Hollow fiber membrane for cell cryopreservation

Abstract

A hollow fiber membrane for cell cryopreservation made of a cellulose ester, which inhibits a decrease in strength at the time of cryopreservation of cells. A hollow fiber membrane for cell cryopreservation, which is a hollow fiber membrane containing a cellulose ester, has a breaking strength after being thawed after being frozen by a vitrification freezing method of 80% or more of the breaking strength at the time of wetting before being frozen.

Description

TECHNICAL FIELD

[0001] The present application relates to a hollow fiber membrane for cell cryopreservation. BACKGROUND

[0002] As a method for cryopreservation of cells such as oocytes and embryonic cells, vitrification freezing is known. In vitrification freezing, an instrument called Cryotop (registered trademark) is used. Cryotop is a dedicated instrument in which a very thin long strip-shaped sheet is attached to the tip of a handle. With respect to cells, in a state where the cells are placed on the sheet at the tip of Cryotop together with a liquid for freezing such as a vitrification solution, cryopreservation is performed using liquid nitrogen or the like.

[0003] In recent years, in such cryopreservation of cells, a method using a hollow fiber membrane (hollow fiber cryopreservation method) has also been studied. In the hollow fiber cryopreservation method, with respect to cells, freezing is performed and maintained in a state where the cells are accommodated inside a hollow fiber membrane formed of cellulose acetate. In this method, an increase in the survival rate of the cells after freezing is expected.

[0004] However, the hollow fiber membrane is a raw material that is relatively easy to damage, and there are concerns about breakage during cryopreservation, and handling is problematic. For example, Patent Literature 1 (Japanese Patent No. 5252556), Patent Literature 2 (Japanese Patent No. 5051716), and Patent Literature 3 (Japanese Patent No. 6667903) disclose a support instrument for a hollow fiber or the like for improving the handling of the hollow fiber during cryopreservation.

[0005] Note that, as a conventional hollow fiber membrane, for example, a hollow fiber membrane containing cellulose acetate for hemodialysis, hemodiafiltration, or the like as disclosed in Patent Literature 4 (Japanese Patent No. 5440332) and Patent Literature 5 (Japanese Patent No. 5212837) is known.

[0006] Prior Art Documents

[0007] Patent Literature

[0008] Patent Literature 1: Japanese Patent No. 5252556

[0009] Patent Literature 2: Japanese Patent No. 5051716

[0010] Patent Literature 3: Japanese Patent No. 6667903

[0011] Patent Literature 4: Japanese Patent No. 5440332

[0012] Patent Literature 5: Japanese Patent No. 5212837 SUMMARY

[0013] Problem to be Solved by the Invention

[0014] As described above, there is a concern that hollow fiber membranes made of cellulose esters such as cellulose acetate are reduced in strength and broken due to volume change and the like at the time of exposure to extremely low temperatures such as for the cryopreservation of cells, freezing, and subsequent thawing.

[0015] Therefore, an object of the present invention is to suppress reduction in strength at the time of cryopreservation of cells for hollow fiber membranes made of cellulose esters for the cryopreservation of cells.

[0016] Means for Solving the Problem

[0017] (1) A hollow fiber membrane that is a hollow fiber membrane for the cryopreservation of cells containing a cellulose ester,

[0018] The breaking strength at the time of thawing after freezing by a vitrification freezing method is 80% or more of the breaking strength at the time of wetting before freezing.

[0019] (2) The hollow fiber membrane according to (1), having a non-uniform structure in the thickness direction.

[0020] (3) The hollow fiber membrane according to (1) or (2), wherein,

[0021] The average pore diameter of the outer surface of the hollow fiber membrane is 1.1 times or more of the average pore diameter of the inner surface.

[0022] (4) The hollow fiber membrane according to any one of (1) to (3), wherein,

[0023] The average area ratio of the thickness direction cross section is 40% or more and 70% or less.

[0024] (5) The hollow fiber membrane according to any one of (1) to (4), wherein,

[0025] The variation in the area ratio of the thickness direction cross section in the thickness direction is less than 5%.

[0026] (6) The hollow fiber membrane according to any one of (1) to (3), wherein,

[0027] The arithmetic average roughness when the inner surface of the hollow fiber membrane is measured by an atomic force microscope is 20 nm or less.

[0028] Effects of the Invention

[0029] According to the present invention, reduction in strength at the time of cryopreservation of cells for hollow fiber membranes made of cellulose esters for the cryopreservation of cells can be suppressed. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a graph showing the measurement results of the inner diameter of the hollow fiber membrane.

[0031] Figure 2 is a graph showing the measurement results of the membrane thickness of the hollow fiber membrane.

[0032] Figure 3 is a graph showing the measurement results of the breaking strength of the hollow fiber membrane.

[0033] Figure 4 is a graph showing the measurement results of the breaking elongation of the hollow fiber membrane.

[0034] Figure 5 is a graph showing the measurement results of the yield strength of the hollow fiber membrane.

[0035] Figure 6 is a schematic diagram for explaining an example of a method for manufacturing a hollow fiber membrane. DETAILED DESCRIPTION

[0036] Hereinafter, an embodiment of the present application will be described, but the present application is not limited thereto. Note that, in the present specification, the expression in the form of "A to B" means the upper limit and the lower limit of the range (i.e., A or more and B or less), and in the case where A is not described with a unit and only B is described with a unit, the unit of A is the same as that of B.

[0037] Hollow Fiber Membrane

[0038] The hollow fiber membrane of the present embodiment can be suitably used for the cryopreservation of cells.

