WO2023243709A1 - Living cell freezing method and living cell freezing system - Google Patents

Abstract

Provided are a method and a living cell freezing system for freezing an unfrozen specimen comprising living cells in such a manner as to keep the cells alive. The method comprises: causing an unfrozen specimen to reach a frozen state by placing the unfrozen specimen, that includes a freezing solution containing a freezing protection agent and living cells immersed in the freezing solution, in an electrical field, generated by an electrode to which an alternating current with a frequency of 10-200 Hz is applied, so that said specimen is cooled while conduction electrical current flows. The electrical field is continuously generated without any interruption for a duration that the specimen goes from an unfrozen state to a frozen state. Also provided is a method for forming spheroids, the method comprising: freezing living cells having a spheroid-forming function by the foregoing freezing method; thawing the frozen living cells; and cultivating the thawed living cells to form spheroids.

Description

Live cell freezing method and live cell freezing system

The present disclosure relates to a technique for freezing living cells while preserving their biological abilities.

In the field of life science, which broadly includes medicine, veterinary medicine, biology, drug discovery, etc., it is essential to use in vitro living cells for treatment, research, new product development, etc. However, it is often not practical to carry out these activities immediately at the time and location where living cells are obtained. Therefore, techniques for cryopreserving the obtained living cells until use are also essential. Cryopreservation techniques are also important, for example, to enable the storage and transportation of collections of useful cell lines by cell banks, allowing technicians around the world to share biologically identical cells.

However, it is known that when cells are frozen, their original biological abilities and even their viability are often significantly lost, mainly due to the formation of microscopic ice crystals that occur inside and outside the cells. It is being Although studies have been carried out on the optimal freezing solution composition and cooling rate conditions for many years, the current state of living cell freezing technology is far from complete. There are cell types that are relatively difficult to freeze, such as nervous system cells and induced pluripotent stem (iPS) cells, whose applications have been rapidly increasing in recent years.

In particular, three-dimensional cell aggregates such as spheroids can provide conditions closer to in-vivo cells than single cells or two-dimensional cultured cells, and are therefore highly useful in medicine, drug discovery, etc. It is difficult to freeze cells that have the ability to form cells while preserving that ability. That is, in many cases, cells that have undergone freezing and thawing significantly lose their ability to form spheroids.

Currently widely used live cell freezing techniques are broadly divided into slow freezing methods and vitrification methods based on different cooling rate conditions.

The slow freezing method is a method in which an unfrozen frozen solution containing living cells is gradually frozen at a cooling rate of approximately -1°C to -2°C/min. According to this method, although ice crystals can be formed, ice crystal formation occurs in a manner that causes less damage to the cells than when simply freezing without controlling the cooling rate, so cell viability is relatively maintained. be able to. The concentration of cryoprotectant (and potentially associated toxicity) in the freezing solution used in slow freezing methods can be kept relatively low. Slow freezing is widely used for cryopreservation of common cultured cell lines including iPS cells. The slow freezing method has a relatively simple procedure and is suitable for freezing a large number of samples in large quantities, and it is also possible to transport the frozen samples in dry ice.

Slow freezing is most preferably carried out using a programmed freezer. A program freezer, also called a controlled rate freezer, is a freezer that can execute a user-specified cooling rate and cooling temperature profile under computer control. While program freezers are expensive, simple slow-freezing containers that can approximate a cooling rate of approximately -1°C/min at a much lower cost are also available and are in fact widely used. Examples of simple slow freezing containers include Nippon Freezer Co., Ltd.'s BiCell™, Corning International Co., Ltd.'s CoolCell™, and Thermo Fisher Scientific's Mr. Frosty™. Each of these containers for simple slow freezing is made of a material with a specific thermal conductivity, so you can program it by simply leaving the container containing an unfrozen sample in a deep freezer at approximately -80°C. It can approximate controlled cooling rates such as those obtained with freezers. However, this is only an approximation, and it is generally understood that freezing in a programmed freezer is inferior in terms of preserving cell properties and viability.

The vitrification method is a method in which an unfrozen solution containing cells is immersed in liquid nitrogen to extremely rapidly lower the temperature and freeze. The vitrification method avoids the formation of ice crystals and allows rapid freezing as an amorphous glassy solid. Vitrification is primarily used to freeze sperm, eggs, and fertilized eggs. The concentration of cryoprotectants (and potentially associated toxicity) in freezing solutions used in vitrification processes can be relatively high. The vitrification method is complicated and requires skill, and is not suitable for freezing a large number or large amount of samples. Furthermore, it is usually impossible to transport samples frozen by vitrification in dry ice while keeping them alive.

