JP2010213692A - Method and container for vitrifying preservation of cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell preservation method that suppresses cytotoxicity by a cytoprotective liquid and increases a cell survival rate by efficiently and stably vitrifying and preserving cells by a very small volume of cytoprotective liquid. <P>SOLUTION: The method includes holding cells in ≤50 nL volume of a preservation solution of a perfect non-serum (containing no serum component) having and preserving the cells in the presence of a refrigerating agent. Preferably, the cells are preserved in the presence of a refrigerating agent by using a cell protective liquid containing a cryoprotective agent having concentration of ≤15%. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cell storage technique by vitrification. In particular, the present invention relates to a container suitable for cell storage by vitrification.

  In addition to being used for artificial insemination and in vitro fertilization in the field of reproductive medicine, cryopreservation of cells is required for preservation of genetic resources and breed improvement. Conventionally, as a cryopreservation method for mammalian cells, glycerol, dimethyl sulfoxide (DMSO), propanediol, and sucrose are used as cryoprotectants, and they are immersed in a frozen solution stepwise and frozen over time. Slow freezing has been used. However, when the slow freezing method is used, in addition to the problem that it takes time (2-3 hours), a dedicated device such as a program freezer is required, and there is a problem that it is difficult to spread widely. .

  On the other hand, the vitrification preservation method that freezes and preserves the solution inside and outside the cell in a glass state by rapid cooling does not require an expensive cooling device and the processing time is short, so it is currently widely used as a method to replace the slow freezing method. It has been put into practical use. However, the vitrification preservation method was reported in 1937 (see Non-Patent Document 1), and it took a long time to develop technology for practical use. In 1985, a high concentration of cryoprotectant (ice crystal formation) Development of a technique using an inhibitor) has finally been put into practical use as a high-viability, simple process, and rapid cryopreservation method (see Non-Patent Document 2).

  However, in the vitrification preservation method, since it is necessary to contain a high concentration of cryoprotectant, toxicity to cells has been a problem. In order to solve this problem, it was found that the cryoprotectant concentration can be lowered by reducing the amount of liquid to be cooled and accelerating the cooling rate, and a method for preserving cells with an ultrafine liquid amount was developed. ing. For example, nylon loops, straw outer walls, chips, plates, films, and the like are used as freeze protection containers (see, for example, Patent Documents 1 to 3). However, in all cases, the amount of the cell-containing preservation solution is at the microliter level, and it is necessary to contain at least about 30% of a cytoprotective substance (see Non-Patent Document 3).

  Demirici et al. Directly drop liquid protective liquid droplets containing cells such as lymphocyte cells, mouse ES cells, fetal skin-derived cell lines, hepatic cell lines, monkey atrial cell lines, etc. into liquid nitrogen to obtain the desired size. It has been reported that the cells can be preserved in a small amount of liquid by virtue of filtration with a filter, and can be vitrified at a lower concentration of cytoprotective substance (see Non-Patent Document 4). However, since this method drops minute droplets directly into liquid nitrogen, a special device is required, it is difficult to equalize the volume of all droplets, and the filter was not filtered. Droplets exceeding the above capacity are discarded, resulting in problems such as a low cell utilization rate and a time required for collecting droplets of a desired size by filtering the droplets. Further, in vitrification and preservation of germ cells and fertilized egg cells, it is known as a problem that even if the survival rate is good, the arrival of blastocysts may be poor, but Demirici et al. There is no mention about.

  Furthermore, although the conventional method uses serum (bovine serum albumin), in recent years, there has been a demand for a method capable of achieving a high survival rate without using serum for the purpose of avoiding the risk of infection and the like.

International Patent Publication No. WO2004 / 098285 International Patent Publication No. WO00 / 21365 JP 2002-315573 A

Luyet, B.M. J., Biodynamica, 29, 1-14 (1937). Rall, W.M. F. et al. , Nature, 313, 573-575 (1985). Kuwayama, J. et al. Mamm. Ova Res. , 22, 193-197 (2005) Demirci et al. , Lab Chip, 7, 1428-1433, 2007

  As a result of studying a method that enables stable and simple vitrification storage at a low volume, the present inventors have found that a small volume of cells can be efficiently and stably formed by opening a hole of a specific volume in a cell support carrier. It has been found that the protective liquid can be retained. In addition, the present inventors have found that cells stored using the container not only have a high survival rate but also have good growth in germ cells.

  In one embodiment, the present invention is a method for preserving cells by vitrification, the step of retaining the cells in a serum-free storage solution having a volume of 50 nL or less, and the step of storing the cells in the presence of a freezing agent. Relates to a method comprising: In another aspect, the present invention is a method for preserving cells by vitrification, the step of retaining a serum-free preservative in a container for vitrification storage of cells having a volume of 50 nL or less, the storage The present invention relates to a method comprising the step of adding the cells to a liquid and the step of storing the cells in the presence of a freezing agent.

  The amount of the preservation solution used in the present invention is preferably 10 nL or less, more preferably 5 nL or less. Moreover, the quantity of a preservation | save liquid can be 0.5-50 nL, 1-10 nL, and 2-5 nL. Moreover, the preservation solution used in the present invention does not contain any serum-derived component. Therefore, the possibility of infection can be denied.