[0039] As the cells to be the object, there can be mentioned: pluripotent stem cells including oocyte (fertilized egg or the like), blastocyte, iPS cell, ES cell, and the like, artificial tissues (cell aggregates) such as organelles from pluripotent stem cells, and the like. The cell aggregate can be composed of a plurality of cell types. Note that, the cells are cryopreserved in a state of being disposed inside the hollow fiber membrane. A cell suspension containing dispersed cells can also be cryopreserved inside the hollow fiber membrane.

[0040] Cellulose ester

[0041] The hollow fiber membrane of the present embodiment contains cellulose ester. The ratio of the cellulose ester in the material constituting the hollow fiber membrane is preferably 90 mass% or more, more preferably 95 mass% or more, and further preferably 98 mass% or more. The hollow fiber membrane can be composed of only cellulose ester.

[0042] It is preferable that the hollow fiber membrane have transparency to the extent that cells housed inside thereof can be seen. Cellulose ester is a material having high transparency, and therefore, it is preferable that the ratio of cellulose ester in the material constituting the hollow fiber membrane be high.

[0043] In addition, it is preferable that the hollow fiber membrane not be dissolved in the vitrification solution, and that the resin component not be eluted into the vitrification solution. From this viewpoint, it is also preferable that the ratio of cellulose ester in the material constituting the hollow fiber membrane be high.

[0044] As the cellulose ester, for example, cellulose acetate, cellulose phthalate, cellulose succinate, and the like can be given. The cellulose ester is preferably cellulose acetate. Cellulose acetate is resistant to chlorine as a sterilizing agent, and can be sterilized using chlorine.

[0045] As the cellulose acetate, for example, triacetate cellulose, monoacetate cellulose, diacetate cellulose, acetate butyrate cellulose, acetate propionate cellulose, and the like can be given. From the viewpoint of durability and the like, the cellulose acetate is preferably triacetate cellulose.

[0046] As the cellulose acetate, for example, various cellulose acetates having different degrees of acetylation, degrees of polymerization, and the like (L-20, 30, 40, 50, 70, LT-35, 55, 105, and the like) are commercially available from Daicel Chemical Industries, Ltd. It is preferable to use cellulose acetate having a viscosity of more than 140 mPa-s and less than 200 mPa-s, and having a relatively low viscosity (a relatively low viscosity polymer).

[0047] The degree of acetylation of the cellulose acetate is preferably 53 to 62%, more preferably 55 to 61.5%, and further preferably 58 to 61.5%. Note that the degree of acetylation indicates the degree of substitution of acetic acid groups to hydroxyl groups in cellulose. Note that the theoretical upper limit of the degree of acetylation is 62.5%, but if the degree of acetylation is too high, there is a possibility that the solubility and moldability will decrease.

[0048] (Breaking strength and the like)

[0049] The breaking strength of the hollow fiber membrane of the present embodiment when melted after being frozen by the vitrification freezing method is 80% or more, preferably 90% or more, and more preferably 95% or more of the breaking strength when wet before freezing.

[0050] In addition, the breaking elongation of the hollow fiber membrane of the present embodiment when melted after being frozen by the vitrification freezing method is preferably 80% or more, more preferably 90% or more, and further preferably 95% or more of the breaking elongation when wet before freezing.

[0051] In addition, the yield strength at the time of melting after the hollow fiber membrane of the present embodiment is frozen by the vitrification freezing method is preferably 80% or more, more preferably 90% or more, and further preferably 95% or more, relative to the yield strength at the time of wetting before freezing.

[0052] In this way, the hollow fiber membrane containing the cellulose ester of the present embodiment suppresses a decrease in strength at the time of cryopreservation of cells.

[0053] Note that the breaking strength, the elongation at break, and the yield strength are measured by the method for measuring the strength (breaking strength, elongation at break, and yield strength) described below in the Examples.

[0054] The vitrification freezing method is a method in which cells are rapidly cooled by immersion in liquid nitrogen or the like, thereby preventing so-called crystallization of water that easily occurs at -60°C to -15°C, and the cells are frozen in a non-crystalline glass state. This method is superior to the slow freezing method in that cell damage caused by the formation of ice crystals is less likely to occur, the time taken for the process is short, no special machine is required, long-term preservation of cells is good, and the like. Various specific methods for vitrification freezing of cells have been developed, and as an example, a method in which cells are immersed in a cryoprotective solution, and rapidly frozen at -80°C or lower, more preferably at -190°C or lower, in liquid nitrogen or in a super-low-temperature freezer can be given.

[0055] Note that the conditions of the vitrification freezing method when comparing the strength (breaking strength, elongation at break, and yield strength) before and after freezing and melting are as follows.

[0056] [Conditions of the vitrification freezing method]

[0057] (Equilibration solution)

[0058] Composition: Ethylene glycol (7.5 mass%), dimethyl sulfoxide (7.5 mass%), and water

[0059] Immersion time: 4 minutes

[0060] (Vitrification solution)

[0061] Composition: Ethylene glycol (15 mass%), dimethyl sulfoxide (15 mass%), sucrose (0.5 M in the vitrification solution), and water

[0062] Immersion time: 30 seconds

[0063] (Cryopreservation conditions)

[0064] Freezing method: Immersion in liquid nitrogen (LN2)

[0065] Preservation method: Preservation for 1 week in the state of immersion in liquid nitrogen (LN2)

[0066] (Melting method)

[0067] Dipping in a melting solution (1M aqueous sucrose solution) for 1 minute

[0068] Dipping in a dilution solution (0.5M aqueous sucrose solution) for 3 minutes

[0069] Dipping in a washing solution (a commercially available TCM199 medium (GIBCO)) for 5 minutes, and further dipping in another washing solution of the same composition for 5 minutes

[0070] (Hollow fiber membrane shape, etc.)

