JP2019110799A - Vitrification freezing preservation method of animal cell - Google Patents

Abstract

To provide a vitrification freezing preservation method of an animal cell capable of achieving a high survival ratio after freeze thawing and excellent cell activity in freezing preservation of an animal cell for freezing preservation of many cells.SOLUTION: There is provided a vitrification freezing preservation method for freezing preservation of 1×10pieces or more pieces of cells in a vitrification liquid, at cell density of 1 to 1×10pieces/μl or more, in a melting operation of the thawed cells, the cells are exposed to a culture medium containing a ROCK inhibitor while melting, then the culture medium is removed from cells, then the cells are exposed to a fresh culture medium.SELECTED DRAWING: None

Description

  The present invention relates to a method for vitrifying cryopreservation of animal cells.

  Excellent preservation techniques for cells or tissues of organisms are required in various industrial fields. In general, long-term culture of cells or tissues in vitro is not preferable because cells or tissues collected from in vivo gradually lose their activity even in the medium. Therefore, techniques for preserving for a long time without losing bioactivity are important. Superior storage technology enables more accurate analysis of harvested cells or tissues, and the use of cells or tissues for transplantation while maintaining higher bioactivity improves the survival rate after transplantation. I can expect it. Furthermore, it is possible to sequentially produce and store artificial tissues for transplantation such as cultured skin cultured in vitro and so-called cell sheets constructed in vitro, and use them when necessary. In addition to research and medical care, significant benefits can be expected from the industrial side as well.

  For example, a slow freezing method is known as a method for preserving cells. In this method, first, cells or tissues are immersed in a preservation solution obtained by adding a cryoprotectant to a physiological solution such as phosphate buffered saline. As the antifreeze agent, compounds such as glycerol and ethylene glycol are used. After immersing cells or tissues in the storage solution, cool to -30 to -35 ° C at a relatively low cooling rate (eg, 0.3 to 0.5 ° C / min) to The solution inside and outside is sufficiently cooled and the viscosity is high. In such a state, when the cells or tissues in the storage solution are further cooled to the temperature of liquid nitrogen (-196 ° C.), any minute solutions in the cells or in the tissues and their surroundings become noncrystalline and solid. Vitrification takes place. By vitrification, when the inside and outside of the cell or the inside and outside of the tissue solidifies, the movement of the molecule substantially disappears, so it is considered that the preservation of the cell or tissue made of glass in liquid nitrogen can be semipermanently preserved.

  However, in the slow freezing method, since it is necessary to cool at a relatively slow cooling rate, operation for cryopreservation takes time. In addition, there is a problem that an apparatus or jig for temperature control is required. In addition, in the slow freezing method, since ice crystals are formed in the extracellular or extracorporeal storage solution, cells or tissues may be physically damaged by the ice crystals.

  As a method for solving the problems in the above-mentioned slow freezing method, a vitrification freezing storage method has been proposed. The vitrification cryopreservation method is based on the principle that ice crystals are difficult to form even at freezing point due to freezing point depression of an aqueous solution containing a large amount of freeze-resistant agent such as glycerol, ethylene glycol and dimethyl sulfoxide. When this aqueous solution is rapidly cooled in liquid nitrogen, it can be solidified without generating ice crystals. Such solidification is called vitrification freezing. In addition, an aqueous solution containing a large amount of a freeze-resistant agent is called a vitrification solution.

  As a specific operation of the said vitrification method, a cell is immersed in a vitrification liquid, and it cools by the temperature (-196 degreeC) of liquid nitrogen after that. The vitrification method has such an advantage that it does not require a long time for operation for cryopreservation and does not require an apparatus or jig for temperature control, because it is such a simple and rapid process. is there.

  Use of the vitrification cryopreservation method can avoid physical damage (freeze damage) to cells during freezing and thawing because ice crystals are not generated either inside or outside the cells, but it is included in the vitrification solution. These high concentrations of cryoprotectants are chemically toxic.

  The application of the above-described vitrification cryopreservation method is generally limited to cells which are unsatisfactory in terms of survival rate and cell activity after freezing and thawing by the slow freezing method from the viewpoint of low freeze resistance. Currently, specific cell types are still limited to embryos, eggs, sperm, ES cells, iPS cells and the like. For other cultured cells, the slow freezing method is a general freezing method, and even if the viability after freezing and thawing by the slow freezing method is poor, it can be used for industry and research by culture and growth. It is. From such a background, reports on vitrification cryopreservation in cultured cells are extremely limited.