  The preservation solution used in the present invention may contain a cryoprotectant. Examples of cryoprotective agents include cell membrane-permeable cryoprotectants such as ethylene glycol, dimethyl sulfoxide, glycerol, propanediol, acetamide, propylene glycol, butanediol; and sucrose, trehalose, lactose, raffinose, percoll, Examples include cell membrane impermeable cryoprotectants such as Ficoll 70, Ficoll 70000, polyvinyl pyrrolidone, and polyethylene glycol. The concentration of the cryoprotectant in the preservation solution used in the present invention is desirably as low as possible for cell survival or growth, for example, 30% or less, preferably 15% or less, More preferably, it is 12% or less. The density | concentration of the cryoprotectant in a preservation | save liquid can be 1-30%, 5-15%, 9-12%, for example.

  The preservation solution used in the present invention may contain a cytoprotective substance. Examples of cytoprotective substances include hyaluronan, polyvinyl alcohol, polyvinyl pyrrolidone, fibronectin and the like. The concentration of the cytoprotective substance contained in the preservation solution used in the present invention is not particularly limited as long as the cell is protected and can survive or grow. For example, the concentration may be 0.005 to 1%. Preferably, it is 0.001 to 0.5%, more preferably 0.05 to 0.1%.

  The preservation solution used in the present invention is a nutrient source, pH adjustment and other purposes, for example, sodium chloride, potassium chloride, calcium chloride, potassium dihydrogen phosphate, sodium bicarbonate, magnesium sulfate heptahydrate, ethylenediamine. It may contain tetrasodium tetraacetate dihydrate, gentamicin sulfate, amino acid, alanyl-L-glutamine, lactate such as D-glucose DL-sodium lactate, pyruvate such as sodium pyruvate, and the like.

  The cells used in the present invention are not particularly limited as long as they can be vitrified, but are preferably eukaryotic cells, more preferably animal cells such as mammals and insects, and plant cells. Mammalian cells are preferred. Examples of cells include sperm, oocytes, amniotic mesenchymal cells, unfertilized egg cells, fertilized egg cells, embryonic cells, embryonic stem cells (ES cells), hematopoietic stem cells, mesenchymal stem cells, neural stem cells, cancer stem cells, Or undifferentiated cells such as induced pluripotent stem cells (iPS cells); and endometrial cells such as endometrial cells; fallopian tube epithelial cells; amniotic epithelial cells; bile duct epithelial cells and other epithelial cells; fibroblasts; Examples thereof include endothelial cells such as sinusoidal endothelial cells and vascular endothelial cells, and differentiated cells such as hepatocytes, preferably undifferentiated cells, more preferably sperm, oocytes, amnion mesenchymal cells, Fertilized egg cells, fertilized egg cells, embryonic cells, or germline undifferentiated cells such as embryonic stem cells (ES cells). The method used in the present invention not only has a high survival rate of these cells, but also can maintain the differentiation and development ability of undifferentiated cells.

The freezing agent used in the present invention is not particularly limited as long as it can cause cell vitrification, and is preferably a highly safe material. Examples of the freezing agent include liquid nitrogen, slush nitrogen, liquid helium, liquid propane, and ethane slush, and preferably liquid nitrogen or slush nitrogen. Slush nitrogen is nitrogen in which the liquid nitrogen temperature is lowered to −205 to −210 ° C., which is lower than normal pressure −196 ° C. by holding the liquid nitrogen under reduced pressure (Huang et al., Human Reproduction, Vol. 20). , No. 1, pp. 122-128 (2005)). When slush nitrogen is used as a freezing agent, vitrification storage can be performed by an apparatus such as Vit-Master ^™ (IMT, Nes Ziona, Israel).

  The cell vitrification storage container used in the present invention has a volume of 50 nL or less, preferably 0.5 to 50 nL, more preferably 1 to 10 nL or less, and still more preferably 2 to 5 nL or less. Through hole or one opening. “Through hole” means a hole penetrating one surface of the vitrification storage container. “One-sided opening” refers to a recess that opens on only one side of the cell vitrification storage container and does not open on the other. The volume of the through hole is a volume of a region surrounded by the side surface of the through hole and the top and bottom surfaces of the through hole parallel to the surface of the cell vitrification storage container. On the other hand, the volume of the opening is the volume of the region surrounded by the top surface of the one opening parallel to the surface of the cell vitrification storage container and the side and bottom surfaces of the one opening. The shape of the through hole or the one opening is a cylindrical shape, a rectangular parallelepiped or a cube whose cross-sectional area parallel to the opening surface is constant in the direction perpendicular to the opening surface, and a shape in which the cross-sectional area differs in the vertical direction. May be. The vitrification storage container for cells is formed of a known material such as resin, metal, ceramics (including glass and carbon). Furthermore, the form of the container for vitrification storage of cells can adopt a thin tubular form, a net-like form, etc., in addition to a plate formed with a through hole or one opening.

FIG. 1 is a perspective view showing an example of a cell vitrification storage container according to the present embodiment. FIG. 2 is an enlarged view of a region A surrounded by an alternate long and short dash line shown in FIG.

