9 nm, 3.7-5.7 nm, 3.7-5.5 nm, 3.7-5.3 nm, 3.9-6.3 nm, 3.9 -6.1 nm, 3.9-5.9 nm, 3.9-5.7 nm, 3.9-5.5 nm, 3.9-5.3 nm, 4.1-6.3 nm, 4.1-6 .1 nm, 4.1-5.9 m, 4.1-5.7 nm, 4.1-5.5 nm, 4.1-5.3 nm, 4.3-6.3 nm, 4.3-6.1 nm, 4.3-5.9 nm, 4.3 to 5.7 nm, 4.3 to 5.5 nm, 4.3 to 5.3 nm, 4.5 to 6.3 nm, 4.5 to 6.1 nm, 4.5 to 5.9 nm, 5 to 5.7 nm, 4.5 to 5.5 nm, 4.5 to 5.3 nm, 4.7 to 6.3 nm, 4.7 to 6.1 nm, 4.7 to 5.9 nm, 4.7 to 5.7 nm, 4.7-5.5 nm, 4.7-5.3 nm, 4.9-6.3 nm, 4.9-6.1 nm, 4.9-5.9 nm, 4.9-5. Although 7 nm, 4.9-5.5 nm, 4.9-5.3 nm, etc. may be sufficient, it is preferable that it is 4.9-5.3 nm.

In the present invention, “RMS of surface roughness” is a square root of a value obtained by averaging the squares of deviations from the reference surface to the designated surface, and is calculated by the following equation.
RMS = [1 / S ₀ ∬ {F (X, Y) −Z ₀ } ² dXdY] ^1/2
Here, S ₀ represents the area of the reference surface, Z ₀ represents the height of the reference surface, and F (X, Y) represents the height of the designated surface at the coordinates (X, Y).

  The surface roughness RMS of the inner surface of the bag-like container in the cell culture bag of the present invention may be in the range of 4.5 to 8.0 nm, for example, 4.5 to 7.8 nm, 4.5 to 7 .6 nm, 4.5 to 7.4 nm, 4.5 to 7.2 nm, 4.5 to 7.0 nm, 4.5 to 6.8 nm, 4.5 to 6.6 nm, 4.5 to 6.4 nm 4.7 to 8.0 nm, 4.9 to 8.0 nm, 5.1 to 8.0 nm, 5.3 to 8.0 nm, 5.5 to 8.0 nm, 5.7 to 8.0 nm, 5 9.9-8.0 nm, 6.1-8.0 nm, 4.7-7.8 nm, 4.7-7.6 nm, 4.7-7.4 nm, 4.7-7.2 nm, 4.7 -7.0 nm, 4.7-6.8 nm, 4.7-6.6 nm, 4.7-6.4 nm, 4.9-7.8 nm, 4.9-7.6 nm, 4.9-7 4 nm, 4.9-7. nm, 4.9-7.0 nm, 4.9-6.8 nm, 4.9-6.6 nm, 4.9-6.4 nm, 5.1-7.8 nm, 5.1-7.6 nm, 5.1-7.4 nm, 5.1-7.2 nm, 5.1-7.0 nm, 5.1-6.8 nm, 5.1-6.6 nm, 5.1-6.4 nm, 5. 3-7.8 nm, 5.3-7.6 nm, 5.3-7.4 nm, 5.3-7.2 nm, 5.3-7.0 nm, 5.3-6.8 nm, 5.3- 6.6 nm, 5.3-6.4 nm, 5.5-7.8 nm, 5.5-7.6 nm, 5.5-7.4 nm, 5.5-7.2 nm, 5.5-7. 0 nm, 5.5-6.8 nm, 5.5-6.6 nm, 5.5-6.4 nm, 5.7-7.8 nm, 5.7-7.6 nm, 5.7-7.4 nm, 5.7-7.2 nm, 5.7-7.0 nm 5.7 to 6.8 nm, 5.7 to 6.6 nm, 5.7 to 6.4 nm, 5.9 to 7.8 nm, 5.9 to 7.6 nm, 5.9 to 7.4 nm, 5. 9 to 7.2 nm, 5.9 to 7.0 nm, 5.9 to 6.8 nm, 5.9 to 6.6 nm, 5.9 to 6.4 nm, 6.1 to 7.8 nm, 6.1 to 7.6 nm, 6.1-7.4 nm, 6.1-7.2 nm, 6.1-7.0 nm, 6.1-6.8 nm, 6.1-6.6 nm, 6.1-6. Although 4 nm etc. may be sufficient, it is preferable that it is 6.1-6.4 nm.

