JP6220108B2 - Cryopreservation solution and cryopreservation method for pluripotent stem cells and other cells that can be suspended in suspension - Google Patents

Description

  The present invention is for cryopreserving cells that can be suspended and dispersed in a dispersion in colony or single cell units, such as human and animal pluripotent stem cells, other stem cells, germ cells, islet cells, etc. The present invention relates to a method and a cryopreservation solution therefor. In particular, the present invention relates to a cryopreservation method capable of reducing freezing and thawing damage when storing cells suitable for culture and storage in colonies, such as stem cells, and a storage solution therefor.

  With the development of regenerative medicine in recent years, medical application research of human-derived pluripotent stem cells has become active. Human pluripotent stem cells mainly include embryonic stem (ES) cells and artificial pluripotent stem (iPS) cells.In particular, the invention of iPS cells enables various aspects such as drug discovery screening as well as regenerative medicine applications. The application of has become realistic. For example, elucidation of the mechanism of genetic diseases and creation of therapeutic agents are expected by using disease-specific iPS cells. In application to regenerative medicine, research into differentiation into various useful cells has been actively conducted, and transplantation after differentiation into, for example, the retina and nerves has become a reality. However, these regenerative medicine applications are the first step as clinical research, and will be tailor-made medicine for specific patients, and will be cleared as clinical applications in a broad sense, that is, medical care accessible to everyone. There are many issues to be solved.

  For example, a period of 1 to 2 months is required from the collection of cells such as skin to the establishment of iPS cells, and then a considerable waiting time is required to induce differentiation into the necessary stem cells. . However, in that case, there is a problem that it is not possible to cope with a sudden disease or accident. Therefore, banking of iPS cells has been proposed. In that case, there are two problems. Immune rejection and preservation technology. When iPS cells are established from the patient's cells, rejection will not occur because they are the cells of the person. However, banking means that someone else's cells are used for transplantation, so an immune response is sure to occur. However, it is possible to reduce the immune response as much as possible by collecting and banking iPS cells established from cells of different HLA types to some extent. Another problem is storage efficiency. Human ES cells and iPS cells are highly sensitive to freezing and are considered difficult to cryopreserve. Usually, human ES cells and iPS cells are cultured in colonies because they cause apoptosis when dispersed in single cells. Therefore, it is common to freeze in units of colonies when freezing. However, since colonies are a cell population, it is difficult to freeze them uniformly. Thawing with a slow freezing method that leaves them in a -80 ° C freezer with a 10% dimethyl sulfoxide (DMSO) solution as in a normal cell line. The survival rate afterwards is less than 1%, so that it is not practical enough. On the other hand, a method of freezing in a single cell state by inhibiting apoptosis of ES cells and iPS cells using a ROCK inhibitor has also been proposed. However, ROCK inhibitors do not function normally to control the quality of the cell, in which damaged cells are eliminated by apoptosis, so that the problematic cells can survive and the quality of the cells may be reduced. Therefore, there is a demand for a method for cryopreserving with high efficiency in a colony state without using a ROCK inhibitor. Until now, vitrification has been developed as a method for preserving fertilized eggs, which are also difficult to freeze. In this method, the solute concentration and freezing speed are increased, and the crystallization of water is suppressed, thereby suppressing the physical damage associated therewith. In human ES cells, there is a report that a recovery rate of around 20% was obtained by a method using a vitrification solution called DAP213 which is a mixed solution of DMSO, acetamide and propylene glycol. Recovery rate refers to the total number of cells in a frozen system that was cultivated for several days after thawing the colony suspension into an unfrozen system and the frozen system, and divided by the total number of cells that were cultured in the unfrozen system for the same period of time. The value indicates the freezing efficiency is higher as this value is larger. The vitrification solution using DAP213 is much higher than 1% or less of the slow freezing method, and it is a number that can withstand practicality, so this method is used for cryopreservation of human ES and iPS cells in Japan. It is often used.

  However, the problem with this method is that the recovery rate of 20% is still low and the procedure is difficult. A recovery rate of 20% means that as many as 80% of cells die, that is, cells that are resistant to freezing are selected. By repeating this, it cannot be excluded as a possibility that changes accumulate in the population of cells. Therefore, it is desirable to improve the freezing method so that a majority of cells survive. Also, the difficulty of the procedure means that DAP213 is a solution with high concentrations of DMSO 2M, acetamide 1M, and propylene glycol 3M, so the osmotic pressure is high and the damage to the cells is very high. This is due to the strong correlation between contact time and cytotoxicity. In other words, if ES / iPS cell colonies are suspended in the DAP213 solution and then immersed in liquid nitrogen as soon as possible in the vial, the recovery rate after thawing is affected, so skill is required. In general, the time required for suspension is preferably about 10 seconds, and it is said that the results differ in units of 1 second. It is difficult to generalize with such a technique that must be cryopreserved under strict conditions. Furthermore, DMSO is said to affect the differentiation of ES cells, and acetamide is a carcinogen. Thus, there is still room for improvement in the composition, and the current preservation solution and preservation method are not necessarily optimal.