  Next, an embodiment of a method for vitrifying and storing cells of the present invention and a container for storing vitrification of cells will be described.

(1) Cell Vitrification Storage Container A suitable cell vitrification storage container used in this embodiment is a resin-made flat plate having a thickness of 30 to 300 μm, and a through-hole whose upper and lower opening surfaces are circular. 1 or more are formed. When two or more through holes are present, the through holes may have the same capacity or different capacities. The plate | board thickness of the said vitrification storage container becomes like this. Preferably it is 50-200 micrometers, More preferably, it is 80-150 micrometers. When the plate thickness is 100 μm and a cylindrical through hole is formed in the vitrification storage container, the volume of the vitrification storage container is a suitable size when the diameter of the through hole is designed in the range of 40 to 400 μm. It can be in the range of 0.5-50 nL. The through hole can be formed by various processing methods such as laser processing, processing using a machine tool, and water jet processing, but it is preferable to use laser processing that can form a fine hole most easily. For laser processing, known lasers such as gas lasers, solid state lasers, and semiconductor lasers can be used. Among them, carbon dioxide gas lasers (a type of gas laser) and excimer lasers (a type of gas laser) that are more suitable for processing. YAG laser (a kind of solid-state laser) can be preferably used.

  FIG. 1 is a perspective view showing an example of a cell vitrification storage container according to the present embodiment. FIG. 2 is an enlarged view of a region A surrounded by an alternate long and short dash line shown in FIG.

  The cell vitrification storage container 1 includes a long and thin flat plate 2 and a gripping portion 3 that is fixed to one end of the flat plate 2 and has a thickness T larger than the thickness t of the flat plate 2. The flat plate 2 has three through-holes 4 (both having a diameter D (μm)) having the same diameter at the tip portion in the length direction. The preferred thickness t and diameter D are 100 μm and 90-100 μm, respectively. The flat plate 2 is made of a colorless and transparent acrylic resin. When the flat plate 2 is made of a colorless and transparent material, a preservation solution containing cells can be more easily put into the through-hole 4 while looking at the microscope. However, the flat plate 2 is not limited to a colorless and transparent material, and may be made of a colored transparent, translucent, or opaque material. Moreover, the material of the vitrification storage container 1 of this invention will not be limited if it is a raw material safe with respect to a cell.

  The grip portion 3 may be made of any known material such as resin, metal, ceramics, or wood. Since the thickness T of the grip portion 3 is sufficiently larger than the thickness t of the flat plate, the cell vitrification storage container 1 in a state where the serum-free storage solution having cells is held in the through-holes 4 is flat. When mounted on the flat plate 2, the through-hole 4 of the flat plate 2 does not directly contact the flat base. That is, the preservation solution having cells remains in the through hole 4 and floats in the space. For this reason, it can prevent that the said preservation | save liquid comes out from the inside of the through-hole 4 carelessly.

(2) Cell adjustment method Cells to be vitrified can be appropriately adjusted by methods well known to those skilled in the art. For example, the target cell is an ovum or embryo (eg, germinal vesicle (GV), pronuclear stage embryo (PN), 2 cell stage embryo, 4 cell stage embryo, 8-16 cell stage embryo, BL (blastocyst)) In this case, it can be performed according to the method of Quinn et al. (Quin et al, Journal of Reproduction and Fertility, 66, 161-168, 1982). GV is collected directly from the ovary, PN, 2 cell stage embryo, 4 cell stage embryo, 8-16 cell stage embryo, and BL are oviducts at 14, 36, 48, 60 and 84 hours after HCG injection, respectively. Alternatively, the uterus can be adjusted by perfusion with a culture medium and collected.

(3) Vitrification preservation method of cell Vitrification preservation | save of a cell can be preserve | saved in a desired vitrification preservation solution after acclimatizing a cell to a vitrification preservation solution. For example, move the cells into 0.5-1 mL of the basic culture and let them acclimate by leaving them at room temperature for 5 minutes, and then move the cells into 0.5-1 mL of vitrification stock solution at room temperature. The cells for vitrification preservation can be prepared by allowing to stand for 3-15 minutes to equilibrate. A vitrification preservation solution can be accurately injected with a glass pipette into a hole of each size of a vitrification storage container, and the vitrification storage container can be immediately poured directly into liquid nitrogen for vitrification preservation. . In addition, since the vitrification preservation solution of the present invention has low toxicity to cells, it is possible to lengthen the time until the freezing solution is charged after acclimating the cells to the vitrification preservation solution, compared to the usual vitrification preservation method. it can. In addition, the acclimatization process may be performed in a shorter time.

  Hereinafter, examples will be shown to describe the present invention in more detail, but the present invention is not limited to these examples. It should be noted that all documents cited throughout this application are incorporated herein by reference in their entirety.

Example 1. Production of container for vitrification preservation A plastic plate (Cryotop, Kitasato Supply) with a thickness of 100 μm is irradiated with laser light, and the diameter is 150 μm (volume 1.8 nL), 200 μm (volume 3.1 nL), and 250 μm (volume 4). .9 nL) through-holes were formed.