  The measurement value necessary for calculating Ra and RMS of the surface roughness is, for example, a dynamic force using a high precision large probe microscope unit AFM5200S (manufactured by HITACHI High-Tech) as an atomic force microscope (AFM). In mode, the surface of the film is measured under the conditions of a measurement area of 3 × 3 μm square, a scanning speed of 1 Hz, a 256 × 256 division in the xy direction, and a cantilever SI-DF-20 (Si, f = 134 kHz, C = 16 N / m). It can obtain | require by performing the inclination automatic correction process about the measured AFM topography image.

  In the present invention, “surface free energy” refers to a value calculated by calculation of two components of water / n-hexadecane according to the Owens-Wendt theory. Such a surface free energy value can be calculated, for example, by measuring a static contact angle with respect to water and n-hexadecane having a droplet volume of 2 μL using DropMatter 701 (manufactured by Kyowa Interface Science Co., Ltd.).

The surface free energy of the inner surface of the bag-like container in the cell culture bag of the present invention may be in the range of 16.5 to 18.5 (mJ / m ² ), for example, 16.5 to 18.3 (mJ). / M ² ), 16.5 to 18.1 (mJ / m ² ), 16.5 to 17.9 (mJ / m ² ), 16.5 to 17.7 (mJ / m ² ), 16.7 ˜18.5 (mJ / m ² ), 16.9 to 18.5 (mJ / m ² ), 17.1 to 18.5 (mJ / m ² ), 17.3 to 18.5 (mJ / m) ² ), 17.5 to 18.5 (mJ / m ² ), 16.7 to 18.3 (mJ / m ² ), 16.7 to 18.1 (mJ / m ² ), 16.7 to 17 .9 (mJ / m ² ), 16.7 to 17.7 (mJ / m ² ), 16.9 to 18.3 (mJ / m ² ), 16.9 to 18.1 (mJ / m ² ) , 16 ^{9~17.9 (mJ / m 2),} 16.9~17.7 (mJ / m 2), 17.1~18.3 (mJ / m 2), 17.1~18.1 (mJ / m ² ), 17.1 to 17.9 (mJ / m ² ), 17.1 to 17.7 (mJ / m ² ), 17.3 to 18.3 (mJ / m ² ), 17.3 ^{18.1 (mJ / m 2),} 17.3~17.9 (mJ / m 2), 17.3~17.7 (mJ / m 2), 17.5~18.3 (mJ / m 2 ) 17.5 to 18.1 (mJ / m ² ), 17.5 to 17.9 (mJ / m ² ), 17.5 to 17.7 (mJ / m ² ), etc. It is preferable that it is 17.5-17.7 (mJ / m < ² >).

  The bag-like container in the cell culture bag of the present invention usually contains an FEP material at least partially so that the inside and the outside are separated from each other via the FEP in a sealed state. For example, when a culture solution is filled in a bag-like container, at least 30%, preferably 50% or more, more preferably 80% or more, more preferably 90% or more, more preferably 90% or more of the surface that can come into contact with the culture solution. Preferably 95% or more, particularly preferably 98% or more, most preferably substantially 100% is constituted by FEP. In the present specification, “sealing” is performed, for example, by a method of closing the tube with a clamp or the like, a method of closing the tube by heat welding, or the like.

  The bag-like container is preferably made of a flexible FEP sheet (film). Examples of the shape of the bag-like container include a square, a rectangle, a rhombus, a circle, and an ellipse. A rectangle is preferable from the viewpoint of appearance and ease of manufacture.

  In the bag-like container, at least one port made of FEP or the like for introducing or discharging a cell suspension or a culture solution can be provided in an airtight manner. The port can be provided with a closing means so that the bag-like container can be sealed. Examples of the closing means include a holding body such as a clip, a communication piece (folding bar), and a cock.

  The bag-like container in the cell culture bag of the present invention is manufactured by stacking a TFE-HFP copolymer film (sheet) and then heat-sealing the edge using an impulse sealer or the like. Can do.

  The cell culture bag of the present invention may be subjected to sterilization treatment such as ethylene oxide gas sterilization and autoclave sterilization as necessary.