  On the other hand, for islet cells or islet-like cells that are similarly stored or cultured in colonies, better storage methods and storage solutions for islet banking and the like are desired.

  On the other hand, better preservation methods and preservation solutions are desired for human and animal germ cells, various stem cells, and also hepatocytes.

JP2011-030557 JP2010-213692 JP2010-273549

  With the current cryopreservation method, especially the cryopreservation of human ES / iPS cells has a low recovery rate after thawing, requires skill in the technique, and is difficult to generalize. In many cases, substances that are highly toxic and affect differentiation are used. Accordingly, the present invention provides a cryopreservation solution composed of components that have a higher recovery rate after thawing than before, have low toxicity and do not affect differentiation, and using this makes the procedure simple and easy. An object of the present invention is to provide a cryopreservation method that allows cryopreservation.

  The cryopreservation method of the present invention comprises 1 to 20% by weight of an amphoteric polymer compound having an amino group and a carboxylic acid group in each repeating unit, 0.25M to 1.5M saccharides having a low molecular weight (molecular weight less than 1000), and a low molecular weight. Using a physiological solution in which 5M to 7M of non-saccharide polyhydric alcohol (molecular weight less than 1000) is dissolved as a cryopreservation solution, cell colonies or single cells (individually released cells) are dispersed in the preservation solution, Cooling is performed at -70 ° C / min to -300 ° C / min. Here, as the amphoteric polymer compound, a polyamino acid having a carboxylic acid group and an amino group in a repeating unit or a derivative thereof is preferably used. Particularly preferably, carboxylated polylysine obtained by partially carboxylating (esterifying) the side chain amino group of polylysine by addition of carboxylic anhydride is used. In the present application, the high molecular compound means a compound having a number average molecular weight of 1000 or more, and the low molecular weight in saccharides and non-saccharide polyhydric alcohols is a compound having a molecular weight distribution such as polyglycerol. The molecular weight shall mean less than 1000. The term carboxylic acid group means a carboxylate group including those in the form of carboxylates.

  The cryopreservation solution of the present invention contains 1 to 20% by weight of the amphoteric polymer compound, saccharides 0.25M to 1.5M, and non-saccharide polyhydric alcohols 5M to 7M.

  By using a cryopreservation solution consisting of an amphoteric polymer compound as a water recrystallization inhibitor and a low molecular weight polyhydric alcohol, in particular, by using a combination of a non-saccharide polyhydric alcohol and a saccharide as the polyhydric alcohol. Even if cooling is performed more slowly than the conventional vitrification method, a stable glass state can be obtained. For this reason, vials called cryotubes or cryovials, or other containers, can be used in liquid nitrogen vapor or in a low-temperature atmosphere created using a program freezer, etc., without performing rapid cooling by directly immersing them in liquid nitrogen. The glass state can be formed by freezing. In particular, by utilizing the ice recrystallization inhibitory effect of amphoteric polymer compounds such as carboxylated polylysine and stabilizing the glass state of the preservation solution, crystallization and recrystallization during quenching and thawing are suppressed. For this reason, the effect of reducing the damage to the cells during freezing and thawing and increasing the freezing efficiency is expected.

  Such freezing technology can provide a safe and highly efficient cryopreservation technology for human iPS cells and other cells. In other words, human iPS cells that can be applied to regenerative medicine are immersed in the preservation solution and put into a glass state in an atmosphere of -150 ° C or in liquid nitrogen or liquid nitrogen vapor, and stored in a frozen state. It can be stored while maintaining the survival rate and differentiation ability without using serum proteins that have sex problems. Since dimethyl sulfoxide, which is an existing freezing protection agent, is not used, toxicity to frozen cells is kept low, and it can be stored frozen for a long time while maintaining its function. In addition, since protein components such as fetal bovine serum and albumin are not used, there is no concern about infectious diseases and the difference between lots due to biologics is not affected. Furthermore, human ES / iPS cells and other cells that can be automated can be cryopreserved safely and efficiently, without being affected by the experimenter's procedure.