Example 2 Effect of vitrification stock volume on cell viability at 15% concentration of protective agent To confirm the amount of preservation solution that can reduce the concentration of cytoprotective agent from 30% currently used to 15%, The relationship between the amount of the preservation solution and the cell viability when the preservation solution containing 15% of the protective agent was used was examined.
(1) Collection of mouse ovum and embryo Mouse ovum and embryo (germ follicle (GV), pronuclear stage embryo (PN), 2 cell stage embryo, 4 cell stage embryo, 8-16 cell stage embryo, BL (blastocyst) Cell)) was performed according to the method of Quin et al. (Quin et al, Journal of Reproduction and Fertility, 66, 161-168, 1982). GV is collected directly from the ovary, PN, 2 cell stage embryo, 4 cell stage embryo, 8-16 cell stage embryo, and BL are oviducts at 14, 36, 48, 60 and 84 hours after HCG injection, respectively. Alternatively, the uterus was collected by perfusion with a culture solution.

(2) Preparation of vitrification preservation solution (protective agent concentration 15%) According to the drying method (Dry, Journal of the Japan Society for Fertilization and Implantation, 25, 23-26, 2008) Street: sodium chloride, potassium dihydrogen phosphate, potassium chloride, calcium chloride, magnesium sulfate heptahydrate, 19 amino acids, sodium hydrogen carbonate, disodium ethylenediaminetetraacetate dihydrate, gentamicin sulfate, polyvinyl alcohol, Alanyl-L-glutamine, lactate such as D-glucose DL-sodium lactate as energy source, pyruvate such as sodium pyruvate) and 12 mM HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) and a medium containing 8 mM sodium HEPES). Ethylene glycol was added to the basic culture solution to a final concentration of 7.5%, and dimethyl sulfoxide (DMSO) was further added to a final concentration of 7.5%. Furthermore, sucrose, polyvinyl alcohol, ficoll and hyaluronan were added to final concentrations of 0.5M, 0.1%, 1% and 0.05%, respectively, and vitrification storage solution (protective agent concentration 15%) and did.

(3) Procedure for vitrification storage 0.5-1 mL of basic culture solution was placed in the first well of a 4-well IVF dish (Nunk, 176740 or 144444), and the mouse 2 cell stage embryo collected as described above was placed therein. Was moved with a glass pipette and allowed to acclimate by leaving it at room temperature for 5 minutes. Then, 0.5-1 mL of the vitrification preservation solution (protective agent concentration 15%) prepared as described above was placed in the second well of a 4-well dish (Nunk, 144444), and a mouse 2-cell embryo was placed in a glass pipette. And allowed to equilibrate for 3-15 minutes at room temperature. A vitrification storage solution (protective agent concentration 15%) is accurately injected into a hole of each size of the vitrification storage container prepared in Example 1 with a glass pipette, and the volume is 1.8 nL, 3.1 nL, and A 4.9 nL stock solution was prepared. The desired volumes were adjusted by injecting the storage solutions having a volume of 10 nL, 25 nL, 50 nL, 100 nL, 250 nL, 500 nL, and 1000 nL in the same manner with a glass pipette. Mouse 2 cell stage embryos after equilibration were removed with a glass pipette and moved one by one into a storage solution in a storage container under a microscope. After adding the mouse 2-cell stage embryo, the vitrification storage container was immediately put into liquid nitrogen for vitrification storage.

(4) Melting method and confirmation of survival rate Melting solution 1 (sucrose 0.7-1.0M, polyvinyl alcohol 0.1%, in the first well of a 4-well IVF dish (Nunk, 176740 or 144444) in advance, 0.5-1 mL of basic culture solution containing 0.05% hyaluronan) and second well containing lysate 2 (sucrose 0.35-0.5M, 0.1% polyvinyl alcohol, 0.05% hyaluronan) 0.5-1 mL of basic culture solution), and 0.5-1 mL of lysis solution 3 (basic culture solution containing only 0.1% polyvinyl alcohol and 0.05% hyaluronan) in the third and fourth wells Got ready. The melting method is that after storage in liquid nitrogen for 1-10 days, the vitrified mouse 2 cell stage embryo is directly immersed in the well containing the first melting solution 1 together with the storage container, and after 1 minute the glass Pipette to the second well of Melt 2 and equilibrate for 3 minutes. Thereafter, the cryoprotectant was removed stepwise without giving an osmotic shock by transferring to the wells of the third and fourth melts 3 at intervals of 3 minutes. The mouse 2-cell stage embryo after the thawing operation was collected and cultured in BM medium (dried, Journal of Japanese Society of Fertilization and Implantation, 25, 23-26, 2008) to confirm cell survival. For confirmation of cell survival, morphological observation was performed with an inverted microscope every 12 or 24 hours, photographs were taken as necessary, and viability was evaluated by morphological classification.

(5) Results Table 1 shows the results. As shown in the table, it was shown that when the cytoprotective agent was reduced to 15%, the viability of the cells was increased by reducing the volume of the vitrification preservation solution to 50 nL or less.