[0071] The inner diameter of the hollow fiber membrane is preferably 30 μm or more and 300 μm or less, and more preferably 35 μm or more and 260 μm or less.

[0072] The thickness of the hollow fiber membrane is preferably 20 to 200 μm, and more preferably 30 to 150 μm. Note that the film thickness can be calculated by "(outer diameter - inner diameter) / 2".

[0073] The hollow fiber membrane (membrane of a hollow fiber type) is preferably formed of a semipermeable membrane. This is because, for cells that are stored by being housed in the hollow fiber membrane, the cells are not allowed to pass through, but the culture solution, the cryopreservation solution, the cryoprotective substances contained in the above-mentioned preservation solution, and the like are allowed to pass through, and thus the state in which the cells are enclosed in the internal space of the hollow fiber membrane can be maintained. That is, there is an advantage that the culture solution, the intracellular solution, and the like in the internal space of the hollow fiber membrane can be easily replaced with the cryopreservation solution.

[0074] The hollow ratio of the hollow fiber membrane is preferably 10 to 65%, and more preferably 12 to 55%. Note that the hollow ratio is the proportion of the area of the hollow portion in the cross section of the hollow fiber membrane, and is represented by "hollow portion cross-sectional area / (membrane portion cross-sectional area + hollow portion cross-sectional area) x 100 (%)".

[0075] The average pore diameter (average pore diameter of fine pores of the entire membrane) of the hollow fiber membrane is preferably 10 μm or less. As a method for measuring the average pore diameter, for example, a bubble point method, a mercury intrusion method, and the like can be given.

[0076] The hollow fiber membrane of the present embodiment is preferably a membrane having a non-uniform structure (asymmetric structure) in the thickness direction. It is considered that, in the case where the hollow fiber membrane has an asymmetric structure, the effect of suppressing the decrease in the strength of the hollow fiber membrane due to the volume change and the like at an extremely low temperature such as for the cryopreservation of cells, freezing, and the like after the freezing, is high. The reason for this is not clear, but as one reason, it is considered that the buffering ability of the polymer chain of the non-uniform structure is higher than that of a uniform structure, and is less affected by the volume change and the like of the hollow fiber membrane.

[0077] As the hollow fiber membrane having an asymmetric structure, for example, a hollow fiber membrane in which density (porosity, cross-sectional opening ratio) and the like are different in the thickness direction can be mentioned. As such a hollow fiber membrane, for example, a membrane having a dense layer on one surface side, which becomes a separation active layer substantially defining the pore size of the hollow fiber membrane, and a lower density on the other surface side than the dense layer can be mentioned.

[0078] It is preferable that in the hollow fiber membrane having an asymmetric structure, the opening ratio of one surface is different from the opening ratio of the other surface. The opening ratio of the one surface having a large opening ratio is preferably 1.1 times or more, more preferably 1.3 times or more, of the opening ratio of the other surface. It is considered that by having such an asymmetric structure, heat transfer from the solution on the outside of the hollow fiber membrane to the solution inside the hollow fiber membrane rapidly occurs in the vitrification freezing, and therefore, the generation of ice crystals can be suppressed, and thus damage to the membrane structure and cells is suppressed.

[0079] Note that, in the measurement of the opening ratio of the membrane surface, first, a hollow fiber membrane is imaged using a scanning electron microscope (SEM) at 10,000 times. After cutting out a region of 762 pixels in the vertical direction x 620 pixels in the horizontal direction from the obtained image, the image is binarized into white / black using an image analysis software (for example, WinROOF 2013), and the opening ratio of the inner surface and the outer surface of the hollow fiber membrane is calculated. This is performed for 10 fields of view, and the average thereof is calculated as the surface opening ratio.

[0080] The area ratio of the hollow fiber membrane is the area ratio of the solid portion (portion where the membrane exists) other than the hollow portion in the cross section (cross section) in the thickness direction of the hollow fiber membrane. The area ratio is determined by analyzing the image obtained by photographing the cross section of the hollow fiber membrane using SEM. Specifically, the membrane cross section is divided into three regions in the membrane thickness direction as described later, and the area ratio is measured for each of them. The three regions are a region A including the outer surface, a region B including the inner surface, and a region between the region A and the region B (central region C). The magnification of the SEM can be any magnification that allows the hollow portion and the solid portion to be distinguished, and for example, 5,000 times to 20,000 times is appropriate for measuring the hollow fiber membrane of the present application. The area ratio is calculated by a method using image analysis. Specifically, binarization processing of the hollow portion and the solid portion (polymer portion) is performed using an image analysis software (for example, WinROOF 2013). After the binarization processing, the area ratio is calculated from the ratio of the total area of the hollow portion to the total area of the polymer portion.

[0081] In the present embodiment, the hollow fiber membrane has the following characteristics: it has the above-described asymmetric structure, and the variation in the volume density (area ratio in the thickness direction cross section) in the thickness direction of the membrane is small (preferably almost fixed). The reason is not clear, but it is considered that by having the above-described structural characteristics, it is possible to withstand drastic temperature changes, volume changes of vitrified liquid. Therefore, it is considered that it is possible to suppress the embrittlement and destruction of the hollow fiber membrane structure caused by freezing and thawing, and it is possible to maintain the membrane strength.

