JP7520398B2 - Cell cryopreservation solution and cell freezing method - Google Patents

Description

The present invention relates to a cell cryopreservation solution and a cell freezing method.

In recent years, cell freezing technology has been attracting attention, in which cellular tissues and pluripotent cells from various organs are frozen and stored, and then thawed for use at the time of treatment such as transplantation. A typical method for freezing cellular tissues involves, for example, preparing a cell suspension by adding a cryopreservation solution, which is physiological saline with various additives, to the cellular tissues, placing a container (such as a cryotube) containing the cell suspension in a cell freezing container filled with 2-propanol, and storing the cell freezing container in the freezer of a freezing device.

More specifically, this is done by using a programmable freezer or the like to cool the cells to a predetermined temperature (e.g., -80°C) at a predetermined cooling rate (e.g., 1°C/min, etc.). Alternatively, the cell freezing container is kept in a freezer set to a predetermined temperature (e.g., -80°C) for a predetermined time (e.g., 24 hours). The container containing the frozen cell suspension is then immersed in liquid nitrogen to preserve the frozen cells.

The following non-patent documents 1 and 2 describe techniques for cryopreserving iPS cells. The following non-patent document 3 describes a method for cryopreserving cellular tissues such as iPS cells and an overview of cryopreservation solutions for cellular tissues. The following patent documents 1 and 4 describe a composition for cell cryopreservation, which is a cell cryopreservation solution with low cytotoxicity.

In addition, the following Patent Documents 2 and 3 describe cell cryopreservation solutions containing sugars, and the following Non-Patent Document 5 describes, in relation to the present invention, the cryoprotection effects of various sugars when cryopreserving boar sperm.

JP 2006-115837 A JP 2022-79113 A JP 2022-104240 A

Kyoto University, "Establishment of an effective cryopreservation method for human iPS cells", [online], [searched on September 15, 2022], Internet <URL: http://www.kyoto-u.ac.jp/static/ja/news_data/h/h1/news6/2010/101201_3.htm> The Society for Biotechnology, Japan, "Cryopreservation of Primate ES/iPS Cells", [online], [searched September 15, 2022], Internet <URL: https://www.sbj.or.jp/wp-content/uploads/file/sbj/9009/9009_tokushu-1_3.pdf> Zenogen Pharma Co., Ltd., "STEM-CELLBANKER GMP grade product guide ("STEM-CELLBANKER" is a registered trademark)", [online], [searched on September 15, 2022], Internet <URL: https://www.zenogenpharma.com/stem-cellbanker.html> Nacalai Tesque, Inc., "Cell Reservoir One", [online], [searched on September 15, 2022], Internet <URL: https://www.nacalai.co.jp/products/entry/d001007.html> Japan Swine Society, "Academic Journal Vol. 30, No. 1: "Effect of fructooligosaccharides on survival of frozen pig spermatozoa and maintenance of morphology of the cap", [online], [searched on September 13, 2022], Internet <URL: https://www.jstage.jst.go.jp/article/youton1987/30/1/30_1_11/_pdf/-char/ja>

As described in Non-Patent Document 3, typical cell cryopreservation solutions contain dimethyl sulfoxide (DMSO) as an additive. However, DMSO is known to be cytotoxic. Therefore, in order to transition cell cryopreservation technology for medical use, which is currently at the experimental and clinical levels, to a practical level in the future, a higher level of safety is required for cell cryopreservation solutions. Of course, in addition to safety, cell cryopreservation solutions are also required to have a high cell viability when thawed.

The cell cryopreservation solutions described in Patent Document 1 and Non-Patent Document 4 above contain additives mainly consisting of the silk protein sericin, and furthermore, these documents also disclose solutions that do not contain DSMO. However, since sericin is made from silk protein derived from cocoons and is obtained by extracting and purifying it from wastewater generated in the smelting process of the silk-making process, it is difficult to produce it at a low cost. In addition, sources from which the raw material can be obtained are also limited.