Example 3 FIG. Effect of cytoprotective agent concentration on cell viability From the above examples, it was shown that when the volume of the preservation solution was small, the viability was high even when the protective agent concentration was low. When the volume of the preservation solution was 5 nL or less, the influence of the concentration of the cytoprotective agent on the cell viability was examined.

(1) Collection of mouse ovum and embryo Mouse ovum and embryo (germ follicle (GV), pronuclear stage embryo (PN), 2 cell stage embryo, 4 cell stage embryo, 8-16 cell stage embryo, BL (blastocyst) Cell)) was performed according to the method of Quin et al. (Quin et al, Journal of Reproduction and Fertility, 66, 161-168, 1982). GV is collected directly from the ovary, PN, 2 cell stage embryo, 4 cell stage embryo, 8-16 cell stage embryo, and BL are oviducts at 14, 36, 48, 60 and 84 hours after HCG injection, respectively. Alternatively, the uterus was collected by perfusion with a culture solution.

(2) Preparation of vitrification preservation solution (protective agent concentration 30%, 15%, 12%, 9%, 6%) Drying method (Dry, Journal of Japanese Society of Fertilization and Implantation, 25, 23-26, 2008) ), Basic culture solution (basic components are as follows: sodium chloride, potassium dihydrogen phosphate, potassium chloride, calcium chloride, magnesium sulfate heptahydrate, 19 amino acids, sodium bicarbonate, ethylenediaminetetraacetate disodium Dihydrate, gentamicin sulfate, polyvinyl alcohol, alanyl-L-glutamine, lactate such as D-glucose DL-sodium lactate as energy source, pyruvate such as sodium pyruvate) (4- (2-hydroxyethyl) ) -1-piperazineethanesulfonic acid (HEPES) -containing medium). Ethylene glycol and dimethyl sulfoxide (DMSO) were added to the basic culture solution so that the final concentration was 15%, respectively, and a vitrification stock solution having a protective agent concentration of 30% was prepared. Similarly, ethylene glycol and dimethyl sulfoxide (DMSO) are added to a final concentration of 7.5%, 6%, 4.5%, or 3%, respectively, and the protective agent concentration is 15%, 12%. 9% or 6% vitrification stock solutions were prepared. Further, sucrose, polyvinyl alcohol, ficoll and hyaluronan were added to all vitrification preservatives so that the final concentrations were 0.5M, 0.1% and 1%, respectively, and vitrification preservatives (protective agent concentration 30) were added. %, 15%, 12%, 9%, 6%).

(3) Vitrification preservation and confirmation of melting method and survival rate Vitrification preservation and survival rate confirmation were performed according to the method of Example 1 above.

(4) Results Table 2 shows the results. As shown in the table, when stored in a vitrification preservation solution of 5 nL or less, a high survival rate of 90% or more is maintained even when the cytoprotective agent is reduced to 12%, and the cytoprotective agent is 6%. It was shown that the survival rate of 70% can be maintained even when the level is reduced to a low level.

Example 4 Effect of storage solution equilibration time during vitrification storage on survival rate From the above experimental results, by reducing the amount of vitrification storage solution to 5 mL or less, sufficient survival rate can be obtained even with 15% or less of cytoprotective agent. It was found. Therefore, next, the time required for equilibration of the cells in the preservation solution before vitrification preservation due to the decrease in the concentration of the cytoprotective agent was examined.

(1) Collection of mouse ovum and embryo Mouse ovum and embryo (blastocyst (GV), pronuclear stage embryo (PN), 2 cell stage embryo, 4 cell stage embryo, 8-16 cell stage embryo, BL (blastocyst) Cell)) was performed according to the method of Quinn et al. (Quinn et al, Journal of Reproduction and Fertility, 66, 161-168, 1982). GV is collected directly from the ovary, PN, 2 cell stage embryo, 4 cell stage embryo, 8-16 cell stage embryo, and BL are oviducts at 14, 36, 48, 60 and 84 hours after HCG injection, respectively. Alternatively, the uterus was collected by perfusion with a culture solution.

(2) Production of Vitrification Preservation Solution (Protective Agent Concentration 15%) The vitrification preservation solution was performed in the same manner as described in Example 2 above.

(3) Vitrification preservation and confirmation of melting method and survival rate Vitrification preservation and survival rate confirmation were performed according to the method of Example 1 above.

(4) Results Table 3 shows the results. As shown in the table, when the cytoprotective agent was reduced to 15%, a high survival rate was maintained even when the equilibration time was 1 minute. This shows that equilibration time is hardly required if the cytoprotective agent is 15%.

Embodiment 5 FIG. Preliminary test using human waste (development-stopped) egg Use this low-toxicity and complete serum-free vitrification storage system (15% cryoprotectant concentration) to test human waste with informed consent (development-stop) Eggs were stored vitrified and their viability was evaluated.

(1) Preparation of human discarded (development-stopped) ovum The development-stopped ovum determined to be discarded after in vitro fertilization treatment was obtained from the owner's patient for informed consent, and then subjected to the test.

(2) Preparation of Vitrification Preservation Solution (Protective Agent Concentration 15%) and Preservation of Vitrification Preparation of vitrification storage solution and vitrification preservation were performed in the same manner as described in Example 2 above.