  Vitrification and cryopreservation of embryos and eggs are carried out by the straw method or cryotop method. In any method, embryos and eggs are obtained together with a very small amount of vitrification solution as compared with the slow freezing method. Freezes to improve the freezing rate of embryos and eggs, shortens the time during which cells are exposed to the vitrification solution, that is, the time to freezing, and avoids the chemical toxicity of the vitrification solution. It is done. These methods are generally applied to the cryopreservation of several cells (at most 20 or less).

In the vitrification cryopreservation of animal cells represented by ES cells and iPS cells, etc., animal cells are dispersed in a vitrification solution containing a cryoprotectant, filled in a freezing vial, the freezing vial is immersed in liquid nitrogen, and freezing is performed. The way to do it is done. Generally, when using the vitrification cryopreservation method, the survival rate after freeze-thawing of animal cells is 50% or less and is still low. However, when the above-mentioned slow freezing method is used, the survival rate is several%, and therefore, vitrification occurs despite the high chemical toxicity when storing animal cells such as ES cells and iPS cells described above. The cryopreservation method is used. Under such a background, development of a cryopreservation method with higher survival rate is desired. In addition, the cryopreservation method using a cryovial is generally used for cryopreservation of 1 × 10 ³ or more animal cells, and the cell density of 1 to 1 × 10 ⁶ cells / μl with respect to the preservation solution in the cryovial Saved with

  Patent Document 1 discloses mammalian embryos or oocytes using a vitrification solution containing about 30% by volume ethylene glycol and 1 M sucrose or a vitrification solution containing about 40% by volume ethylene glycol and 1 M sucrose. How to save is described. Also described is a method of equilibrating with about 15% by volume ethylene glycol solution or about 10% by volume glycerol solution for 5-10 minutes prior to exposure to the vitrification solution.

  In Patent Document 2, an egg cryopreservation device using a strip-like flexible and colorless transparent film as a egg attachment holding strip is used in the method of the above-mentioned cryotop method used in the field of human infertility treatment. There has been proposed a method of attaching an ovum or an embryo under a microscope with a very small amount of vitrification solution on the film and cryopreserving.

  In Patent Document 3, as a medium for cryopreserving pluripotent stem cells, a medium containing dimethylsulfoxide, propylene glycol, acetamide, and a medium corresponding to the pluripotent stem cells intended for storage is proposed.

  Patent Document 4 proposes a method in which 10 to 20 dispersed mammalian anterior alveolar follicles are embedded in a collagen gel, immersed in a cryoprotectant solution, and then cryopreserved.

In Patent Document 5, it is described that slow freezing storage can be suitably performed using a medium containing hydroxyethyl starch, dimethylsulfoxide and ethylene glycol in slow freezing storage method of a large number of stem cells of 1 × 10 ³ or more. In addition, it is also described that suitable cell viability is obtained by culture | cultivating in the culture medium which added the ROCK inhibitor after the freezing and thawing process.

JP 2000-189155 A JP, 2006-271395, A Patent No. 4705473 JP 2001-226201 A International Publication No. 2013/187077 brochure

The method proposed in Patent Document 1 is not suitable for cryopreservation of animal cells, which usually cryopreserves 1 × 10 ³ or more cells, because the inner diameter of the straw is limited. Moreover, in patent document 1, freezing becomes quick by setting the liquid volume of a vitrification liquid to 0.5 microliter or less per embryo or egg, and it is described that survival rate is maintained high. The rapid freezing rate can be obtained by using a method proposed in Patent Document 1 and simply freezing a large number of animal cells of 1 × 10 ³ or more at one time, since the volume of the vitrification liquid used increases. The toxicity of the vitrification solution is strongly expressed, and a satisfactory survival rate can not be obtained.

  The method proposed in Patent Document 2 is a method of freezing embryos with a very small amount of vitrification liquid on a resin film having a width of about 0.5 to 2.0 mm, and as in the case of Patent Document 1, a large number of them are used. Is not suitable for cryopreservation of animal cells, such as cryopreservation of animal cells.