  The cell culturing method of the present invention is not particularly limited as long as it is a method of culturing the above-mentioned suspension cells or adhesive cells in the culture solution held in the bag-like container of the cell culture bag of the present invention, The culture medium for such cells is not particularly limited as long as the cells can survive and proliferate, and 0.1 to 30 (v / v)% serum (Fetal bovine serum; FBS), calf Culture medium for animal cell culture (DMEM, EMEM, IMDM [for example, manufactured by gibco)], RPMI1640, αMEM, F-12, F-10, M-199, AIM- containing serum [Calf bovine serum; CS], etc. V, MSCBM [for example, made by Lonza] etc.), commercially available B27 supplement (-insulin) (made by Life Technologies), N2 supplement (made by Life Technologies), B27 supplement (made by Life Technologies), Knockout Se Examples include the above-mentioned culture medium for animal cell culture to which serum substitutes such as rum Replacement (manufactured by Invitrogen) and mesenchymal stem cell additive factor set (manufactured by Lonza) are added in an appropriate amount (for example, 1 to 30%). . In order to further improve the floating properties of the cells, the culture solution may be supplemented with a high molecular polymer such as methylcellulose, gellan gum, guar gum, xanthan gum, dextran as appropriate. However, due to the effect of the cell culture bag of the present invention. The cells can be effectively cultured in suspension even in the absence of the polymer. For this reason, the culture solution preferably does not contain the above polymer.

The culture temperature of the cells is usually in the range of about 30-40 ° C, preferably 37 ° C. The CO ₂ concentration at the time of culture is usually in the range of about 1 to 10%, preferably about 5%. Moreover, the humidity at the time of culture | cultivation is in the range of about 70-100% normally, Preferably it exists in the range of about 95-100%.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

1. Manufacture of cell culture bag Three types of 5cm x 10cm size films (50μm thick FEP material, 25μm thick FEP material, and tetrafluoroethylene [TFE] -perfluoro) using impulse sealer By heat-sealing [alkyl vinyl ether] [PAVE] copolymer [PFA] film having a thickness of 50 μm under the conditions of a sealing temperature of 370 ° C., a sealing time of 50 seconds, a sealing pressure of 0.2 MPa, and a sealing width of 5 mm. Four types of cell culture bags (cell culture bag B [FEP material thickness 50 μm bag; example sample], cell culture bag C [FEP material thickness 25 μm bag; comparative example sample 2], Cell culture bag D [PFA material 50 μm thick bag; comparative sample 3], and cells [Bag PFA material thickness 50 [mu] m; Comparative Sample 4] Yoyo bag E) was prepared.

2. Surface analysis of the inner surface of a cell culture bag 2-1 Surface roughness In addition to the four types of cell culture bags produced in Example 1, using a high precision large probe microscope unit AFM5200S (manufactured by HITACHI High-Tech) Then, the surface roughness of the bag inner surface of two types of commercially available cell culture bags (cell culture bags A and F [Comparative Example Samples 1 and 5]) was measured (see Table 1). When the measurement range is 3 μm square, Ra and RMS on the inner surface of the cell culture bag A (Comparative Example Sample 1) are 3.0 nm and 3.8 nm, respectively (see FIG. 1A, Table 1). The Ra and RMS on the inner surface of the cell culture bag C (Comparative Sample 2) were 4.0 nm and 5.1 nm (see Table 1), respectively, whereas the Cell Culture Bag B (Example Sample) ) Of the bag inner surface was 5.1 nm and 6.3 nm (see FIG. 1B, Table 1), respectively. These results indicate that the surface roughness of the inner surface of the FEP material cell culture bag B (Example sample) is larger than that of the FEP material cell culture bag A and C (Comparative Samples 1 and 2). Is shown. Moreover, Ra and RMS of the bag inner surface of the cell culture bag D of PFA material (Comparative Example Sample 3) are 5.5 nm and 7.5 nm, respectively, and the cell culture bag E of PFA material (Comparative Example Sample 4). Ra and RMS on the inner surface of the bag are 4.5 nm and 5.7 nm, respectively, and Ra and RMS on the inner surface of the cell culture bag F (Comparative Example Sample 5) made of ethylene-vinyl acetate copolymer (EVA) material. Were 3.0 nm and 3.7 nm, respectively (see Table 1).