[0082] Note that the small variation in the area ratio in the thickness direction cross section means, for example, that for a region A including the outer surface, a region B including the inner surface, and a central region C (a region between the region A and the region B) in the thickness direction cross section, the difference between the area ratios (%) is small. Specifically, when the area ratios of the regions A, B, and C are measured, among all combinations of two area ratios selected from a (area ratio of the region A), b (area ratio of the region B), and c (area ratio of the region C), the absolute value of the difference between the two area ratios is preferably less than 5%, and more preferably less than 3% (see the following equation). Note that, for example, the region A is a region from the outer surface to a depth of 30% of the membrane thickness, and the region B is a region from the inner surface to a depth of 30% of the membrane thickness.

[0083] Preferably, |a-b| < 5%, and |b-c| < 5%, and |c-a| < 5%

[0084] More preferably, |a-b| < 3%, and |b-c| < 3%, and |c-a| < 3%

[0085] The average area ratio (for example, "(a+b+c) / 3") of the thickness direction cross section is preferably 40% or more and 70% or less (see the following equation).

[0086] 40% ≤ (a+b+c) / 3 ≤ 70%

[0087] In addition, in the present application, it is preferable that the smoothness of the inner surface of the hollow fiber membrane be high. By making the smoothness of the inner surface high, even if the egg cells, the embryonic cells come into contact with the inner surface of the hollow fiber membrane, it is possible to minimize the risk of causing damage or the like to the cell surface. Here, the high smoothness means that the arithmetic average roughness Ra value is 20 nm or less. If the smoothness is high, the damage to the cells becomes small, and therefore, the Ra value is more preferably 10 nm or less, and further preferably 1 nm or more and less than 8 nm. The arithmetic average roughness Ra value can be measured using an atomic force microscope (AFM).

[0088] <Method for producing a hollow fiber membrane>

[0089] The present application also relates to a method for producing a hollow fiber membrane including the above-described cellulose ester.

[0090] The method for producing a hollow fiber membrane of the present embodiment is, for example, the following method.

[0091] The method for producing a hollow fiber membrane described above includes:

[0092] A spinning process in which a spinning dope and an inner liquid are ejected from a nozzle in a sleeve shape through an in-air travel section into a coagulation liquid, the spinning dope is coagulated in the coagulation liquid, and a coagulum of the spinning dope is pulled out from the coagulation liquid, thereby obtaining a hollow fiber membrane.

[0093] The spinning dope described above contains a resin raw material containing a cellulose ester, a solvent, and a non-solvent,

[0094] The inner liquid described above contains water,

[0095] The temperature of the spinning dope in the nozzle is 70 to 110°C, and the temperature of the inner liquid is 40 to 70°C,

[0096] The ratio of the amount of the solvent in the spinning dope to the non-solvent is 60 / 40 to 80 / 20,

[0097] The nozzle draw ratio is 0.4 to 0.9.

[0098] The concentration of the cellulose ester in the spinning dope is preferably 10 to 30 mass%.

[0099] In the spinning dope, the ratio of the amount of the solvent to the total amount of the solvent and the non-solvent is preferably 60 to 80 mass%.

[0100] The solvent is preferably an aprotic polar solvent.

[0101] The non-solvent is preferably a glycol ester.

[0102] The straight-line distance of the in-air travel section is preferably 10 to 50 mm.

[0103] [Spinning process]

[0104] Reference Figure 6 In the spinning process, a spinning dope 10a and an inner liquid 10b are ejected from a nozzle 11 in a sleeve shape through an in-air travel section (air gap) 20 into a coagulation liquid 21, the spinning dope is coagulated in the coagulation liquid 21, and a coagulum of the spinning dope is pulled out from the coagulation liquid 21, thereby obtaining a hollow fiber membrane 16. The pulling out of the hollow fiber membrane and the like is performed, for example, by a liquid guide 12 and rollers 13, 14, and 15.

[0105] The nozzle 11 is a sleeve type having an outer tube and an inner tube provided inside the outer tube. The dope is ejected from the gap (slit) between the outer tube and the inner tube, and the inner liquid is ejected from the inside of the inner tube. The ratio of the diameter of the outer tube (outer diameter of the slit) to the diameter of the inner tube (inner diameter of the slit) is preferably 110 to 300%, more preferably 110 to 200%. The diameter of the outer tube is preferably 220 to 400 μm, more preferably 220 to 330 μm. The diameter of the inner tube is preferably 150 to 330 μm, more preferably 150 to 270 μm. Note that the diameter of the inner tube is preferably the same degree as the diameter of the hollow fiber membrane.

[0106] The ratio of the cross-sectional area of the inner tube to the cross-sectional area of the slit is preferably 80 to 120%. By using such a nozzle, the ejection linear velocity of the membrane-forming dope and the extraction velocity can be made the same degree, and thus, the interfacial friction of the ejected membrane-forming dope and the inner liquid can be reduced, and the roughness of the inner surface of the hollow fiber membrane can be prevented.

[0107] Note that the ejection linear velocity of the membrane-forming dope is calculated by dividing the amount of the ejected membrane-forming dope by the cross-sectional area of the slit calculated from the outer diameter (a) and the inner diameter (b) of the nozzle slit [π (a / 2) 2 - π (b / 2) 2 ].

[0108] Ejection linear velocity of membrane-forming dope [m / min] = amount of ejected membrane-forming dope / cross-sectional area of slit

[0109] The extraction velocity is the rotation velocity (surface velocity) of the roller 13 provided at the outlet of the coagulation bath (refer to Figure 6 ).