  Even when the medium proposed in Patent Document 3 is cryopreserved using the animal cell vitrification cryopreservation method, the cell viability after freeze-thaw was insufficient.

  The method proposed in Patent Document 4 has a cell recovery rate as high as about 90%, but the survival rate is extremely low as about 20%, and was not sufficiently satisfactory.

  The method proposed in Patent Document 5 relates to the slow freezing method, which requires a special apparatus such as a program freezer as compared with the vitrification freezing method, and it is a complicated step such that the freezing step takes time. Describe that the addition of a ROCK inhibitor at the time of culture suitably acts on cell viability.

  The present invention mainly provides a method for vitrifying cryopreservation of animal cells in which high survival rate and recovery rate are achieved after vitrification freeze-thaw in vitrification cryopreservation of animal cells for cryopreservation of a large number of animal cells. To be a challenge.

  MEANS TO SOLVE THE PROBLEM This inventor discovered that the said subject could be solved with the following method, as a result of earnestly examining in order to solve the said subject.

A vitrification cryopreservation method of cryopreserving 1 × 10 ³ or more cells at a cell concentration of 1 to 1 × 10 ⁶ cells / μl in a vitrification solution, wherein ROCK inhibition is caused in the thawing operation of the frozen cells. A method for vitrifying cryopreservation of animal cells, which comprises thawing while exposing cells to a culture solution containing an agent, and thereafter removing the culture solution from the cells and exposing the cells to fresh culture solution.

According to the present invention, it is possible to provide a method for vitrifying cryopreservation of animal cells, which achieves high cell viability and excellent cell activity when cryopreserving 1 × 10 ³ or more of a large number of cells. In addition, the vitrification cryopreservation method of the present invention can be conveniently performed using a device / device used for conventional culture of mammalian cells without the need for a special device / device. Furthermore, using the vitrification cryopreservation method of the present invention, the cryopreservation operation of cells can be performed in a shorter time than the conventional slow freezing method.

  The vitrification cryopreservation method of the present invention can be used for cryopreservation of animal cell colonies or single cells. Examples of the cells that form colonies or single cells can include, for example, cultured cells of animals. The cultured cells are human cells or cells of animals other than human, and examples thereof include primary cultured cells, subcultured cells, and cell line cells. Cell types include cancer cells such as fibroblasts and liver cancer cells, epithelial cells, endothelial cells, endothelial cells, lymphatic endothelial cells, nerve cells, tissue stem cells, embryonic stem cells, and adherent cells such as immune cells. Can be mentioned. The vitrification cryopreservation method of the present invention can be suitably used particularly for cryopreservation of hepatocytes. In the present invention, a colony refers to a cell population consisting of several or more cells, and a single cell refers to one cell. Multiple types of cells may be contained in a single stock solution in the form of colonies or single cells. Also, multiple types of cells may be contained in a single storage solution in the form of a cluster-like cell mass consisting of multiple types of cells. In addition to cells, it may contain noncellular substances such as extracellular matrix.

  The vitrification cryopreservation method in the present invention includes "freeze operation" in which cells are frozen at cryogenic temperature, "storage operation" in which cells are stored at cryogenic temperature, and thawing for reusing stored cells. Alternatively, it shall include a series of operation procedures of "thawing operation" to thaw.

  The vitrification freezing in the present invention is different from the so-called slow freezing method in which freezing is performed at a relatively slow cooling rate (for example, a rate of 0.3 to 0.5 ° C./min). It refers to a method of freezing using a fast cooling rate and suppressing the formation of ice crystals. The cooling rate in vitrification freezing is, for example, 200 ° C./min or more from 0 ° C. to −150 ° C., which is measured using a sheath type thermocouple (tip outer diameter 0.3 mm) manufactured by CHINO corporation.

Vitrification cryopreservation method of the invention, as cryopreserved using conventional slow freezing method, for example, 1 × 10 ³ or more animal cells per freezing vial, and more preferably 1 × 10 ³ ~ It can be used for cryopreservation of 1 × 10 ⁸ animal cells. Moreover, the cell density at the time of cryopreservation is 1 to 1 × 10 ⁶ cells / μl with respect to the preservation solution (vitrification solution) in the freezing vial, as in the normal slow freezing method. When the cell density is smaller than this range, loss of cells mainly in the steps of centrifugation, filtering and the like increases, and a satisfactory cell number (recovery rate) can not be obtained after cryopreservation and thawing. On the other hand, when the cell density is larger than this range, it is difficult to disperse the cells in the vitrification solution, or the penetration of the vitrification solution into the cells becomes insufficient, and a satisfactory survival rate and recovery rate I can not get it.