2-2 Contact angle Moreover, about the contact angle of the bag inner surface of the bags A to F for cell culture when water and n-hexadecane (n-HD) are used by the droplet method, a fully automatic contact angle meter DM-701 ( (Manufactured by KYOWA) (number of measurements: 5 times each) (see Table 1). The surface free energy was calculated based on the average value of the obtained measurement data (see Table 1). As a result, the surface free energies of the bag inner surfaces of the cell culture bags A and C (Comparative Samples 1 and 2) were 19.5 and 22.6 (mJ / m ² ), respectively. The surface free energy of the bag inner surface of the culture bag B (Example sample) was 17.6 (mJ / m ² ) (see Table 1). This result shows that the surface free energy on the inner surface of the FEP material cell culture bag B (Example sample) is smaller than that of the FEP material cell culture bag A and C (Comparative sample 1 and 2). Indicated. The surface free energy of the inner surface of the cell culture bag D made of PFA material is 17.5 (mJ / m ² ), and the surface of the bag inner surface of the cell culture bag E made of PFA material (Comparative Sample 4). The free energy was 22.5 (mJ / m ² ), and the surface free energy on the inner surface of the EVA cell culture bag F (Comparative Sample 5) was 30.0 (mJ / m ² ). (See Table 1).

3. Culturing hMSC cells using cell culture bag In Example 1, cell culture bag B (Example sample) is more surface than cell culture bags A and C (Comparative Example Samples 1 and 2) of the same FEP material Since it was shown that the roughness was high and the surface free energy was small, when cells were cultured using the cell culture bag B, it was analyzed whether cell adhesion was suppressed and suspension culture was possible.

3-1 Method 3-1-1 Cell culture [1] MSCBM (Mesenchymal Stem with hMSC cells (Lonza, PT-2501) added with mesenchymal stem cell additive factor set (Lonza, PT-4105) Cell Basal Medium) (manufactured by Lonza, PT-3238) was suspended in a culture solution at a concentration of 1 × 10 ⁵ cells / mL, and 4 mL was seeded in six types of cell culture bags A to F.
[2] After culturing in an incubator (37 ° C., 5% CO ₂ ), the cells were observed under a microscope (IX-70, Olympus) on the first day (see FIG. 2).
[3] After further culturing for 6 days, gene expression analysis was performed according to the method described in “3-1-2 mRNA expression analysis” below.

3-1-2 mRNA expression analysis [1] Cells are collected in a 15 mL centrifuge tube, and the total RNA of the cells is obtained using the RNeasy Mini Kit (Qiagen) and QIA shredder (Qiagen) according to the protocol attached to the product. Extracted.
[2] The concentration of the extracted total RNA was measured using NanoDrop 2000 (Thermo Fisher Scientific).
[3] Total RNA was adjusted to 20 μg / mL, and dispensed to a 96-well plate (Fast 96 well Reaction plate [Applied Biosystems, # 4309169]) at 3 μL / well.
[4] TaqMan One-Step RT-PCR Master Mix Reagents Kit (manufactured by Applied Biosystems) in order to detect mRNA expression levels of two types of pluripotent stem cell marker genes (Nanog and Oct3 / 4) by RT-PCR , # 4309169) was used to prepare a reaction solution consisting of the following 1) to 6) and added dropwise to a 96-well plate into which total RNA was dispensed. The GAPDH gene was used as an internal standard.
1) Rnase-free water; 0.5 μL
2) 2X Master Mix without UNG; 10 μL (1 ×)
3) 40X MultiScribe and RNase Inhibitor Mix; 0.5 μL
4) Forward Primer; 2.0 μL (300 nM)
5) Reverse Primer; 2.0 μL (900 nM)
6) TaqMan Probe; 2.0 μL (200 nM)

  Base sequences of primer sets (above “Forward Primer” and “Reverse Primer”) for amplifying cDNAs of the above two types of pluripotent stem cell marker genes and probes that hybridize to the amplification (PCR) products (above “ The base sequence of “TaqMan Probe”) is shown in Table 2.

[5] Real-time RT-PCR by ABI PRISM 7500FAST Sequence Detection system (Applied Biosystems) using the mixture of total RNA and reaction solution prepared in step [4] is performed in the following 1) to 3). Performed under the conditions indicated.
1) One cycle of 48 minutes at 48 ° C (reverse transcription reaction from mRNA to cDNA)
2) 95 ° C., 10 minutes, 1 cycle (polymerase activation)
3) 40 cycles of 95 ° C, 15 seconds and 60 ° C, 1 minute reciprocation (cDNA amplification by "Forward Primer" and "Reverse Primer")
[6] The PCR cycle number (threshold cycle; Ct value) in which the PCR product becomes a constant amount is measured using Baseline software (Applied Biosystems), and cDNA of the GAPDH gene is determined by the comparative Ct method (delta delta Ct method). Relative values of Ct values of cDNA amplification products of the above-mentioned two types of pluripotent stem cell marker genes based on Ct values of the amplified products are obtained, and the above two types of pluripotent stem cell marker genes are determined from the relative values of the Ct values. The relative amount of cDNA, that is, the relative amount of mRNA of the above two types of pluripotent stem cell marker genes was calculated (mean ± standard deviation, N = 3) (see Table 3).