Effect of PLL (0.65) and ethylene glycol concentrations and cooling temperature on the recovery rate of human iPS cells using vitrification solution (containing sucrose 0.75M). Expression of undifferentiated markers in human iPS cells cryopreserved in ethylene glycol 6.5M, sucrose 0.75M, PLL (0.65) 7.5% at -99 ° C / min. Alkaline phosphatase, Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 are all positively expressed. Polylysine in which 65 mol% of ethylene glycol 6.5M, sucrose 0.75M, PLL (0.65) 7.5% (ε-poly-L-lysine free amino group (side chain and terminal amino group) is succinate ester) In vitro pluripotency evaluation of human iPS cells cryopreserved in a cooling condition of -99 ° C / min. After the embryoid body was created, differentiation induction was performed, and the expression of endoderm-derived (AFP), ectoderm-derived (nestin), and mesoderm-derived (αSMA) proteins was positive. In vivo pluripotency evaluation of human iPS cells cryopreserved in ethylene glycol 6.5M, sucrose 0.75M, PLL (0.65) 7.5% at -99 ° C / min. Teratoma was formed by subcutaneous implantation in the back of the mouse. Formation of tissues derived from mesoderm (Cartilage), ectoderm (neuroepithelium), and endoderm (endodermal epithelium) is observed. Chromosome aberration evaluation of human iPS cells cryopreserved in ethylene glycol 6.5M, sucrose 0.75M, PLL (0.65) 7.5% at -99 ° C / min. According to the chromosome image by G-Band method, no chromosomal abnormality was observed.

  Examples of cells to be preserved include pluripotent stem cells such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells, and various stem cells, islet cells and islet progenitor cells extracted from islets Islet-like cells obtained from the above, various germ cells and reproductive organ cells. Various stem cells include hematopoietic stem cells, mesenchymal stem cells, neural stem cells and the like. Examples of germ cells and reproductive organ cells include sperm, oocytes, endometrial cells, fallopian tube epithelial cells, amniotic cells, unfertilized egg cells and fertilized egg cells. In addition to the cell types listed above, in some cases, intimal cells, epithelial cells, fibroblasts, hepatocytes, etc. can be stored frozen. That is, the freezing technique of the present invention can be applied as long as it can be dispersed in a preservation solution or a culture solution in a colony or single cell state.

  Preferably, the cryopreservation solution of the present invention substantially contains a water-soluble amphoteric polymer having a cationic group and an anionic group, particularly an amino group and a carboxylic acid group, as a water recrystallization inhibitor. The other composition is a physiological solution obtained by adding 0.25 to 1M saccharides to physiological saline and 6 to 8M low molecular weight polyhydric alcohols such as ethylene glycol, glycerin and propylene glycol. The amphoteric polymer as a water recrystallization inhibitor is particularly preferably ε-poly-L-lysine into which a carboxyl group has been introduced.

  The low molecular weight saccharide is particularly preferably sucrose (sucrose; non-reducing disaccharide, 8 hydroxyl groups), trehalose (non-reducing disaccharide), glucose (monosaccharide), raffinose (non-reducing trisaccharide), etc. One or more selected from the group consisting of these can be used. As low molecular weight sugars, lactose (reducing disaccharide), maltose (maltose; reducing disaccharide), mannose (monosaccharide), galactose (monosaccharide), fructose (fructose; monosaccharide) and the like are also suitable. One or more selected from the group consisting of the saccharide compounds mentioned above can be used as appropriate. The low molecular weight saccharides are not limited to these, and can be appropriately selected from monosaccharides, disaccharides, and trisaccharides. In some cases, oligosaccharides and the like can be partially blended. The concentration of the low molecular weight saccharide in the cryopreservation solution of the present invention can be said to be a suitable range of 0.25M to 1.5M, preferably 0.5M to 1M, and particularly preferably 0.6 to 0.9M. be able to. In addition, since the hydroxyl equivalent varies greatly depending on whether it is a monosaccharide, a disaccharide, or a trisaccharide, these concentration ranges can be a concentration converted to a disaccharide. That is, in the concentration based on the number of moles of hydroxyl group, 0.03M to 0.2M can be considered as a suitable range, preferably 0.06M to 0.13M, and 0.07M to 0.11M can be mentioned as a particularly preferred range.

  Particularly preferably, the low molecular weight polyhydric alcohol is one or more selected from the group consisting of ethylene glycol, glycerin, and propylene glycol. Among these, ethylene glycol can be mentioned as the most preferable one. As the low molecular weight polyhydric alcohol, in addition to the above, sugar alcohols such as xylitol, sorbitol, erythritol and mannitol can be suitably used, and diethylene glycol and triethylene glycol can also be used. That is, one or a combination of two or more selected from the group consisting of the polyhydric alcohols listed above can be used. In some cases, a polyhydric alcohol compound having a relatively high molecular weight such as polyglycerin can be partially blended. The concentration of the low molecular weight polyhydric alcohol in the cryopreservation solution of the present invention is from about 6M to about 7M, particularly from 5.8 to about 5% when ethylene glycol, glycerin and propylene glycol are used. It is preferably 7.3M, most preferably 6.3M to 7.3M. By setting such a concentration range, on the one hand, it is possible to obtain a sufficient effect for stabilizing the vitrification state, and on the other hand, it is possible to sufficiently reduce the adverse effects on the cells due to the high concentration of polyhydric alcohol. . However, particularly when sorbitol or the like is used, the hydroxyl group equivalent is greatly different. Therefore, the concentration based on the number of moles of hydroxyl group is preferably about 3M to 3.5M, more preferably 2.9 to 3.7M, most preferably 3.1M to 3.7M. However, depending on the type of polyhydric alcohol, even if the number of hydroxyl groups is the same, there is a difference in hydration and the like, so it is estimated that some deviation occurs.