  The freezing operation in the vitrification freezing storage method of the present invention will be described. In the freezing operation in the vitrification cryopreservation method of the present invention, cultured cells are detached / dispersed by trypsin treatment or the like, and then the cells are immersed / dispersed in a vitrification liquid containing a high concentration of a cryoprotectant, and further, This is a step of immersing cells or cells-containing containers (for example, frozen vials) in a cryogenic refrigerant, and vitrifying them.

  The vitrification liquid used in the freezing operation in the vitrification freezing storage method of the present invention contains a high concentration of a freeze resistant agent. As a freezing-proof agent which the vitrification liquid used for the vitrification freezing preservation method of the present invention contains, dimethyl sulfoxide, glycerin, ethylene glycol, propylene glycol, propanediol etc. can be used. Moreover, it can be preferably used also about the antifreeze agent which consists of non-cell-permeable polylysine as described in the international publication 2009/157209 pamphlet. Preferred antifreeze agents are dimethyl sulfoxide, ethylene glycol. The freezing resistance used in the vitrification liquid may contain one type, or two or more types. The total concentration of the freeze resistant agent is preferably 20 to 50% by volume, more preferably 25 to 45% by volume.

  As other composition contained in the vitrification liquid used for the vitrification freezing preservation method of the present invention, about 0.2-1 M of saccharides may be contained in culture fluid or physiological saline. Examples of saccharides that may be contained in the vitrification liquid include sucrose, trehalose, glucose, raffinose, lactose, maltose, mannose, galactose and fructose. When the vitrification liquid contains a saccharide, it may contain one type, or two or more types of saccharides may be contained.

  The vitrification liquid used for the vitrification freezing storage method of the present invention can contain a polymer compound. Examples of the polymer compound include polyethylene glycol, ficoll (copolymer of sucrose and epichlorohydrin), dextran, polyvinyl alcohol, polyvinyl pyrrolidone, a polymer compound such as albumin, or hyaluronic acid, gellan gum, xanthan gum, Polymeric compounds such as polysaccharides such as carrageenan, alginic acid derivatives such as sodium alginate and salts thereof, cellulose derivatives such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose and carboxymethyl cellulose and salts thereof can be contained. It is known that the viability of cells after freezing and thawing is improved by the cytoprotective effect and the viscosity increase of the vitrification liquid due to the inclusion of the above-mentioned polymer compound.

  The vitrification liquid used in the vitrification cryopreservation method of the present invention may additionally contain a biological material such as serum. Serum is widely used for the purpose of cytoprotective effect at the time of freezing, although its constituent components have not been completely elucidated yet.

  In the freezing operation in the vitrification cryopreservation method of the present invention, the time for which the cells are immersed in the vitrification solution (the time from the contact of the cells with the vitrification solution to the start of cooling with a cooling solvent) Although depending on the composition of the solution, in most cases, within 1 minute is preferable in view of avoiding chemical toxicity of the vitrification solution. More preferably, it is within 45 seconds.

  In the freezing operation in the vitrification cryopreservation method of the present invention, before soaking and dispersing the cells in the vitrification liquid, so-called equilibration treatment in which the cells are dipped and dispersed in the pretreatment liquid having a lower concentration of the freezing resistance than the vitrification liquid. You can also do The equilibration treatment is particularly useful for maintaining the survival rate after freezing and thawing for cells which are less resistant to osmotic shock and rapid changes in the concentration of the cryoprotectant.

  At the time of liquid exchange in the freezing operation, it is preferable to separate the solution and the cells by a technique such as centrifugation or filtering, and immerse the cells in the next solution. It is preferable to use centrifugation from the viewpoint that cells can be separated more easily. For example, cultured cells are detached by trypsinization, and cells dispersed in a culture solution containing trypsin are centrifuged to separate the cells and the solution, and then the solution is discarded and then vitrified. The solution can be added to immerse and disperse the cells in the vitrification solution.