(4) Melting method and confirmation of survival rate Melting solution 1 (sucrose 0.7-1.0M, polyvinyl alcohol 0.1%) in the first well of a 4-well IVF dish (Nunk, 176740 or 144444) in advance 0.5-1 mL of basic culture solution containing 0.05% hyaluronan) and 2nd well containing melt 2 (sucrose 0.35-0.5M, 0.1% polyvinyl alcohol, 0.05% hyaluronan) 0.5-1 mL of the basic culture solution) and 0.5-1 mL of the melting solution 3 (basic culture solution containing only 0.1% polyvinyl alcohol and 0.05% hyaluronan) in the third and fourth wells Got ready. The melting method is that after storage in liquid nitrogen for 1-10 days, the vitrified mouse 2 cell stage embryo is directly immersed in the well containing the first melting solution 1 together with the storage container, and after 1 minute the glass Pipette to the second well of Melt 2 and equilibrate for 3 minutes. Thereafter, the cryoprotectant was removed stepwise without giving an osmotic shock by transferring to the wells of the third and fourth melts 3 at intervals of 3 minutes. Eggs that had been thawed were collected and cultured in BM medium (dried, Journal of the Japan Fertilization and Implantation Society, 25, 23-26, 2008), and their survival was confirmed by morphological evaluation.

(5) Results Table 4 shows the results. As shown in the table, even when the concentration of the cytoprotective agent was reduced to 15%, the survival of 91.7% (11/12) was confirmed, indicating that application to human eggs is possible.

Example 6 Survival rate of vitrification storage of various cells The difference in the viability of various cells when using a slow cryopreservation method, a conventional vitrification storage method, and a low volume vitrification storage method was measured.

(1) Preparation of cells (1-1) Human endometrial cells Human endometrial cells were prepared according to the method of Mizuno et al. (12th International Academy of Human Reproduction, Venice, Italy, 502-505 (2005)) The culture solution was adjusted with MEMα (Gibco, 12571-063) + 5% umbilical cord serum, seeded in a plastic flask (IWAKI, Tissue Culture Flask, 3113-025) and cultured. Cells that reached confluence by culturing for 2-3 days were detached with trypsin solution (Sigma, T3924), adjusted to a concentration of 1-8 × 10 ⁶ cells / ml, and used for vitrification storage.
(1-2) Human fallopian tube epithelial cells Human fallopian tube epithelial cells were prepared according to the method of Mizuno et al. (12th International Academy of Human Reproduction, Venice, Italy, 502-505 (2005)), and the culture solution was MEMα (Gibco , 12571-063) + 5% umbilical cord serum, seeded in plastic flasks (IWAKI, Tissue Culture Flask, 3113-025) and cultured. Cells that reached confluence by culturing for 2-3 days were detached with trypsin solution (Sigma, T3924), adjusted to a concentration of 1-8 × 10 ⁶ cells / ml, and used for vitrification storage.
(1-3) Human amniotic cells Human amniotic cells were prepared according to the method of Mizuno et al. (12th International Academy of Human Reproduction, Venice, Italy, 502-505 (2005)), and the culture solution was MEMα (Gibco, 12571-063). ) Adjusted with + 5% umbilical cord serum, seeded in a plastic flask (IWAKI, Tissue Culture Flask, 3113-025) and cultured. Cells that reached confluence by culturing for 2-3 days were detached with trypsin solution (Sigma, T3924), adjusted to a concentration of 1-8 × 10 ⁶ cells / ml, and used for vitrification storage.
(1-4) Normal human dermal fibroblasts As normal human dermal fibroblasts, a commercially available cell line NHDF-Ad (Takara Corporation, CC-2511) was used. The culture solution was adjusted with MEMα (Gibco, 12571-063) + 5% umbilical cord serum, seeded in a plastic flask (IWAKI, Tissue Culture Flask, 3113-025) and cultured. Cells that reached confluence by culturing for 2-3 days were detached with trypsin solution (Sigma, T3924), adjusted to a concentration of 1-8 × 10 ⁶ cells / ml, and used for vitrification storage.
(1-5) Buffered Lart Liver-Derived Cell As a buffered Lartert liver-derived cell, a commercially available cell line (buffalo rat liver cells, BRL3A) was used. The culture solution was adjusted with MEMα (Gibco, 12571-063) + 5% umbilical cord serum, seeded in a plastic flask (IWAKI, Tissue Culture Flask, 3113-025) and cultured. Cells that reached confluence by culturing for 2-3 days were detached with trypsin (Sigma, T3924), adjusted to a concentration of 1-8 × 10 ⁶ cells / ml, and used for vitrification storage.
(1-6) Mouse ovary cell The mouse | mouth ovary cell used the tissue piece which shredded the ovarian tissue extract | collected from the ICR type | system | group female mouse | mouth (Clea Japan) of 6-10 weeks into 0.1 mm square.
(1-7) Human sperm Human sperm was tested using discarded sperm from which informed consent was obtained after semen examination.