  In addition to the above, the physiological solution constituting the cryopreservation solution of the present invention includes polyethylene glycol, ficoll (Ficoll; copolymer of sucrose and epichlorohydrin), dextran, hydroxyethyl starch, polyvinyl alcohol, polyvinyl High molecular compounds such as pyrrolidone, Percoll (aqueous colloidal silica dispersion having a particle size of 15 to 30 nm coated with polyvinyl pyrrolidone), and albumin can also be added.

  Amphoteric polymer compounds such as ε-poly-L-lysine into which a carboxyl group is introduced have their own cryoprotective effect on cells. There are several possible causes such as membrane protection during freezing, among which the ice recrystallization inhibitory effect is mentioned. Although specific data is omitted, the present inventors examined ice recrystallization at low temperatures of a 30% sucrose solution to which polylysine having a carboxyl group introduced at various ratios was added. A strong correlation was observed between the group introduction ratio and the ice recrystallization inhibition effect. From the research progress of the present inventors so far, the introduction ratio of carboxyl groups is 50 to 80 mol%, which has a high cryoprotective effect and ice recrystallization inhibitory effect, and the most suitable introduction ratio is 60 mol% to 70 mol%.

  In the cooling process for preserving general animal cultured cells, established cells, cancer cells, etc., a freezing method called a slow freezing method is generally employed. The slow freezing method freezes at a cooling rate of about -1 ° C / min in the presence of about 10% by weight of a cryoprotectant. This allows free water in the cell to be dehydrated and stored frozen without destroying the intracellular microstructure. However, human ES cells and iPS cells have a low freezing effect by this method, so vitrification, which is a completely different freezing method, is recommended. The vitrification method is a method of suspending cells in a vitrification solution with an increased solute concentration and immersing the whole vial in liquid nitrogen, for example, by rapidly freezing at about 300 ° C / min. This is a method of preventing crystallization of water and solidifying in a glass state. By this method, the toxicity to cells due to the expansion of water is suppressed. One of the problems of vitrification preservation is that if it is not thawed rapidly even during dissolution, recrystallization occurs and damages the cells. Therefore, it is generally recommended to dissolve as rapidly as possible in a medium warmed to + 37 ° C. after removing from liquid nitrogen at the time of dissolution. However, skilled techniques have been required to prevent recrystallization as much as possible. According to the present invention, by utilizing the ice recrystallization inhibitory effect of amphoteric polymer compounds such as carboxylated polylysine and stabilizing the glass state of the preservation solution, crystallization and recrystallization during quenching and thawing can be suppressed. Therefore, the effect of reducing the damage to the cells at the time of freezing and thawing and increasing the freezing efficiency is expected.

  In addition, the vitrification solution is usually a very concentrated aqueous solution having a solute concentration of 6M or more, and thus has a problem of strong cytotoxicity due to osmotic pressure. For this reason, vitrification of existing ES / iPS cells such as DAP213 causes problems such as cell death if stirring is not performed for about 15 seconds before freezing. come. In the present invention, for example, it was confirmed that the influence of osmotic pressure was reduced by increasing the viscosity of the solution by adding carboxylated polylysine and sucrose, and the effect of protecting the cell membrane. From this result, there is a margin in the time required for suspension, and stable vitrification can be performed with little influence on the skill of the experimenter. Furthermore, although it is difficult to automate the method by directly immersing in liquid nitrogen at the time of freezing, in the present invention, an atmosphere of −150 ° C. to −90 ° C. prepared in advance by a program freezer by using the storage solution. It was shown that vitrification occurred even under a somewhat slow cooling rate obtained by putting a vial in and maintaining the same atmospheric temperature, and that highly effective storage was performed. In other words, it was confirmed that the glass was solidified in a glass state even at a cooling rate half that of the cooling rate when the vial was directly put into liquid nitrogen, which was about -200 ° C./min. Therefore, by using the preservation solution of the present invention and the freezing conditions for storage, it is safe and highly efficient, and it is possible to cryopreserve human ES / iPS cells that can be automated without being affected by the procedure of the experimenter. Become.