  Next, the storage operation in the vitrification cryopreservation method of the present invention will be described. The cells vitrified and frozen by the freezing operation can be stored semipermanently by storing them in a cryogenic environment where the vitrified state of the cells is maintained. The temperature at which the vitrified state is maintained varies depending on the composition of the vitrification liquid, but in most cases -150 ° C. or less is preferable. As an environment in which such temperature is maintained, it is preferable to be in a liquid nitrogen storage container or in a gas phase nitrogen storage container.

  Next, the thawing operation in the vitrification cryopreservation method of the present invention will be described. In the thawing operation in the vitrification cryopreservation method of the present invention, the frozen cells are removed from the cryogenic environment, thawed while exposing the cells to a medium containing ROCK inhibitor, and then the medium was removed from the cells , Exposing the cells to fresh culture fluid. As an example of the operation, for example, the vitrification solution and cells frozen in a freezing vial are brought into direct contact with a culture solution containing a temperature-controlled ROCK inhibitor, and the cells are treated in a culture solution containing a ROCK inhibitor. Upon exposure, the vitrification solution and cells are thawed, and the vitrification solution is diluted. By such an operation, re-ice crystal formation and toxicity of the vitrification liquid can be alleviated, and at the same time, damage to cells can be suppressed. Thereafter, the culture solution containing the cells and the ROCK inhibitor is separated by centrifugation or the like, and after removing the culture solution, the cells are dispersed in a fresh culture solution to expose the cells to the fresh culture solution. . Cells exposed to fresh media can be used depending on the subsequent application. For example, when culture and growth are attempted after freezing and thawing, it can be added to the culture petri dish together with the culture solution, and static culture can be performed.

  The culture solution constituting the culture solution containing the ROCK inhibitor according to the present invention is not particularly limited as long as it does not show toxicity to the activity or proliferation of the target cells, but the culture solution used for the subsequent culture Is preferred. In this case, the composition of the culture solution used for the subsequent culture may be a composition excluding serum and growth factors.

  In the present invention, any substance that inhibits Rho-associated coiled-coil kinase (ROCK) can be used as a ROCK inhibitor in a culture solution containing a ROCK inhibitor, for example, Y-27632, Fasudil (HA1077) H-152, Wf-536 (all available from Wako Pure Chemical Industries, Ltd.) and their derivatives, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids, and vectors containing these.

  The concentration of the ROCK inhibitor in the culture solution containing the ROCK inhibitor in the present invention is preferably 1 to 100 μM, more preferably about 10 μM.

  The ROCK inhibitor used in the vitrification freezing preservation method of the present invention is, for example, as described in JP-A-2011-519576 or WO2011 / 00090068, a comparison such as adding to a culture medium and maintaining culture It is known that exposure to cells for a prolonged period of time suitably affects cell proliferation. Moreover, by adding to the vitrification liquid at the time of slow freezing, the cells are exposed for a relatively long time until they are frozen (In addition, phenomenologically, the cells are still exposed in the vitrified state after freezing) It is generally known that the survival rate after freezing and thawing is suitably affected.

  On the other hand, substances involved in the control of intracellular signal transduction such as ROCK inhibitors can not completely rule out the possibility of unintended effects on cells, so exposure in a short time if possible Is desirable.

  In the thawing operation in the vitrification cryopreservation method of the present invention, after the frozen cells are removed from the cryogenic environment, it takes a short time to thaw the cells with a medium containing a ROCK inhibitor preferable. The time for removing the cells from the extremely low temperature environment and completely dispersing the cells in the culture solution is preferably within 2 minutes, more preferably within 1 minute. In particular, when using a rapid vitrification method such as the so-called cryotop method, it is important to melt as quickly as possible to avoid ice crystal re-formation.

  In the thawing operation in the vitrification freezing storage method of the present invention, the addition amount of the culture solution containing the ROCK inhibitor is a vitrification liquid frozen in a vitrified state together with the cells during the storage operation from the viewpoint of increasing the melting rate. The larger the amount of liquid, the better. Preferably, it is 4 times or more, more preferably 40 times or more with respect to the amount of vitrification liquid.

  In the thawing operation in the vitrification cryopreservation method of the present invention, it is preferable that the liquid temperature of the culture solution containing the ROCK inhibitor be temperature-controlled from the viewpoint of enhancing the melting rate. Preferably it is 20 degreeC or more, More preferably, it is 35 degreeC or more.