[0110] The ratio of the extraction velocity to the ejection linear velocity (extraction velocity / ejection linear velocity), that is, the nozzle draw ratio, is 0.4 to 0.9, preferably 0.5 to 0.9. In this way, by making the ejection linear velocity faster than the extraction velocity of the membrane-forming dope, the characteristic membrane structure of the hollow fiber of the present application can be obtained.

[0111] The straight-line distance of the above-described in-air traveling section 20 (the distance between the front end of the nozzle 11 and the liquid surface of the coagulation liquid 21) is preferably 10 to 50 mm, more preferably 10 to 40 mm.

[0112] Note that a washing process (water washing process) using pure water can be further performed on the hollow fiber membrane obtained in the spinning process. The flow of water in the water washing process is preferably a flow in the opposite direction to the moving direction of the hollow fiber membrane (counter flow), but can be a flow in the same direction as the moving direction of the hollow fiber membrane (forward flow).

[0113] (Dope)

[0114] The dope 10a contains a resin raw material containing the above-described cellulose ester, a solvent, and a non-solvent.

[0115] The temperature (set temperature) of the dope in the nozzle 11 is 70 to 110°C, preferably 70 to 100°C.

[0116] The concentration of the cellulose ester in the above-described dope is preferably 10 to 30 mass%, more preferably 10 to 25 mass%. If the concentration of the cellulose ester is too low, the strength of the hollow fiber membrane becomes low. On the other hand, if the concentration of the cellulose ester is too high, the viscosity of the dope becomes too high, and there is a case where the implementation of the spinning becomes difficult.

[0117] The solvent is a liquid capable of dissolving the cellulose ester. The solvent is preferably a polar solvent, and is preferably water-soluble. The polar solvent is preferably an aprotic polar solvent. As the aprotic polar solvent, for example, N-methylpyrrolidone (NMP), dimethylformamide (DMF), dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), acetonitrile, and the like can be given.

[0118] The non-solvent is a liquid (except water) that cannot dissolve the cellulose ester. As the non-solvent, for example, glycol ester, glycerol, alcohol, and the like can be given, and the glycol ester is preferable. As the glycol ester, for example, ethylene glycol, triethylene glycol (TEG), polyethylene glycol (polyethylene glycol 200, polyethylene glycol 400, and the like), propylene glycol, and the like can be given.

[0119] In the above-described dope, the ratio of the amount of the solvent (S) to the non-solvent (NS) (S / NS ratio) is 60 / 40 to 80 / 20, more preferably 65 / 35 to 75 / 25. If the S / NS ratio in the dope is too small, the dissolution of the cellulose ester becomes unstable, and thus, sometimes the spinning stability is reduced, or an asymmetric structure suitable for the use of the present application cannot be obtained. If the S / NS ratio is too large, sometimes a hollow fiber membrane of an asymmetric structure cannot be obtained, or the spinning stability is reduced.

[0120] Note that the dope can further contain water in addition to the solvent and the non-solvent.

[0121] The order of addition of the materials and the mixing method when the resin raw material containing the cellulose ester, the solvent, and the non-solvent, which become the constituent materials of the hollow fiber membrane, are mixed are not particularly limited.

[0122] (Inner liquid)

[0123] The inner liquid contains water. The content rate of the water in the inner liquid is 95 to 100 mass%, preferably 98 to 100 mass%.

[0124] The inner surface of the hollow fiber membrane is preferably high in smoothness. This is because damage caused by contact with cells subjected to cryopreservation can be reduced.

[0125] In order to improve the smoothness of the inner surface of the hollow fiber membrane, it is preferable that the inner surface be rapidly solidified (fixed) before being affected by disturbance after the spinning dope is ejected from the nozzle, and that phase separation not be excessively performed. In addition, it is preferable that no external force such as stretching be applied to the inner surface during and after solidification, and that the inner diameter variation after the structure of the hollow fiber membrane is fixed and the like be suppressed as much as possible.

[0126] In order to rapidly solidify the inner surface, it is preferable to use an inner liquid 10b that is high in solidification property with respect to the spinning dope 10a, or to adopt a spinning dope composition, temperature conditions that easily solidify.

[0127] Therefore, in the production method of the present embodiment, an inner liquid containing water that is high in solidification property with respect to a spinning dope containing a cellulose ester is used.

[0128] In addition to water, ethylene glycol, triethylene glycol, polyethylene glycol 200 or 400, glycerol, propylene glycol, and the like, which are generally used as non-solvents of cellulose esters, can also be used alone or in a mixture. In the case where an inner liquid with water as the main component is used, as a component other than water, the above non-solvents, N-methylpyrrolidone, dimethylacetamide, dimethylformamide, dimethyl sulfoxide, and the like, which are solvents of cellulose triacetate-based polymers, can be added up to 5% by weight as the upper limit.

[0129] The temperature (set temperature) of the inner liquid in the nozzle 11 is 40 to 70°C, and is preferably 45 to 65°C. As described above, the temperature (set temperature) of the spinning dope in the nozzle 11 is 70 to 110°C, and it is preferable that the inner liquid be set to a lower temperature than this. The temperature of the inner liquid in the nozzle 11 is preferably 10°C or more lower than the temperature of the spinning dope in the nozzle 11, more preferably 20°C or more lower, and further preferably 30°C or more lower.