  As described above, in the cryopreservation solution of the present invention, 1 to 50 w / w% of a water-soluble polymer compound having a cationic group and an anionic group in a repeating unit, such as carboxylated polylysine, is dissolved in a physiological solution. Being done. Preferably it is dissolved 2-20 w / w%, particularly preferably 3-15 w / w%. Further, a polyhydric alcohol such as ethylene glycol is dissolved in 3M-9M, preferably 5M-8M, more preferably 6M-7M. In addition, saccharides such as sucrose are contained at 0.25M to 1.5M, preferably 0.5M to 1M.

  As a physiological solution, a general culture solution for various cells or tissues can be used in addition to physiological saline. For example, Dulbecco's modified Eagle MEM medium (DMEM) can be mentioned as a preferable one.

  The amphoteric polymer compound as a cryoprotective component preferably has a number average molecular weight of 1,000 to 100,000, more preferably 1,000 to 20,000. The carboxylated polyamine polymer, which is a preferred amphoteric polymer compound, is one in which a carboxyl group is partially introduced into the amino group side chain contained in the repeating unit of the polyamine polymer. In introducing the carboxyl group, it is preferable to react with dicarboxylic anhydride, but in some cases, carboxymethylation or the like may be used. Of the polyamines, polylysine is most preferred.

  The polylysine may be any one of ε-poly-L-lysine or ε-poly-D-lysine, α-poly-L-lysine, and α-poly-D-lysine. Among these, the most preferable polymer species includes ε-poly-L-lysine having a number average molecular weight of 1,000 to 20,000, particularly 1,000 to 10,000 produced by a microorganism or an enzyme. ε-Poly-L-lysine is produced exclusively by actinomycetes belonging to the genus Streptomyces and is used exclusively as a food additive (http://www.chisso.co.jp/fine/jp/ polylisin / index.html) and those having a degree of polymerization of 15 to 35, and those having a degree of polymerization of 20 or less have also been attempted (for example, JP 2003-171463, JP 2005-318815). The number average molecular weight or the number average degree of polymerization is measured by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), for example, using an electrophoresis apparatus manufactured by Ato Co., Ltd. and a densitograph (AE-6920V type). And can be easily measured. At this time, a standard protein marker is used. Polylysine and other polyamino acids can be used with a molecular weight of 30,000 or more by increasing the molecular weight by heat treatment. However, the above molecular weight range is preferable from the viewpoint of preventing an increase in viscosity. Polylysine, which has a free carboxyl group only at the terminal, has only a primary amino group in the side chain, but has excellent miscibility by partially amidating with carboxylic anhydride as described later. Or it is thought that solubilization performance is exhibited. In particular, excellent performance can be exhibited by reacting dicarboxylic anhydride or the like to partially carboxylate.

  The amino groups of polylysine and other polyamines are preferably partially carboxylated with succinic anhydride. At this time, the amino group of the polyamine is preferably 50 to 99 mol%, particularly 50 to 93%, more preferably 50 to 90 mol%, still more preferably 55 to 80 mol%, most preferably 58 to 76 mol%. Is carboxylated. Instead of succinic anhydride used here, dicarboxylic anhydrides such as citric anhydride, glutaric anhydride, malic anhydride, fumaric anhydride, and maleic anhydride are also possible. Among these, succinic anhydride can be mentioned as a particularly preferable one. Here, instead of polylysine, polyamino acids composed of amino acids having amino group side chains such as polyarginine and polyaspartic acid can also be used. In some cases, chitosan can be used.

  Furthermore, instead of the carboxylated polyamino acid as described above, a partially aminated carboxylic acid side chain of polyglutamic acid can be used. Moreover, depending on the case, the polyallylamine which partially carboxylated the amino group of polyallylamine similarly to the case of said polylysine can also be used. That is, it is particularly preferable that the repeating unit of the polymer compound has both an amino group and a carboxyl group. In a particularly preferred embodiment, an amide bond is introduced into the side chain by carboxylation or amination. In addition, it can replace with introduction | transduction of the carboxyl group with respect to a polyamine polymer, or can introduce | transduce other anion groups, such as a sulfonate group, with introduction of a carboxyl group.

  Antioxidants and other additives can be added to the cryopreservation solution of the present invention as necessary. It is said that when cells are thawed from freezing, they are damaged by oxidative stress, and the effect is expected to increase by adding antioxidants. Examples of the antioxidant include catalase, peroxidase, superoxide dismutase, vitamin E, vitamin C, polyphenols such as epigallocatechin gallate, glutathione, and the like.