  In the thawing operation in the vitrification cryopreservation method of the present invention, for example, pipetting using a micropipette can be suitably used to disperse cells in a culture solution containing a ROCK inhibitor.

  In the thawing operation in the vitrification cryopreservation method of the present invention, the cells are thawed while being exposed to a medium containing ROCK inhibitor, then the medium is removed from the cells and the cells are exposed to a fresh medium. At the time of solution exchange in these thawing operations, the solution and the cells can be separated by a technique such as centrifugation, filtering and the like, and the cells can be immersed in the next solution (fresh culture solution).

  In the thawing operation in the vitrification cryopreservation method of the present invention, the time during which the ROCK inhibitor is exposed to the cells (ie, the culture solution containing the ROCK inhibitor is added to the frozen cells, and then the culture solution is removed The time to expose the cells to fresh culture fluid is preferably 3 to 30 minutes, more preferably 5 to 10 minutes. If the cells are exposed for less than 3 minutes, good viability after freeze-thaw may not be obtained. On the other hand, when the exposure time is longer than 30 minutes, the activity of the cells is adversely affected due to the problem of cell suspension stress due to the toxicity of the vitrification solution diluted by the culture solution containing the ROCK inhibitor and the dispersed state. There is a case.

  In the thawing operation in the vitrification cryopreservation method of the present invention, the fresh culture solution is not particularly limited as long as it does not show toxicity to the activity or proliferation of the target cells, but the culture solution used for the subsequent culture Is preferred. Serum and growth factors can also be added from the viewpoint of subsequent culture. Furthermore, the fresh culture solution may contain, for example, 1 to 100 μM of ROCK inhibitor.

  EXAMPLES The present invention will be further described in more detail by way of examples to specifically describe the present invention, but the present invention is not limited to the following examples.

Example 1
<Preparation of cells>
Mouse embryonic fibroblasts cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing L-Glutamine supplemented with 10% by volume of bovine serum (The primary culture was followed by about 5 passages) Subcultured cells were collected from plastic petri dishes by trypsinization. The viability of the recovered cells was determined by trypan blue staining and was 95% or more. The collected cells were in a state in which a single cell (one cell) and a cluster-like cell population consisting of a plurality of cells coexist.

<Freezing operation>
A culture solution (DMEM containing L-Glutamine supplemented with 10% of bovine serum) was added to the collected mouse embryonic fibroblasts to prepare a cell dispersion at a cell concentration of 1 × 10 ⁷ cells / ml. 50 μl (5 × 10 ⁵ ) of them were dropped to a 1.5 ml freezing vial. To the cell dispersion in the freezing vial, 200 μl of a vitrification solution of the following composition was added, and the cells were dispersed in the vitrification solution. The cell concentration at this time was 2 × 10 ³ cells / μl. Thereafter, the freezing operation was performed by immersing the freezing vial in liquid nitrogen. After contacting the cells with the vitrification solution, the time to immerse the frozen vial in liquid nitrogen was 30 seconds.

<Composition of Vitrification Liquid of Example 1>
Ethylene glycol 30% by volume
Glycerin 0.5% by volume
Sucrose 500 mM
The total volume was adjusted to 100% by volume by adding DMEM.

<Storage operation>
The frozen vials subjected to the freezing operation were stored in a liquid nitrogen storage container for at least 2 days.

Melting operation
A frozen vial subjected to a storage operation is taken out of liquid nitrogen, and a culture solution containing 10 μM of ROCK inhibitor Y-27632 and thermostated at 37 ° C. (DMEM containing L-Glutamine to which 10% of bovine serum is added) One ml was added directly into the freezing vial and pipetted with a micropipette. After the vitrification solution was completely thawed, the cell dispersion in a freezing vial was added to 9 ml of the culture solution containing the above ROCK inhibitor, which had been thermostatted at 37 ° C. The cell concentration at this time is 5 × 10 ⁴ cells / ml. Then, the cells were pelleted by centrifugation at 320 × g for 5 minutes, and the culture broth containing the ROCK inhibitor was removed from the supernatant. Furthermore, fresh culture solution (DMEM containing L-Glutamine supplemented with 10% of bovine serum) was added to disperse the cells. The time for which the cells were exposed to the medium containing ROCK inhibitor was 8 minutes.