In order to bring cell freezing technology for medical use to a practical level, it is necessary to obtain a cell freezing medium that uses safe, inexpensive, and easily available raw materials while also being expected to improve cell survival rates.

The present invention aims to provide a cell cryopreservation solution that is safe, inexpensive, and made from easily available raw materials, and a cell freezing method that uses this cell cryopreservation solution.

In one aspect of the present invention to achieve the above object, a solution containing D-PBS, which is a physiological saline solution, as a solvent, a main additive and a sub-additive,
The main additive is fructooligosaccharide,
The secondary additive is ovalbumin,
The auxiliary additive is added to a fructooligosaccharide solution in which the fructooligosaccharide is contained in the physiological saline at a ratio of 10 wt % to 30 wt %,
The ovalbumin is contained in an amount of 0.5 vol% or more and 1.0 vol% or less.
It is a cell cryopreservation medium.

The solvent is D-PBS, which is a physiological saline solution, and the solution contains a main additive and a sub-additive.
The main additive is fructooligosaccharide,
The secondary additive is chondroitin sulfate,
The auxiliary additive is added to a fructooligosaccharide solution in which the fructooligosaccharide is contained in the physiological saline at a ratio of 10 wt % to 30 wt %,
The chondroitin sulfate is contained in an amount of 3.0 vol% or more and 10.0 vol% or less.
It may also be used as a cell cryopreservation solution.

The solvent is D-PBS, which is a physiological saline solution, and the solution contains a main additive and a sub-additive.
The main additive is fructooligosaccharide,
The secondary additive is a collagen peptide,
The auxiliary additive is added to a fructooligosaccharide solution in which the fructooligosaccharide is contained in the physiological saline at a ratio of 10 wt % to 30 wt %,
The collagen peptide is contained in an amount of 5.0 vol% or more and 10.0 vol% or less.
It can also be used as a cell cryopreservation medium.

An embodiment of the present invention also includes a method for freezing cells using the cell cryopreservation solution, the method comprising the steps of:
A sample preparation step of preparing a cell suspension by adding the cell cryopreservation solution to cells to be frozen as a sample;
A sample placement step of placing a first container containing the sample in a second container filled with 2-propanol;
a freezing step of placing the second container in a freezer to freeze the sample;
Including,
In the freezing step, the inside of the freezer in which the sample is placed is gradually cooled to a temperature at which the sample is in an overcooled state, and the temperature is maintained for a predetermined period of time.
This is used as a cell freezing method.

The present invention provides a cell cryopreservation solution that is safe, inexpensive, and made from easily available raw materials, and a cell freezing method that uses the cell cryopreservation solution. Other effects will be described below.

This figure shows the cell viability when a sample obtained by adding a cell freezing preservation solution consisting of physiological saline (D-PBS) to which fructooligosaccharides has been added to HEK293 cells (test cells) was frozen and thawed using a new freezing method. FIG. 1 shows the cell viability when a sample obtained by adding a cell cryopreservation solution, which is a physiological saline solution (D-PBS) to which fructooligosaccharides has been added, to HEK293 cells (test cells) was frozen and thawed by a slow freezing method. This figure shows the cell viability when a sample obtained by adding a cell freezing preservation solution consisting of the D-PBS to which the fructooligosaccharide and ovalbumin have been added to the test cells is frozen and thawed. This figure shows the cell survival rate when a sample obtained by adding a cell freezing preservation solution consisting of the D-PBS to which the fructooligosaccharide and chondroitin sulfate have been added to the test cells is frozen and thawed. This figure shows the cell survival rate when a sample obtained by adding a cell freezing preservation solution consisting of the D-PBS to which the fructooligosaccharide and collagen peptide have been added to the test cells is frozen and thawed.

An embodiment of the present invention will be described below with reference to the accompanying drawings.