  Next, according to a preferred embodiment of the cryopreservation method of the present invention, a colony of cells or a single cell is dispersed in the above cryopreservation solution, and a cylindrical tube called a cryotube or a cryovial or a conical vial at the tip side of the cylinder After being put inside and sealed, the vial is placed in an atmosphere of −150 ° C. to −90 ° C. prepared using a temperature control device such as a program freezer, and the same atmospheric temperature is maintained. The vial used here is generally made of plastic such as polypropylene and has a wall thickness of 0.2 to 0.4 mm, for example, but in addition to the straw-like vial for quick freezing having an inner diameter of 2 to 5 mm, the slow freezing described above is used. Vials in the method can be used. That is, a diameter of 10 to 20 mm, for example, 12 to 17 mm, and a volume of, for example, 1 to 10 mL can be used. Therefore, a large amount of cells can be efficiently cryopreserved as compared with the case of using a quick freezing vial.

  In particular, the cell dispersion can be easily cooled at −70 ° C./min to −150 ° C./min, particularly preferably at −80 ° C./min to −120 ° C./min with a program freezer or the like. it can. If the cooling rate is within this range, the vitrification progresses relatively gently and solidifies uniformly, and the vitrification is stabilized by the addition of the amphoteric polymer compound, so damage due to recrystallization during dissolution is also possible. Can be suppressed. In particular, good preservation can be realized for cells that are easily damaged during freezing and thawing, such as stem cells.

  That is, by using the above-described cryopreservation solution of the present invention and maintaining the same atmospheric temperature by placing a vial in an atmosphere of -150 ° C to -90 ° C created using a temperature control device such as a program freezer, It is possible to develop semi-automated and automated freezing methods.

  Here, the program freezer is a device that performs automatic cooling according to a preset cooling program, and generally does not require a cooling bath such as being immersed in liquid nitrogen, and can be automatically cooled by installing a plurality of vials. As a result, the cost and labor involved in cryopreservation can be reduced. For example, the program freezer “CM series” manufactured by Taiyo Nippon Sanso Corporation can be used with appropriate modifications in accordance with the use situation, and extremely uniform cooling can be performed using a vibration function or the like.

  If the above-described cryopreservation solution of the present invention is used, a cooling rate of −200 ° C./min to −300 ° C./min similar to that of the conventional vitrification method may be used, such as by directly immersing the vial in liquid nitrogen. Even if cooling is performed, a considerable cryopreservation effect can be obtained. Therefore, the cryopreservation solution of the present invention can be used for cryopreservation at a cooling rate of -70 ° C / min to -300 ° C / min.

  Examples of the present invention and comparative examples will be described below. In addition, this invention is not limited to the following Example.

<Example 1 Preparation of Cryopreservation Solution>
ε-poly-L-lysine (manufactured by Chisso Co., Ltd., molecular weight 4000) is a 25% aqueous solution, and succinic anhydride (manufactured by Wako Pure Chemical Industries, Ltd.) is 0-100% in mol% with respect to the amino group in the molecule. Was added to prepare polylysine having different carboxyl group introduction rates. The substitution rate of the amino group to the carboxyl group in the polylysine molecule was determined by the TNBS method (quantitative determination of amino group by the trinitrobenzenesulfonic acid method). (Hereinafter, for example, when 65 mol% of the amino group of polylysine is substituted with a carboxyl group, “PLL (0.65)” will be indicated.)) The PLL (0.65) solution is treated with Dulbecco's modified medium (DMEM, Sigma). (Made by Aldrich) to be 0 to 20 w / w%. To this PLL (0.65) solution, ethylene glycol (Wako Pure Chemical Industries) as an additive was added so as to be 5 to 7.5 M and sucrose to 0.75 M. At this time, the solution was neutralized with a 1N or 5N aqueous sodium hydroxide solution so that the pH was in the range of 6.4 to 8.0. On the other hand, DAP213 was prepared as a cryopreservation solution for the comparative example. At this time, DMSO, acetamide, and propylene glycol were all manufactured by Wako Pure Chemical Industries, Ltd., and were prepared by dissolving 2M, 1M, and 3M in physiological saline, respectively.

<Example 2 Measurement of temperature drop rate>
Put a thermocouple into a 1mL cryovial (Simport Plastics) with a vitrification solution of ethylene glycol 6.5M, sucrose 0.75M, PLL (0.65) 7.5%, and immerse the vial in liquid nitrogen, or pre-chamber at -150 ° C The temperature drop rate when the sample was put into a program freezer (Taiyo Nippon Sanso) that had been cooled inside or put into a room temperature chamber and then rapidly cooled to -150 ° C by blowing liquid nitrogen vapor was quantified. The results are shown in Table 1.

  It was found that the cooling rate was -294.8 ° C / min when liquid nitrogen was directly charged, but the cooling rate was reduced by about 1/3 to 1/4 when charged in an atmosphere of -150 ° C and sprayed with liquid nitrogen.