  As described above, the thawing operation, the storage operation, and the thawing operation in the vitrification cryopreservation method of Example 1 were performed.

(Example 2)
In the freezing operation of the vitrification freezing storage method of Example 1, the vitrification freezing storage method of Example 2 was carried out in the same manner as in Example 1 except that the composition of the vitrification liquid was changed as follows.

<Composition of Vitrification Liquid of Example 2>
Ethylene glycol 15% by volume
Dimethyl sulfoxide 15% by volume
Sucrose 500 mM
The total volume was adjusted to 100% by volume by adding DMEM.

(Example 3)
The vitrification freezing preservation method of Example 3 changed the freezing operation in the vitrification freezing preservation method of Example 1 as follows, and was implemented.

5 × 10 ⁵ cells were dispersed in the following pretreatment solution at a cell concentration of 1 × 10 ⁵ cells / ml. This dispersion state was maintained for 3 minutes in a room temperature environment.

<Composition of pretreatment liquid>
Ethylene glycol 10% by volume
Glycerin 0.5% by volume
The total volume was adjusted to 100% by volume by adding DMEM.

  Then, centrifugation was performed for 5 minutes at a centrifugal force of 320 × g, and the mouse embryonic fibroblasts described above were sedimented and separated from the pretreatment solution. After removing the pre-treatment solution of the supernatant, 200 μl of the vitrification solution of Example 1 was added, and the cells were dispersed in the vitrification solution. The subsequent freezing operation, storage operation, and thawing operation were performed in the same manner as in Example 1, and the vitrification freezing storage method of Example 3 was performed.

(Example 4)
<Preparation of cells>
Human hepatoma-derived cell line HepG2 cells cultured in Eagle's Minimum Essential Medium (EMEM) containing L-Glutamine supplemented with 10% by volume of bovine serum are recovered from the plastic petri dish by trypsin treatment, and the culture solution (EMEM containing L-Glutamine supplemented with 10% of bovine serum) was added to prepare a cell dispersion at a cell concentration of 1 × 10 ⁷ cells / ml. The viability of the recovered cells was determined by trypan blue staining and was 95% or more. The collected cells were in a state in which a single cell (one cell) and a cluster-like cell population consisting of a plurality of cells coexist.

50 μl (5 × 10 ⁵ ) of the collected HepG2 cell dispersion was dropped into a freezing vial. To the cell dispersion in the freezing vial, 200 μl of a vitrification solution of the following composition was added, and the cells were dispersed in the vitrification solution. The cell concentration at this time was 2 × 10 ³ cells / μl. Thereafter, the freezing operation was performed by immersing the freezing vial in liquid nitrogen. After contacting the cells with the vitrification solution, the time to immerse the frozen vial in liquid nitrogen was 30 seconds.

<Composition of Vitrification Liquid of Example 4>
Ethylene glycol 30% by volume
Glycerin 0.5% by volume
Sucrose 500 mM
EMEM was added to adjust the total volume to 100% by volume.

<Storage operation>
The frozen vials subjected to the freezing operation were stored in a liquid nitrogen storage container for at least 2 days.

Melting operation
A frozen vial subjected to a storage operation is removed from liquid nitrogen and a culture solution containing 10 μM of ROCK inhibitor Y-27632 and temperature-controlled at 37 ° C. (EMEM containing L-Glutamine to which 10% of bovine serum is added) One ml was added directly into the freezing vial and pipetted with a micropipette. After the vitrification solution was completely thawed, the cell dispersion in a freezing vial was added to 9 ml of the culture solution containing the above ROCK inhibitor, which had been thermostatted at 37 ° C. The cell concentration at this time is 5 × 10 ⁴ cells / ml. Then, the cells were pelleted by centrifugation at 320 × g for 5 minutes, and the culture broth containing the ROCK inhibitor was removed from the supernatant. Furthermore, fresh culture fluid (EMEM containing L-Glutamine supplemented with 10% of bovine serum) was added to disperse the cells. The time for which the cells were exposed to the medium containing ROCK inhibitor was 8 minutes.