(2) Preparation of preservation solution (2-1) Vitrification preservation solution (protective agent concentration 30%, 15%, 12%, 9%, 6%)
Vitrification stock solutions (protectant concentration 30%, 15%, 12%, 9%, 6%) were performed in the same manner as in Example 3 above.
(2-2) As a stock solution for slow cryopreservation, PBS (phosphate buffer solution, Gibco, 14190) + 20% umbilical cord serum supplemented with 10% DMSO (dimethyl sulfoxide, Sigma, D2438) as a cryoprotectant according to a conventional method is used. Using.

(3) Storage (3-1) Procedure of vitrification preservation Vitrification preservation was performed according to the method of Example 1 above.
(3-2) Slow cryopreservation was performed using PBS (phosphate buffer, Gibco, P14190) + 20% umbilical cord serum supplemented with 10% DMSO (dimethyl sulfoxide, Sigma, D2438) according to a conventional method. Each cell is filled into a cryotube (Nalgen, Vryogenic Vial, 5000-1020) with 50-300 microliters of cryopreservation solution, held at a height of about 2 cm from the liquid nitrogen surface for 30 minutes, slowly cooled, and 30 minutes Later, it was immersed in liquid nitrogen for storage.

(4) Confirmation of melting method and survival rate According to the method of Example 1, the survival rate was confirmed.

(5) Results The results are shown in Tables 5 to 12. Table 5 shows human endometrial cells, Table 6 shows human fallopian tube epithelial cells, Table 7 shows human amniotic cells, Table 8 shows normal human dermal fibroblasts, Table 9 shows cells derived from buffalo rat liver, Table 10 shows mouse ovary cells, Table 11 shows mouse testis cells, and Table 12 shows human sperm results.
As shown in the table, it was shown that the low-volume vitrification storage method maintained the same or better survival rate than the conventional method in various types of cells. In particular, when the concentration of the cytoprotective agent was 15%, the survival rate exceeding the conventional method was obtained for most cells. In particular, in ovarian tissues and sperm germline cells, the survival rate surpassed that of conventional methods when using any concentration of cytoprotective agent. It has been shown to be effective against cell lines.

Example 7 Survival rate of cells with different developmental stages To confirm the survival rate of mouse ova with different developmental stages, immature ovum, pronuclear ovum, 2 cell stage, 4 cell stage, 8-16 cell stage, blastocyst stage The survival rate after low-volume vitrification storage was examined using cells.

(1) Collection of mouse ovum and embryo Mouse ovum and embryo (blastocyst (GV), pronuclear stage embryo (PN), 2 cell stage embryo, 4 cell stage embryo, 8-16 cell stage embryo, BL (blastocyst) Cell)) was performed according to the method of Quinn et al. (Quinn et al, Journal of Reproduction and Fertility, 66, 161-168, 1982). GV is collected directly from the ovary, PN, 2 cell stage embryo, 4 cell stage embryo, 8-16 cell stage embryo, and BL are oviducts at 14, 36, 48, 60 and 84 hours after HCG injection, respectively. Alternatively, the uterus was collected by perfusion with a culture solution.

(2) Preparation of vitrification preservation solution (protective agent concentration 30%, 15%, 12%, 9%, 6%) Drying method (Dry, Journal of Japanese Society of Fertilization and Implantation, 25, 23-26, 2008) ), Basic culture solution (basic components are as follows: sodium chloride, potassium dihydrogen phosphate, potassium chloride, calcium chloride, magnesium sulfate heptahydrate, 19 amino acids, sodium bicarbonate, ethylenediaminetetraacetate disodium Dihydrate, gentamicin sulfate, polyvinyl alcohol, alanyl-L-glutamine, lactate such as D-glucose DL-sodium lactate as an energy source, pyruvate such as sodium pyruvate) and 12 mM HEPES (4- ( Contains 2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES) and 8 mM sodium HEPES To prepare the ground). Ethylene glycol was added to the basic culture solution to a final concentration of 7.5%, and dimethyl sulfoxide (DMSO) was further added to a final concentration of 7.5%. Furthermore, sucrose, polyvinyl alcohol, ficoll and hyaluronan were added to final concentrations of 0.5M, 0.1%, 1% and 0.05%, respectively, and vitrification storage solution (protective agent concentration 15%) and did.

(3) Vitrification preservation and confirmation of melting method and survival rate Vitrification preservation and survival rate confirmation were performed according to the method of Example 1 above.

(4) Results Tables 13 and 14 show the results. As shown in Table 13, it was shown that a high survival rate can be maintained even if the cryoprotectant (CPA) concentration is decreased at each development stage. Further, as shown in Table 14, it was shown that a high incidence can be maintained even when the cryoprotectant (CPA) concentration is lowered.

Example 8 FIG. Viability and normal spindle formation rate of vitrification storage using slush nitrogen Low-volume vitrification using slush nitrogen to confirm whether low-capacity vitrification storage can be performed using slush nitrogen The survival rate of storage and normal spindle formation were investigated.

(1) Collection of mouse ovum and embryo Mouse ovum and embryo (blastocyst (GV), pronuclear stage embryo (PN), 2 cell stage embryo, 4 cell stage embryo, 8-16 cell stage embryo, BL (blastocyst) Cell)) was performed according to the method of Quinn et al. (Quinn et al, Journal of Reproduction and Fertility, 66, 161-168, 1982). GV is collected directly from the ovary, PN, 2 cell stage embryo, 4 cell stage embryo, 8-16 cell stage embryo, and BL are oviducts at 14, 36, 48, 60 and 84 hours after HCG injection, respectively. Alternatively, the uterus was collected by perfusion with a culture solution.