In the table, “Before” indicates a cell sample immediately after culture (0 hour) in a cell culture bag.

3-2 Results As a result of microscopic observation, when hMSC cells were cultured using four types of cell culture bags C to F (Comparative Samples 2 to 5), the cells adhered, while the other two types When hMSC cells were cultured using the cell culture bag A (Comparative Sample 1) and the cell culture bag B (Example Sample), cell adhesion was suppressed and floated (see FIG. 2). The present inventors have found that when the floating state of hMSC cells is improved, the expression of pluripotent stem cell markers is increased and the pluripotency acquisition efficiency of hMSC cells is increased. Thus, when the expression of pluripotent stem cell markers was analyzed, when hMSC cells were cultured using cell culture bag B (Example sample), than when hMSC cells were cultured using other cell culture bags. Was also shown to increase the mRNA expression level of the pluripotent stem cell marker gene (see Table 3). These results indicate that pluripotent stem cells can be efficiently induced by suspension culture of hMSC cells using the cell culture bag B (Example sample).

4). HPBMC culture using cell culture bag 4-1 Method (cell culture and trypan blue staining)
[1] 2 hPBMC cells (Lonza, CC-2702), which are floating cells, were added to a 2% IMDM (gibco, 12440-053) culture solution containing 10% FBS (obtained from ATCC, 30-20202). It was suspended at a concentration of × 10 ⁵ cells / mL, and 5 mL was seeded in six types of cell culture bags A to F.
[2] Cultivation in an incubator (37 ° C., 5% CO ₂ ). After 1 hour, a part (10 μL) of the cell culture solution is collected and mixed with 10 μL of 0.4% trypan blue (Gibco). The number of viable cells was counted with a cell counter and the cell viability was calculated (see Table 4).
[3] After culturing for 7 days after seeding in a cell culture bag, the cells are observed under a microscope (IX-70, Olympus) (see FIG. 3), and include both suspended cells and adhered cells. 10 μL of the cell suspension was collected, mixed with 10 μL of 0.4% trypan blue (Gibco), the number of viable cells was counted with a cell counter, and the cell viability was calculated (see Table 4).

4-2 Results As a result of microscopic observation, when hPBMC cells were cultured using cell culture bag B (Example sample), the same level of cells were cultured as in the case of culturing hPBMC cells using commercially available cell culture bags. Adhesion was suppressed (see FIG. 3). Furthermore, when cell viability was analyzed, when hPBMC cells were cultured using cell culture bag B (Example sample), hPBMC cells were obtained using commercially available cell culture bag F (Comparative sample 5). It was shown that a decrease in cell viability was significantly suppressed as compared to the case of culturing (see Table 4).

  According to the present invention, it is possible to prepare a high concentration and a large amount of suspension suspension and pluripotent stem cell suspension, so that it is possible to provide a high quality cell suspension for transplantation in regenerative medicine. .

Claims (6)

A bag-shaped container made of a tetrafluoroethylene-hexafluoropropylene copolymer is provided, and the arithmetic average roughness (Ra) of the surface roughness of the inner surface of the bag-shaped container is 3.5 to 6.5 nm. The root mean square roughness (RMS) of the inner surface of the container is 4.5 to 8.0 nm, and the surface free energy of the inner surface of the bag-shaped container is 16.5 to 18.5 (mJ / m ² ). A cell culture bag characterized by being.   The bag for cell culture according to claim 1, wherein the bag-like container has a thickness of 10 to 100 µm.   The cell culture bag according to claim 1 or 2, wherein the cells are mesenchymal stem cells or peripheral blood-derived mononuclear cells. A bag-shaped container made of a tetrafluoroethylene-hexafluoropropylene copolymer is provided, and the arithmetic average roughness (Ra) of the surface roughness of the inner surface of the bag-shaped container is 3.5 to 6.5 nm. The root mean square roughness (RMS) of the inner surface of the container is 4.5 to 8.0 nm, and the surface free energy of the inner surface of the bag-shaped container is 16.5 to 18.5 (mJ / m ² ). A method for culturing cells, comprising subjecting the cells to suspension culture in the bag-like container of a cell culture bag.   The culture method according to claim 4, wherein the bag-like container has a thickness of 10 to 100 μm.   The culture method according to claim 4 or 5, wherein the cells are mesenchymal stem cells or peripheral blood-derived mononuclear cells.