<Example 3 Vitrification state evaluation of various vitrification liquids under each cooling condition>
Each of the vitrification solutions of ethylene glycol 5 to 7.5M, sucrose 0.75M, PLL (0.65) 0 to 7.5% and DAP213 vitrification solution as a comparative example were obtained in Example 2 with 200 μL added to a 1.5 mL microtube. We examined whether or not vitrification occurred by cooling under the three cooling conditions. In addition, it was allowed to stand at room temperature during thawing and evaluated whether recrystallization occurred. Evaluation method is determined visually, and if it remains transparent, it is evaluated as ○ if it is not vitrified or recrystallized. If it becomes cloudy, it is considered that crystallization has occurred x evaluation, cracks or partial cloudiness is evaluated △ It was. The results are shown in Table 2.

  DAP213 was vitrified at a cooling rate of -294.8 ° C / min, but was incompletely vitrified at -99.3 ° C / min, and no vitrification occurred at a cooling rate of -72.7 ° C / min. Further, it was confirmed that recrystallization progressed and left cloudy when left at room temperature. On the other hand, in the system of vitrification solution with varying ethylene glycol concentration, 5M does not add PLL (0.65). Was not. Further, recrystallization occurred under any conditions and became cloudy. However, it was confirmed that the addition of PLL (0.65) stabilized the vitrification, and the 7.5% addition system vitrified under any conditions. However, the EG concentration could not prevent recrystallization even under the condition of adding PLL (0.65). Moreover, it can be seen that the vitrification becomes stable by increasing the concentration of EG, and stabilization of the glass state was observed depending on the addition concentration of PLL (0.65) under any condition. Especially with EG6M, the effect of adding PLL (0.65) is remarkable. Even if it is not added, recrystallization occurs under any condition, but addition of 7.5% causes vitrification under any condition and recrystallization. It was confirmed that does not occur. In addition, the glass state was quite stable at EG7M or higher, and at 7.5M, the glass state was stabilized under all conditions regardless of the addition of PLL (0.65).

<Example 4 Cryopreservation of human iPS cells under various vitrification conditions>
The human iPS cells used for the cryopreservation experiment were 253G1 strain, passage number 20 to 70, which was donated by iPS Research Institute, Kyoto University.

The cells were treated with human iPS cells (1 mg / mL collagenase type IV, 1 mM CaCl ₂ , 0.25% trypsin / PBS solution) cultured as SNL cells as feeder cells for 5 minutes and collected in a colony state. The collected iPS cell colony suspension was dispensed into a centrifuge tube of the test section, and one was set as an unfrozen section in which freezing was not performed, seeded on feeder cells, and cultured. In addition, each test group was suspended in 200 μL of vitrified solution containing 0.75% sucrose containing DAP213, EG concentration and PLL (0.65) concentration for 30 seconds, and preserved by vitrification under the above three cooling rate conditions. Went. For thawing, each medium warmed to 37 ° C. was added to a 1 mL vial, dissolved rapidly by stirring, washed with 9 mL medium, and cultured on feeder cells. Evaluation was made by comparing the total number of cells for each condition when cultured for 4 days divided by the total number of cells when cultured for 4 days in an unfrozen section in the same experimental group as the recovery rate. . The results are shown in FIG.

  As a result, in DAP213, a recovery rate of about 20% was obtained at -294 ° C./min, but at a cooling rate lower than that, a considerably low recovery rate was obtained. On the other hand, in the EG system, a higher recovery rate than that of DAP213 was obtained at all concentrations, and a high value was obtained depending on the addition concentration of PLL (0.65). However, at high concentrations of EG such as 7.5M, the recovery rate decreased due to high toxicity to cells. Interestingly, EG6.5M showed a higher recovery rate at -99 ° C / min than at -294 ° C / min, especially in the PLL (0.65) addition system. This is often caused by the volume change caused by a temperature change that is too rapid when immersed in liquid nitrogen, and this is considered to be one of the reasons that iPS cells are physically damaged. Therefore, if the cooling rate is -99 ° C / min, vitrification progresses relatively gently and solidifies uniformly, and the vitrification is stabilized by the addition of PLL (0.65). It is possible to suppress damage caused by the above, and it can be said that this technique is superior to existing cryopreservation solutions and methods.