==== Working Example ====
<Cell cryopreservation solution containing sugar>

As mentioned above, in order to put cell cryopreservation technology for medical use into practical use, a cell cryopreservation solution is required to be highly safe, low-cost, and easy to obtain raw materials. Of course, it is also necessary to have a high cell viability. Examples of cell cryopreservation solutions that meet such requirements include the cell cryopreservation solution containing trehalose and ovalbumin described in Patent Document 2 above, and the cell cryopreservation solution containing trehalose and chondroitin sulfate described in Patent Document 3. The cell cryopreservation solutions described in Patent Documents 2 and 3 contain additives derived from food, are safe, and achieve a high cell viability. Patent Documents 2 and 3 also state that an even higher cell viability can be achieved by freezing cells while applying a magnetic field using the cell cryopreservation solutions described in these documents.

The inventors therefore conducted extensive research to develop a new cell cryopreservation solution that, while making safety their number one priority, contains food-derived substances that are inexpensive and easy to obtain as raw materials, and that can provide a cell survival rate that is equal to or higher than that of the cell cryopreservation solutions described in Patent Documents 2 and 3. As a result, they came up with the present invention.

The cell cryopreservation solution according to the embodiment of the present invention contains fructooligosaccharide, a sugar, as the main additive in a solvent made of physiological saline, and also contains one of ovalbumin, chondroitin sulfate, and collagen peptide as a secondary component of the additive (hereinafter, secondary additive).

<Sample preparation procedure>
In order to evaluate the characteristics of the cell cryopreservation solution according to the embodiment, cell suspension samples were prepared using various cryopreservation solutions with different types of main and sub-additives, and the samples were frozen and the cell survival rate after thawing was examined. The cell tissue to be frozen was HEK293 cells obtained by culturing a human fetal kidney cell line (HEK293A). The sample preparation procedure and evaluation procedure are exemplified below.

First, human fetal kidney cell line (HEK293A) is cultured in a basic medium DMEM (Dulbecco's Modified Eagle's Medium) to which fetal bovine serum (FBS) and penicillin streptomycin are added at 10 vol% and 1 vol%, respectively, under an environment of 37°C and 5% _CO2 concentration to obtain HEK293 cells (hereinafter sometimes referred to as "test cells"). Next, a cell suspension obtained by adding a cell cryopreservation solution to the test cells is used as a sample. The cell concentration in the cell suspension is 1 x ¹⁰⁷ (cells/ml). The cell cryopreservation solution used for the sample is mainly composed of D-PBS (Dulbecco's phosphate buffered saline) with sugars added as the main additive.

<Cell freezing test procedure>
The sample prepared by the above-mentioned procedure is frozen by the following procedure: First, 500 μl of the sample is placed in a cryotube (hereinafter sometimes referred to as a "tube"), which is a first container, and then the tube containing the sample is placed in a cryopreservation container (hereinafter sometimes referred to as a "container"), which is a second container filled with 2-propanol, and the sample is frozen in the cryopreservation container (hereinafter sometimes referred to as a "container") by the freezing method described below.

The samples were thawed by holding the cryopreservation container containing the tube containing the frozen sample in a thermostatic bath at 37°C for 1 minute. A freeze-thaw test was then performed on each sample, in which the sample was frozen and thawed according to the procedure described above. The number of surviving cells in the thawed sample was counted, and the cell viability was calculated based on the number of cells before freezing and the number of surviving cells after thawing. The number of surviving cells was measured by performing automatic analysis using a float cytometer to determine whether the test cells in the sample stained with photoreceptor dye (PI) were alive or dead.

Because samples are prepared each time a test is performed, the test results, such as cell viability, may differ for each sample shown below, even if the preparation conditions and test methods are the same, depending on the test occasion. In addition, the above samples used HEK293 cells as test cells, but the cell cryopreservation solution in the examples can also be applied to other cells.

<Freezing method>
A common method for freezing a cell suspension is to place a sample in the freezer of a programmable freezer and to freeze the sample by cooling the inside of the freezer from room temperature to an extremely low temperature of about -80°C at a predetermined temperature drop rate (for example, -1°C/min) (hereinafter referred to as the slow freezing method). Note that there is also a freezing method in which the sample is placed in a freezer set at a temperature of -80°C, but in recent years, the slow freezing method has become mainstream.