(2) Preparation of vitrification preservation solution (protective agent concentration 30%, 15%, 12%) Vitrification preservation solution (protective agent concentration 15%, 12%) is the same as the method described in Example 2 and Example 3 above. It produced by the same method. Also, a vitrification stock solution (30%) was prepared in the same manner as described in Example 3 above for use as a control.
(3) Vitrification preservation and confirmation of melting method and survival rate Vitrification preservation and survival rate confirmation were performed according to the method of Example 1 above.
(4) Confirmation of spindle formation About each cell, it was confirmed with the observation apparatus (MTG company) for confirming a spindle whether the normal spindle was formed.

(5) Results The results are shown in Table 15. As shown in the table, it was shown that the low-capacity vitrification storage method can maintain the same survival rate as the conventional method even when slush nitrogen is used, despite the low concentration of the cytoprotective agent. Moreover, it was shown that a high normal spindle rate can be achieved by combining the low-capacity vitrification preservation method and slush nitrogen. From this, the low-capacity vitrification preservation method makes it possible to form normal spindles that could not be achieved so far by using slush nitrogen. It is thought that it can be used for undifferentiated cells that are indispensable for germ cells, particularly germline cells and fertilized eggs.

Example 9 Production of pups by embryo transfer of vitrified eggs In order to check whether eggs stored with low volume vitrification grow normally after embryo transfer, the vitrified cells are transplanted into recipients and delivered to pups. Observed growth.

(1) Preparation of vitrified and preserved embryos Vitrified and preserved embryos are prepared according to the methods described in Examples 2 and 3 using a vitrification preservation solution (30%, 15%, 12%). The storage (volume is 4.9 nL) was used.
(2) Transplantation Vitrified and preserved embryos were thawed according to the method described in Example 2 and continuously cultured. Of the cells using vitrification solution (15% cytoprotective agent), 30 cells that had grown to blastocysts were transplanted into 3 recipients. Similarly, 28 cells that had grown to blastocysts among cells using vitrification preservation solution (12% cytoprotective agent) were transplanted into 3 recipients. As a control, 40 cells that developed to blastocysts among cells using vitrification preservation solution (30% cytoprotective agent) were transplanted into 3 recipients.

(3) Results Twenty normal fetuses were obtained from 30 cells using vitrification preservation solution (15% cytoprotective agent), and 66% embryos could be developed to fetuses. Twenty normal embryos were obtained from 28 embryos using vitrification medium (12% cytoprotective agent), and 71% embryos could be developed to fetuses. On the other hand, 21 normal fetuses were obtained from 40 cells using vitrification preservation solution (30% cytoprotective agent), and 53% embryos could be developed to fetuses. Therefore, it was shown that the probability of growth of the preserved egg can be increased by reducing the concentration of the cytoprotective agent during low-volume vitrification preservation.

  The vitrification preservation method of the present invention can be used widely in the field of cell preservation because it can preserve cells with low volume and high viability. In particular, the vitrification preservation method of the present invention can be preserved in undifferentiated cells such as germ line cells and egg cells while maintaining its normal spindle formation ability and differentiation / development ability. It can be used in the field of seed preservation and breeding.

Claims (8)

  A method for preserving cells by vitrification, comprising a step of retaining the cells in a serum-free storage solution having a volume of 50 nL or less, and a step of storing the cells in the presence of a freezing agent.   The method according to claim 1, wherein the preservation solution contains a cryoprotectant having a concentration of 15% or less.   Cells are sperm, oocyte, endometrial cell, fallopian epithelial cell, amniotic cell, unfertilized egg cell, fertilized egg cell, embryonic cell, embryonic stem cell (ES cell), hematopoietic stem cell, mesenchymal stem cell, neural stem cell The method according to claim 1, wherein the method is a cancer stem cell or an induced pluripotent stem cell (iPS cell).   A method for preserving cells by vitrification, the step of retaining a serum-free preservative in a container for vitrification of cells having a volume of 50 nL or less, the step of adding the cells to the preservative, the cells A method comprising the step of preserving in the presence of a cryogen.   Cells are sperm, oocyte, endometrial cell, fallopian epithelial cell, amniotic cell, unfertilized egg cell, fertilized egg cell, embryonic cell, embryonic stem cell (ES cell), hematopoietic stem cell, mesenchymal stem cell, neural stem cell The method according to claim 4, wherein the method is a cancer stem cell or an induced pluripotent stem cell (iPS cell), and the preservation solution contains a cryoprotectant having a concentration of 15% or less. .   The method according to claim 1, wherein the cells are mammalian cells other than germ cells, and the preservation solution contains a cryoprotectant at a concentration of 15% or less.   The method according to claim 6, characterized in that the cells are intimal cells, epithelial cells, fibroblasts, liver cells.   A container for storing vitrification of cells, comprising a hole having a volume of 50 nL or less.

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