(Example 5)
In the freezing operation of the vitrification freezing storage method of Example 4, the vitrification freezing storage method of Example 5 was carried out in the same manner as in Example 4 except that the composition of the vitrification liquid was changed as follows.

<Composition of Vitrification Liquid of Example 5>
Ethylene glycol 15% by volume
Dimethyl sulfoxide 15% by volume
Sucrose 500 mM
Bovine serum derived albumin (BSA) 1.5% by mass
EMEM was added to adjust the total volume to 100% by volume.

(Comparative example 1)
In the thawing operation of the vitrification cryopreservation method of Example 1, the frozen vial subjected to the storage operation is removed from liquid nitrogen, and then the ROCK inhibitor, Y-27632, is added to the culture solution to be added and exposed to the cells. The vitrification cryopreservation method of Comparative Example 1 was carried out in the same manner as in Example 1 except that it was not present.

(Comparative example 2)
In the thawing operation of the vitrification cryopreservation method of Example 4, the frozen vial subjected to the storage operation is removed from liquid nitrogen, and then ROCK inhibitor Y-27632 is added to the culture solution added and exposed to the cells. The vitrification cryopreservation method of Comparative Example 2 was carried out in the same manner as in Example 4 except that it was not present.

<Evaluation of cell viability after freezing and thawing>
A part of mouse embryonic fibroblast dispersion (Examples 1 to 3 and Comparative Example 1) obtained by the above operation or a part of human hepatoma cell line HepG2 cell dispersion (Examples 4 and 5 and Comparative Example 2) The viability of the cells was determined on a hemocytometer by trypan blue staining. The survival rate is a ratio of the number of living cells to the total number of cells (the total number of cells in the cell dispersion obtained by the above operation). As for the evaluation of the survival rate, the survival rate of 80% or more was evaluated as 以上, 60% or more and less than 80% as Δ, and less than 60% as x. The results are shown in Table 1.

<Measurement of freezing rate>
When immersing the frozen vial in liquid nitrogen in the freezing operation of the freeze-preservation method of Examples 1 to 5 and Comparative Examples 1 and 2, the state of temperature drop in the freeze vial was measured by a sheath type thermocouple made by CHINO (tip outer diameter It measured using 0.3 mm. The cooling rates from 0 ° C. to −150 ° C. were all 500 ° C./min or more.

<Evaluation of cell activity>
After thawing the mouse embryonic fibroblasts or human hepatoma-derived cell line HepG2 cells subjected to the vitrification cryopreservation method of Examples 1 to 5 and Comparative Examples 1 and 2, 10 ml of culture solution (mouse embryo Morphology of cells after culturing for 24 hours on a plastic petri dish (100 mm dish) containing DMEM containing L-Glutamine supplemented with 10% of bovine serum for fibroblasts and EMEM for human hepatoma cell-derived cell line HepG2 cells And evaluation of cell activity after freeze-thawing. Evaluation of cell activity was performed based on the following criteria, and the results are shown in Table 2.

○: almost no floating cells, cell adhesion and spreading observed in most cells Δ: small number of floating cells, cell adhesion and spreading observed in many cells, ×: many floating cells

  From the above results, when using the vitrification cryopreservation method of the present invention, a large number of animal cells can be vitrified cryopreserved with high survival rate and excellent cell activity. In addition, the freezing operation in the vitrification cryopreservation method of the present invention does not require a special device / apparatus. In the conventional slow freezing method, about half a day to about one day is required using a program freezer until the freezing operation is completed. Therefore, the cell freezing and storage operation can be performed in a shorter time by using the vitrification freezing storage method of the present invention It can be performed.

  The present invention is used for cryopreservation of primary cultured cells, subcultured cells, and cell line cells, which are cells of human cells or non-human animals that can be dispersed in the form of colonies or single cells. Can. Therefore, it is useful in biotechnology fields such as cosmetics, medicines, and pharmaceuticals.

Claims (1)

A vitrification cryopreservation method of cryopreserving 1 × 10 ³ or more cells at a cell concentration of 1 to 1 × 10 ⁶ cells / μl in a vitrification solution, wherein ROCK inhibition is caused in the thawing operation of the frozen cells. A method for vitrifying cryopreservation of animal cells, which comprises thawing while exposing cells to a culture solution containing an agent, and thereafter removing the culture solution from the cells and exposing the cells to fresh culture solution.