The cell cryopreservation solution according to the embodiment differs from the one containing DMSO that penetrates into cells in that it contains fructooligosaccharide, a sugar, as the main additive. As is well known, sugars are not toxic, but are not lipid-soluble, so if samples are frozen using the general freezing methods described above, there is a possibility that the samples will not be evaluated appropriately.

Specifically, when freezing cells, intracellular ice crystal formation has a greater impact on cell viability than extracellular ice crystal formation. Therefore, when freezing samples using a cell cryopreservation solution containing DMSO, it is important to use standard freezing methods to thoroughly penetrate the cells with DMSO, a cryoprotectant, to replace the free water that is the source of intracellular ice with the cryoprotectant, and to prevent damage to intracellular organelles (organizers) caused by ice crystal formation.

The cell cryopreservation solution according to the embodiment uses sugars with large molecular weights as additives, which are difficult to penetrate into cells. Therefore, it is possible to freeze the sample using a method (hereinafter, for convenience, referred to as the "novel freezing method") in which the sample is kept at a temperature (e.g., -30°C) that is lower than the melting point and is cooled to a supercooled state without solidifying, so that the free water in the cell is dehydrated as much as possible or the osmotic pressure in the cell is maintained to a certain extent. In order to properly evaluate the performance of the cell cryopreservation solution according to the embodiment, it is necessary to freeze the sample using an appropriate method that takes into account the physical properties of the additives, so that performance comparisons can be made between different additives.

First, to confirm which freezing method is most effective in improving survival rates, a freeze-thaw test was conducted using the slow freezing method and the new freezing method described above when freezing samples. In the freeze-thaw test, various fructooligosaccharide solutions containing only fructooligosaccharides at a ratio of 10 wt% to 30 wt% in D-PBS were used as cell cryopreservation solutions.

The freezing procedure using the new freezing method involved using a programmed freezer to cool the inside of the freezer to -30°C at a temperature drop rate of -2°C/min and maintaining the temperature at -30°C for 10 minutes to freeze the samples. The freezing procedure using the slow freezing method involved using a programmed freezer to cool the inside of the freezer to -80°C at a temperature drop rate of -1°C/min to freeze the samples. After freezing the samples using the new freezing method or the slow freezing method in this way, the samples were transferred to a -80°C freezer and stored for one week. The stored samples were then thawed using the method described above, and the cell viability was examined.

Figures 1 and 2 show the cell viability after thawing when samples were frozen using a cell cryopreservation solution containing fructooligosaccharides in D-PBS, which is the solvent, using the new freezing method and the slow freezing method. Figure 1 also shows the cell viability of a sample using a cell cryopreservation solution containing trehalose at a concentration of 200 mM in D-PBS as a comparative example. Note that the units of sugar concentration are different (wt% and mM) between the cell cryopreservation solution containing fructooligosaccharides and the cell cryopreservation solution containing trehalose. This is because the powdered fructooligosaccharides (e.g., Fujifilm Wako Pure Chemical Industries, Ltd.) provided by the manufacturer as a sample contain fructooligosaccharides with different molecular weights, and cannot be quantified in M (molar concentration) based on molecular weight. On the other hand, the molecular weight of trehalose is constant, so the concentration is specified in mM.

As shown in Figure 1, when samples using a cell cryopreservation solution containing fructooligosaccharides as a sugar were frozen using the new freezing method, the samples showed higher cell viability than samples using a cell cryopreservation solution containing trehalose when the proportion of fructooligosaccharides in D-PBS was in the range of 10 wt% to 30 wt%. Furthermore, samples using a cell cryopreservation solution containing 15 wt% fructooligosaccharides showed an extremely high cell viability of 84.1%.