[0130] When the dope 10a and the inner liquid 10b are discharged from the nozzle 11 in a sheath shape, it is preferable to set a temperature difference between the dope and the inner liquid from the viewpoint of improving the coagulability (coagulation speed) of the inner surface of the hollow fiber membrane. By increasing the coagulation speed of the inner surface, a hollow fiber membrane having an asymmetric structure (a structure that is not uniform in the thickness direction) can be obtained. Further, on the basis of setting the temperature of the inner liquid to a specific range, adjusting the composition of the coagulation liquid described later to a specific range, or adjusting the nozzle draw ratio, the smoothness of the inner surface of the hollow fiber membrane is improved, and the change in the volume density (area ratio) of the membrane in the thickness direction in the membrane cross section can be reduced even though it is an asymmetric structure. Note that, in order to set a temperature difference between the dope and the inner liquid, it is preferable to use a nozzle having a structure that enables temperature control of the dope and the inner liquid separately until immediately before discharge.

[0131] (coagulation liquid)

[0132] The coagulation liquid preferably contains a solvent and a non-solvent (other than water). Note that, in this case, the coagulation liquid can contain water in addition to the solvent and the non-solvent.

[0133] The ratio of the total amount of the solvent and the non-solvent in the coagulation liquid (the concentration of the coagulation liquid) is 60 to 90% by mass, and is preferably 65 to 90% by mass. Thereby, the characteristic structure of the hollow fiber membrane of the present application can be obtained.

[0134] Further, the temperature of the coagulation liquid is preferably 20 to 60°C, and is more preferably 30 to 50°C.

[0135] Example

[0136] Hereinafter, the present application will be described in more detail by citing examples, but the present application is not limited to these examples.

[0137] [Example 1]

[0138] The hollow fiber membrane of Example 1 was produced by the production method of the hollow fiber membrane described in the embodiment under the following conditions.

[0139] (composition of the dope)

[0140] Raw material resin (cellulose ester): cellulose triacetate (CTA) (LT75, Daicel Chemical Industries)

[0141] Raw material resin concentration (polymer concentration): 17.5% by mass (in the dope)

[0142] Solvent: N-methylpyrrolidone (NMP)

[0143] Non-solvent: triethylene glycol (TEG)

[0144] [Solvent / Non-solvent (S / NS) ratio = 7 / 3]

[0145] Inner liquid: water

[0146] (Preparation of the dope solution)

[0147] The dope solution used in the spinning process was prepared by mixing the above-mentioned powder of the raw resin with other materials.

[0148] (Composition of the coagulation liquid)

[0149] Solvent (S): NMP

[0150] Non-solvent (NS): TEG

[0151] Water

[0152] Concentration of the coagulation liquid [(mass of S + mass of NS) / mass of the coagulation liquid]: 78 mass%

[0153] The S / NS ratio was the same as that of the dope solution.

[0154] (Conditions of the spinning process)

[0155] Discharge temperature of the dope solution (set temperature): 93°C

[0156] Discharge temperature of the inner liquid (water) (set temperature): 55°C

[0157] Nozzle: sleeve-type nozzle (diameter of outer tube: 270 μm, diameter of inner tube: 200 μm)

[0158] Distance of the in-air traveling section (air gap length): 25 mm

[0159] Residence time of the in-air traveling section: 0.025 seconds

[0160] Temperature of the coagulation liquid: 43°C

[0161] Take-up speed: 60 m / minute

[0162] Nozzle draw ratio: 0.78

[0163] [Conditions of the water washing process]

[0164] Flow of the water washing tank: counter flow

[0165] Temperature 98°C

[0166] [Examples 2, 3]

[0167] The hollow fiber membranes of Example 2 and Example 3 were produced in the same manner as Example 1 except that the draw ratio of the nozzle was changed as shown in Table 1. The results of measuring the arithmetic average roughness and the area ratio of the inner surface of the obtained hollow fiber membranes are shown in Table 2.

[0168] [Comparative Examples 1 and 2]

[0169] The composition of each of the dope solution, the inner solution, and the coagulation solution, and each production condition were changed as shown in Table 1. Note that liquid paraffin was used as the inner solution, and the set temperature of the inner solution was not particularly controlled, and was the same degree as the temperature of the dope solution. The hollow fiber membranes of Comparative Examples 1 and 2 were produced in the same manner as Example 1 except for this.

[0170] [Table 1]

[0171]

[0172] [Measurement of Area Ratio]

[0173] The hollow fiber membrane in a wet state was immersed in liquid nitrogen, frozen, and then taken out of the liquid nitrogen, and immediately subjected to bending and cutting, whereby a sample having a smooth cross section (thickness direction cross section) was obtained. The sample was fixed to a sample stage in a manner that the cross section could be observed, and the cross section of the sample was subjected to carbon evaporation. The cross section of the sample after evaporation was photographed using a scanning electron microscope (Hitachi S-2500) at an acceleration voltage of 5 kV and a magnification of 10,000 times. The center portion of a region equivalent to the above-described regions A to C (region A including the outer surface, region B including the inner surface, and region C between region A and region B) in the obtained image was subjected to binarization processing of the hollow portion and the polymer portion using image analysis software WinROOF 2013. After the binarization processing, the area ratio was calculated from the ratio of the hollow portion to the polymer portion. Here, region A is a region from the outer surface to a depth of 30% of the film thickness, and region B is a region from the inner surface to a depth of 30% of the film thickness.

[0174] Note that in some cases, the structure becomes unclear due to cutting, electron beams, and the like, and thus in such cases, the measurement was performed using a portion in which the structure was clear, or the sample was changed and the photograph was taken again.

[0175] The results of the measurement of the area ratio are shown in Table 2.