<Example 5: Characterization of cryopreserved human iPS cells>
It is necessary to confirm that the human iPS cells cryopreserved by the method of the present invention exhibit the function as iPS cells even after thawing. Therefore, immunostaining and alkaline phosphatase staining were first performed to determine whether human iPS cells after freezing and thawing EG6.5M, sucrose 0.75M, PLL (0.65) 7.5% express undifferentiated markers in vitro. The result of the examination is shown in FIG. The confirmed undifferentiated markers are alkaline phosphatase, Oct-4, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. The alkaline phosphatase was observed with a light microscope using a Sigma alkaline phosphatase activity kit. The other five undifferentiated markers were treated with each primary antibody, and then the fluorescence of the secondary antibody suitable for each antibody was observed with a fluorescence microscope. Due to the fluorescent dye bound to the secondary antibody, Oct-4, SSEA-3, TRA-1-60 positive cells emit green fluorescence, and SSEA-4, TRA-1-81 positive cells emit red fluorescence. FIG. 2 shows, after RGB separation, a color image obtained by a fluorescence microscope, the green fluorescence shows only the green component, and the red fluorescence shows only the red component. From FIG. 2, it was confirmed that all the markers were positive, and the human iPS cells cryopreserved by the method of the present invention maintained the undifferentiated ability.

In addition, as an in vitro pluripotency evaluation, embryoid bodies were formed from human iPS cells, and then differentiation induction was performed, thereby confirming protein expression derived from three germ layers. The results are shown in FIG. As in FIG. 2, FIG. 3 also shows a color image obtained by a fluorescence microscope after RGB decomposition, and green fluorescence (Nestin and αSMA) shows only the green component and red fluorescence (AFP) shows only the red component. . α-fetoprotein (AFP) is a liver-derived protein and has shown differentiation into endoderm. Nestin is a nerve-derived protein that shows differentiation into ectoderm. In addition, α-smooth muscle actin (αSMA) is a muscle marker and shows differentiation into mesoderm. Since all these markers are positive, it can be said that iPS cells frozen by the method of the present invention in vitro maintain pluripotency. However, as a method for evaluating the differentiation ability as a pluripotent stem cell, there is a method in which iPS cells are implanted in a mouse and teratoma formation is observed. This is a method for confirming whether iPS cells exhibit pluripotency even in vivo, and is a method often used for confirming the pluripotency of ES and iPS cells. About 1 × 10 ⁶ human iPS cells frozen and thawed by the preservation method of the present invention are suspended in 100 μL of physiological saline and transplanted by subcutaneous injection into the back of SCID mice (CLEA Japan, 5 week-old female, n = 5). did. Ten weeks later, the tumor formed at the transplantation site was taken out and evaluated histologically by hematoxylin-eosin staining. The result is shown in FIG. FIG. 4 shows only the green component after RGB separation of the microscope image. Fig. 4 shows that the formed tumor has mesoderm cartilage, neuroepithelium of ectoderm, endodermal epithelium of endoderm, and malformation in which tissues derived from three germ layers are mixed. It was confirmed to be a tumor. Similar results were obtained for all mice (N = 5). From the results of these undifferentiated and pluripotent evaluations, it was confirmed that the human iPS cells stored under the cryopreservation composition of the present invention and the chilled conditions maintained the function as pluripotent stem cells.

<Example 6: Chromosome aberration evaluation of human iPS cells>
Since human iPS cells may be transplanted, it is not desirable that genetic abnormalities occur due to manipulation techniques or the like. As a means for simply examining gene abnormality, there is a method for evaluating chromosomal abnormality. Thereby, abnormalities such as chromosomal deletions and duplications can be identified. The results of examining the shape of human iPS cells cryopreserved by the preservation solution and method of the present invention by the G-Band method are shown in FIG. From this result, since no abnormality was observed in all chromosomes, human iPS cells could be cryopreserved without causing genetic abnormality by the method of the present invention.

Claims (5)

For polylysine, 5 to 7.5 % by weight of partially carboxylated polylysine in which a carboxyl group in a form obtained by reacting 50 to 99 mol% of an amino group with succinic anhydride is introduced, sucrose 0.25M to 1.5M, ethylene glycol 6.3 Use a physiological solution in which M or more and less than 7.0M are dissolved as a cryopreservation solution, disperse cell colonies or single cells in the preservation solution, and cool at -70 ° C / min to -150 ° C / min. A method for cryopreserving cells characterized in that it is performed.   The cryopreservation method according to claim 1, wherein the cooling is performed at −80 ° C./min to −120 ° C./min.   Cell colonies or single cells are dispersed in the cryopreservation solution, placed in a vial and sealed, and then placed in an atmosphere of −150 ° C. to −90 ° C. prepared using a temperature control device or liquid nitrogen vapor. The cryopreservation method according to claim 1 or 2, wherein the cooling is performed by maintaining an atmospheric temperature.   The cryopreservation method according to claim 3, wherein the vial has a cylindrical shape or a conical shape on the tip side of the cylinder, a diameter of 10 to 20 mm, and a volume of 1 to 10 mL. The cryopreservation method according to any one of claims 1 to 4, wherein the partially carboxylated polylysine is obtained by carboxylating 55 to 80 mol% of an amino group of polylysine with succinic anhydride.