On the other hand, in the samples frozen by the slow freezing method, as shown in Figure 2, the maximum cell viability was 65.2% when the ratio of fructooligosaccharides in D-PBS was 20 wt%. From the above, when using the cell cryopreservation solution according to the embodiment in which fructooligosaccharides is the main additive, the cell viability can be increased more by freezing the samples by the new freezing method. And, for each sample shown below, unless otherwise stated, the samples were frozen by the new freezing method according to the above procedure, and the container containing the tube containing the frozen sample was stored in a -80°C freezer for one week, and then the cryopreservation container was thawed by keeping it in a 37°C incubator for one minute.

<Additive Sub-components>
As shown in Figure 1, it was found that a cell survival rate of at least 70% was obtained by using a cell freezing preservation solution that does not contain any cytotoxic additives and contains fructooligosaccharide, a sugar derived from food, as an additive, and adopting a new freezing method. Next, in order to further improve the cell survival rate, samples were prepared using a cell freezing preservation solution that contains fructooligosaccharide as the main additive and any of ovalbumin (OVA), chondroitin sulfate, and collagen peptide as a secondary additive. Then, as described above, the samples were frozen using the new freezing method and stored at -80°C for one week, and then a freezing and thawing test was performed in which the samples were thawed using the same procedure as above, and the cell survival rate of the samples was examined.

The cell cryopreservation solution containing the auxiliary additives was produced by preparing a fructooligosaccharide solution containing fructooligosaccharides at a specified ratio in D-PBS, and then adding various auxiliary additives to the prepared fructooligosaccharide solution. The content (vol%) of the auxiliary additives in the cell cryopreservation solution was calculated based on a specified volume (volume) of fructooligosaccharide solution and the volume of the cell cryopreservation solution obtained by adding the auxiliary additives to the specified volume of fructooligosaccharide.

OVA and chondroitin sulfate are additives described in the above Patent Documents 2 and 3, respectively, and both are food-derived additives with high safety. As is well known, collagen peptide is a low-molecular-weight water-soluble protein obtained by hydrolyzing gelatin with enzymes or the like. Gelatin can be extracted by applying heat to collagen, an insoluble protein that is abundant in living organisms such as fish, cows, and pigs. Collagen peptide is widely used as a raw material for supplements and the like, and is highly safe, inexpensive, and easily available.

Figure 3 shows the cell viability after a freeze-thaw test for samples prepared using a cell cryopreservation solution containing 15 wt% fructooligosaccharides as the main additive in D-PBS, and 0.1 vol% to 10.0 vol% OVA as a secondary additive. Similarly, Figures 4 and 5 show the cell viability after a freeze-thaw test for samples prepared using various cell cryopreservation solutions containing 0.1 vol% to 10.0 vol% chondroitin sulfate and collagen peptide as secondary additives in the above fructooligosaccharide solution.

As shown in Figures 3 to 5, even if the ratio of fructooligosaccharides in the fructooligosaccharide solution is the same, the content of the secondary additive that increases the cell viability also differs depending on the type of secondary additive. When the secondary additive is OVA, as shown in Figure 3, a cell viability of 65% or more was observed when the content in the fructooligosaccharide solution was in the range of 0.1 vol% to 10.0 vol%, and a high cell viability of 90% or more was observed when the content was in the range of 0.5 vol% to 1.0 vol%, demonstrating the effect of the secondary additive in improving cell viability.

When the secondary additive was chondroitin sulfate, as shown in Figure 4, the cell viability was 76% or more when the content in the fructooligosaccharide solution was in the above-mentioned range, and at 1.0 vol% or more, the cell viability was stable at 80% or more. At 3.0 vol% or more, the cell viability was high at 85% or more, which is higher than the cell viability when a cell cryopreservation solution containing only fructooligosaccharide solution was used.

When the secondary additive was collagen peptide, as shown in Figure 5, the cell viability was consistently 80% or more within the above-mentioned range of content in the fructooligosaccharide solution, and at 5.0 vol% or more, the cell viability was stable at 85% or more. At 5.0 vol% or more, a high cell viability of 85% or more was shown, and at 7.0 vol% and 10.0 vol%, extremely high cell viability of 93.4 vol% and 92.5%, respectively, was shown.