[0176] [Measurement of Arithmetic Average Roughness]

[0177] The hollow fiber membrane prepared so as to be able to observe the inner surface of the hollow fiber membrane by splitting (inclined cutting along the length direction of the hollow fiber membrane) was used as a test sample. Observation was performed in the DFM mode in the atmosphere using an atomic force microscope E-Sweep (Hitachi High-Technologies Corporation). Si-DF3 was used as a cantilever, and a 20 μm scanner was used as a scanner. The observation field was set to 2 μm square, and the arithmetic average roughness (Ra) of the inner surface of the hollow fiber membrane was measured at 256 x 256 pixels. The measurement results of the arithmetic average roughness (Ra) are shown in Table 2.

[0178] [Table 2]

[0179]

[0180]

[0181] Freeze storage tests using the vitrification method were performed for the hollow fiber membranes of Examples 1 to 3 and Comparative Examples 1 and 2. The conditions of the vitrification method were the same as those in the measurement of the breaking strength (breaking strength at the time of thawing after freezing by the vitrification method) described above.

[0182] For each of the hollow fiber membranes of Examples 1 to 3 and Comparative Examples 1 and 2 in the freeze storage test, the following measurements were performed in the initial dry state ("dry state"), the state of wetting by water ("wet state"), the state of immersion in the equilibrium liquid ("equilibrium liquid"), the state of immersion in the vitrification liquid ("vitrification liquid"), and the state after thawing after freezing ("after thawing").

[0183] [Measurement of the inner diameter, outer diameter, and membrane thickness of the hollow fiber membrane]

[0184] The inner diameter, outer diameter, and membrane thickness were measured by the following method.

[0185] An appropriate number of hollow fiber membranes were passed through a 3 mm diameter hole formed in the center of a glass slide to the extent that the hollow fiber membranes did not fall off, and the hollow fiber membranes were cut along the upper and lower surfaces of the glass slide by a razor to obtain a hollow fiber membrane cross-section sample. For the obtained hollow fiber membrane cross-section sample, the inner diameter and outer diameter of the hollow fiber membrane were measured using a projector (Nikon PROFILE PROJECTOR V-12).

[0186] ​Specifically, the dimensions of the hollow fiber membrane outer surface in the X-X direction and the Y-Y direction (two directions orthogonal on the cross section) were measured for each 1 hollow fiber membrane cross section, and the arithmetic mean of the values thereof was taken as the outer diameter of 1 hollow fiber membrane cross section. In addition, the dimensions of the hollow portion in the X-X direction and the Y-Y direction (two directions orthogonal on the cross section) were measured for each 1 hollow fiber membrane cross section, and the arithmetic mean was taken as the inner diameter of 1 hollow fiber membrane cross section. Note that the measurement was performed similarly for 10 cross sections, and the average was taken as the inner diameter and the outer diameter.

[0187] The film thickness (average) was calculated based on the inner diameter of the hollow fiber membrane and the measurement results (average) of the inner diameter, and by the formula "(outer diameter - inner diameter) / 2".

[0188] The measurement results (average) of the inner diameter of the hollow fiber membrane are shown in Table 3 ( Figure 1 ), and the measurement results (average) of the film thickness are shown in Table 4 ( Figure 2 ). Note that, regarding the comparative examples, the strength of the hollow fiber membrane after melting was low, and the hollow fiber membrane cross section sample could not be produced, and thus the inner diameter and the film thickness could not be measured.

[0189] [Table 3]

[0190]

[0191] [Table 4]

[0192]

[0193] [Measurement of tensile strength]

[0194] For the hollow fiber membranes of Examples 1 to 3 and Comparative Examples 1 and 2, the tensile strength (breaking strength, breaking elongation, and yield strength) was measured by the following method.

[0195] For the tensile strength of the hollow fiber membrane, a tensile tester (UTMII, manufactured by Toyo Baldwin Co., Ltd.) was used, 1 hollow fiber membrane was cut to a length of about 15 cm, and was installed between chucks (distance of about 10 cm) in a manner that did not slacken, and the hollow fiber membrane was stretched at a crosshead speed of 10 cm / min in a temperature and humidity environment of 20 ± 5°C, 60 ± 10% Rh, and the measurement was performed.

[0196] From the obtained S-S curve, the load of each filament at the breaking point of the hollow fiber membrane (breaking strength), the elongation (breaking elongation), and the load of each filament at the yield point (yield strength), the elongation (yield elongation) were read. Specifically, the load and the elongation were obtained using the method shown in

[0061] of Japanese Patent Application Publication No. 2011-212638.

[0197] The respective measurement results of the breaking strength, the breaking elongation, and the yield strength are shown in Tables 5 to 7. Figures 3 to 5 Note that the measurement was performed 5 times for each of the examples of the examples and the comparative examples, and the average value thereof was shown as the measurement value.

[0198] [Table 5]

[0199]

[0200] [Table 6]

[0201]

[0202] [Table 7]

[0203]

[0204] As shown in Table 5, Figure 3 for the hollow fiber membrane of the comparative examples, the breaking strength was significantly decreased in the state after being thawed after the freezing, as compared with the value before the freezing (the "dry state", the "wet state", the "equilibrium liquid", and the "vitrified liquid" in the table and the graph). In contrast, in the case of the hollow fiber membrane of the examples, the breaking strength was hardly decreased even in the state after being thawed, as compared with the value before the freezing. Note that for the hollow fiber membrane of the examples, the breaking strength in the state after being thawed after the freezing by the vitrification freezing method was 95% or more of the breaking strength in the wet state ("wet state") before the freezing.

[0205] In addition, as shown in Tables 6 and 7, Figure 4 and Figure 5 for the hollow fiber membrane of the comparative examples, the breaking elongation and the yield strength were significantly decreased in the state after being thawed after the freezing, as compared with the value before the freezing. In contrast, in the case of the hollow fiber membrane of the examples, the breaking elongation and the yield strength were hardly decreased even in the state after being thawed, as compared with the value before the freezing. Note that for the hollow fiber membrane of the examples, the breaking elongation or the yield strength in the state after being thawed after the freezing by the vitrification freezing method was 95% or more of the breaking elongation or the yield strength in the wet state ("wet state") before the freezing.

[0206] According to these results, it was found that the hollow fiber membrane of the examples was able to suppress the decrease in the strength at the time of the cryopreservation of cells.

[0207] <Evaluation of Cell Viability>

[0208] Using the hollow fiber membranes of Examples 1 to 3 and Comparative Example 1, the number of surviving cells was measured after freezing and thawing cells (embryonic cells from pigs) by the vitrification freezing method (hollow fiber freezing preservation method), and the survival rate (ratio of the number of surviving cells to the number of test cells) was calculated.

[0209] In addition, using Cryotop (Cryotop: Registered Trademark, manufactured by Kitasato Corporation) which is a commercially available product as a cell freezing preservation device, the number of surviving cells was measured after freezing and thawing cells by the vitrification freezing method (Cryotop method), and the survival rate was calculated.

[0210] The evaluation results of the survival rate, the number of test cells (number of cells subjected to the test), and the number of surviving cells are shown in Table 8. Note that the conditions of the vitrification freezing method were the same as those when the above-described tensile strength was measured, and in the hollow fiber membrane freezing preservation method, the freezing preservation was performed in a state in which the cells were housed inside the hollow fiber membrane.

[0211] [Table 8]

[0212]

[0213] As shown in Table 8, in the case where the hollow fiber membranes of Examples 1 to 3 were used for the freezing preservation of cells, the ratio of surviving cells after thawing (survival rate) was higher than that of the commercially available product and Comparative Example. From this result, it was found that the hollow fiber membranes of Examples 1 to 3 can be suitably used for the purpose of cell freezing preservation.

[0214] Explanation of Reference Numerals

[0215] 10a Spinning dope, 10b Internal liquid, 11 Nozzle, 12 Liquid guide, 13, 14, 15 Roll, 16 Hollow fiber membrane, 20 Air travel section, 21 Coagulation liquid

Claims

1. A hollow fiber membrane for cell cryopreservation comprising cellulose ester. The ratio of the cellulose ester in the material constituting the hollow fiber membrane is 90% by mass or more, The breaking strength of the material after being frozen by vitrification and then thawed is at least 80% of the breaking strength when wet before freezing. The elongation at break when thawed after being frozen by vitrification is at least 80% of the elongation at break when wet before freezing. The yield strength of the material after being frozen by vitrification method and then thawed is at least 80% of the yield strength in the wet state before freezing. The thickness of the hollow fiber membrane is 20 μm to 200 μm. The hollow fiber membrane has a non-uniform structure in the thickness direction, In the hollow fiber membrane, the opening porosity of one surface is 1.1 times or more the opening porosity of the other surface.

2. The hollow fiber membrane according to claim 1, wherein The average pore diameter of the outer surface of the hollow fiber membrane is 1.1 times or more the average pore diameter of the inner surface.

3. The hollow fiber membrane according to claim 1 or 2, wherein The average area ratio of the cross section in the thickness direction is 40% or more and 70% or less.

4. The hollow fiber membrane according to claim 1 or 2, wherein The variation in the area ratio of the cross section in the thickness direction is less than 5%.

5. The hollow fiber membrane according to claim 1 or 2, wherein The arithmetic mean roughness of the inner surface of the hollow fiber membrane measured with an atomic force microscope was 20 nm or less.

6. A method for manufacturing a hollow fiber membrane, The method comprises a spinning step of ejecting a spinning solution and an inner solution from a sleeve-shaped nozzle through an air-running portion into a coagulation liquid, coagulating the spinning solution in the coagulation liquid, and pulling the coagulated product of the spinning solution out of the coagulation liquid, thereby obtaining a hollow fiber membrane. The spinning solution comprises a resin raw material containing cellulose ester, a solvent and a non-solvent, The coagulation liquid comprises the solvent and the non-solvent, The internal liquid comprises water, The temperature of the spinning solution in the nozzle is 70°C to 110°C. In the spinning solution, the ratio of the amount of the solvent to the amount of the non-solvent is 60 / 40 to 80 / 20, The nozzle stretch ratio is 0.4 to 0.

9. The hollow fiber membrane comprises cellulose ester and is used for cell cryopreservation, The breaking strength of the hollow fiber membrane when thawed after being frozen by vitrification is at least 80% of the breaking strength when wet before freezing. The average area ratio of the cross section in the thickness direction of the hollow fiber membrane is 40% or more and 70% or less.

7. The method for producing a hollow fiber membrane according to claim 6, wherein: The concentration of cellulose ester in the spinning solution is 10% by mass to 30% by mass.

8. The method for producing a hollow fiber membrane according to claim 6 or 7, wherein: The ratio of the total amount of the solvent and the non-solvent in the coagulation liquid is 60% by mass to 90% by mass.

9. The method for producing a hollow fiber membrane according to claim 6 or 7, wherein: The solvent is an aprotic polar solvent, The non-solvent is a glycol ester.

10. The method for producing a hollow fiber membrane according to claim 6 or 7, wherein: The straight-line distance of the aerial traveling portion is 10 mm to 50 mm.

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