From the above, when a sample is frozen using a cell cryopreservation solution in which OVA, chondroitin sulfate, or collagen peptide is added as a secondary additive to a fructooligosaccharide solution, an extremely high cell survival rate can be obtained if the content of OVA, chondroitin sulfate, and collagen peptide in the fructooligosaccharide solution is 0.5 vol% or more and 1.0 vol% or less, 3.0 vol% or more and 10.0 vol% or less, and 5.0 vol% or more and 10 vol% or less, respectively.

In this way, the cell cryopreservation solution according to the embodiment can achieve an extremely high cell viability rate of 90% or more by adjusting the content of the main additive and the sub-additive. Furthermore, if the sample is frozen while applying a magnetic field using the freezing devices described in Patent Documents 2 and 3, it is expected that an even higher cell viability rate can be obtained.

<About fructooligosaccharides>
The cell cryopreservation solution according to the embodiment contains fructooligosaccharide as a main additive. As is well known, fructooligosaccharide is an oligosaccharide in which fructose is bound to the fructose residue of saccharose by β-1,2 bond, and is a mixture of 1-kestose, nystose, and 1-fructofuranosyl-D-nystose. Fructooligosaccharide is used in research on bifidobacteria growth agents, and anti-digestion and anti-caries agents.

Fructooligosaccharides have been investigated as a cryoprotectant for frozen sperm, as described in Non-Patent Document 5 above, but as shown in the document, their effectiveness as a cryoprotectant was inferior to that of lactose. For this reason, the idea of using fructooligosaccharides as an additive to cell cryopreservation solutions has not previously been considered. Of course, there is currently no evidence of research or development into cell cryopreservation solutions containing fructooligosaccharides.

However, the cell cryopreservation solution according to the embodiment contains both fructooligosaccharides and various proteins (OVA, chondroitin sulfate, collagen peptide), which can increase the survival rate of cells frozen using this cell cryopreservation solution after thawing.

Claims (4)

The solvent is D-PBS, which is a physiological saline solution, and the solution contains a main additive and a sub-additive.
The main additive is fructooligosaccharide,
The secondary additive is ovalbumin,
The auxiliary additive is added to a fructooligosaccharide solution in which the fructooligosaccharide is contained in the physiological saline at a ratio of 10 wt % to 30 wt %,
The ovalbumin is contained in an amount of 0.5 vol% or more and 1.0 vol% or less.
Cell cryopreservation medium. The solvent is D-PBS, which is a physiological saline solution, and the solution contains a main additive and a sub-additive.
The main additive is fructooligosaccharide,
The secondary additive is chondroitin sulfate,
The auxiliary additive is added to a fructooligosaccharide solution in which the fructooligosaccharide is contained in the physiological saline at a ratio of 10 wt % to 30 wt %,
The chondroitin sulfate is contained in an amount of 3.0 vol% or more and 10.0 vol% or less.
Cell cryopreservation medium. The solvent is D-PBS, which is a physiological saline solution, and the solution contains a main additive and a sub-additive.
The main additive is fructooligosaccharide,
The secondary additive is a collagen peptide,
The auxiliary additive is added to a fructooligosaccharide solution in which the fructooligosaccharide is contained in the physiological saline at a ratio of 10 wt % to 30 wt %,
The collagen peptide is contained in an amount of 5.0 vol% or more and 10.0 vol% or less.
Cell cryopreservation medium. A method for freezing cells using the cell cryopreservation solution according to any one of claims 1 to 3 , comprising:
A sample preparation step of preparing a cell suspension by adding the cell cryopreservation solution to cells to be frozen as a sample;
A sample placement step of placing a first container containing the sample in a second container filled with 2-propanol;
a freezing step of placing the second container in a freezer to freeze the sample;
Including,
In the freezing step, the inside of the freezer in which the sample is placed is gradually cooled to a temperature at which the sample is in an overcooled state, and the temperature is maintained for a predetermined period of time.
Cell freezing methods.