Several publications describe performing freezing while applying a magnetic field to cell samples in freezers equipped with coils or magnets around or within the walls of the freezing chamber. Patent Document 1 describes a method for cryopreservation of cells characterized by freezing cells in a fluctuating (alternating current) magnetic field (1.5-2.2 G, 5-20 Hz) in the absence of a cryoprotectant. There is. Patent Document 2 and Non-Patent Document 1 disclose that cells are frozen while applying an alternating current magnetic field (0.22 to 0.50 mT=2.2 to 5.0 G, 25 to 35 Hz) to human iPS cell-derived neural stem cells/progenitor cells that form neurospheres. It describes how to do this. Patent Document 3 describes a method of freezing human pluripotent stem cell-derived cardiomyocytes while applying an alternating magnetic field (0.1 to 1.0 mT=1 to 10 G, 30 to 60 Hz). Patent Document 4 and Non-Patent Document 2 describe freezing iPS cell-derived neurospheres in a proton freezer. Proton freezers freeze samples while applying a static magnetic field of 1 to 200 mT (=10 to 2000 G), radio waves of 0.2 to 1 MHz, and cold air.

Thus, various approaches have been attempted with varying degrees of success in order to move closer to the ultimate goal of preserving the exact same biological properties of unfrozen cells after freezing. However, each approach also has drawbacks. For example, vitrification requires skilled techniques and is not suitable for freezing multiple or large amounts of samples. Programmed freezers and magnetic field freezers tend to be expensive, and it is not easy to adapt existing freezer hardware to them. Although simple slow-freezing containers are inexpensive and do not require special laboratory equipment, their preservation of biological properties tends to be inferior to other freezing techniques.

Patent Document 5 discloses a supercooling process in which an electric field and/or a magnetic field is applied to an object to be frozen while cooling the object, and a freezing process in which application of the electric field and/or magnetic field is stopped after the supercooling process is completed. This describes a freezing method characterized by repeating the steps. Patent Document 6 discloses a cooling method in which an alternating electric field is formed in the refrigerator to cool an object placed in the refrigerator, and the alternating electric field suppresses the coarsening of ice crystals in the object during cooling. describes a cooling method for refrigerators and freezers in which the frequency of AC voltage is 500 hertz or higher. The inventions of Patent Documents 5 and 6 attempt to solve problems such as cell destruction and deterioration of color and taste due to coarsening of ice crystals during freezing, and outflow of liquid juice during thawing (called drip in the food freezing field). That is. In other words, it is understood that Patent Documents 5 and 6 are intended to prevent cell destruction to the extent that foods and the like can be kept fresh and drips can be prevented.

Patent Document 7 states, ``Techniques for suppressing quality deterioration (e.g., drips due to oxidation and tissue destruction) during thawing of frozen foods are known. The effectiveness of applying the above voltage to To provide a method for freezing and preserving frozen products in which quality deterioration of the frozen products contained therein can be more appropriately suppressed than in the past." Under this introduction, Patent Document 7 discloses that frozen products are stored frozen at a freezing temperature for a predetermined storage period or longer while applying an alternating current voltage so that a "weak current" (1 μA to 1000 mA) flows through the frozen products. is listed.

However, the cell integrity (or "freshness") required in the food field is different from the life science field's requirement to maintain strict viability and functionality of living cells. In fact, even if the cells contained in food are not "destroyed" enough to cause drips and are apparently intact and fresh, and even if they were alive before freezing, after freezing and thawing, Strict viability and original functionality are usually lost. This is the fundamental reason why, apart from the general technology of food freezing, special technologies have been developed for the distinct field of live cell freezing, such as cryoprotectants and programmable freezers. Patent Documents 5 and 6 do not teach desirable freezing conditions for strictly maintaining survival and function of living cells. Further, Patent Document 7 is a document related to continuing frozen preservation of frozen foods that are already in a frozen state for an extended period of time. Patent Document 7 is not a document that provides any insight into the fundamental and inherent problem of the technical field of live cell freezing, which is de novo ice crystal formation that occurs during the process of freezing unfrozen living cells, but is a document that strictly focuses on keeping cells alive. Nor was it a literature that studied how to do things.

Japanese Patent Application Publication No. 2014-155443 Japanese Patent Application Publication No. 2017-104061 Japanese Patent Application Publication No. 2019-24325 International Publication No. 2021/100829 Japanese Patent Application Publication No. 2007-259709 Japanese Patent Application Publication No. 2011-182700 International Publication No. 2018/185912

Therefore, new types of live cell freezing techniques can be selected depending on the purpose of live cell freezing, the type of cells to be frozen, the practical constraints of the facility (e.g., equipment, financial, or personnel constraints), etc. The need for choice continues to exist. In particular, new freezing method options are desired that can at least partially overcome the shortcomings of conventional methods and provide cells that approximate unfrozen cells to an extent that is comparable to or even better than conventional methods. It is rare.

The present inventors have demonstrated that when unfrozen living cells immersed in a freezing solution containing a cryoprotectant are placed in a freezing chamber that generates an electric field and frozen while an alternating current (AC) is applied, the cells It has been found that the loss of inherent biological properties (expressed by viability, proliferation ability, ability to form three-dimensional cell aggregates, etc.) can be significantly suppressed.

The mechanical principle itself that allows electric current to flow through articles placed in the freezing chamber of a freezer was also described in, for example, Patent Document 7 (International Publication No. 2018/185912). However, as mentioned above, Patent Document 7 is a document relating to a technique for continuing frozen preservation of frozen foods that are already in a frozen state for an extended period of time. This document does not provide any insight into the fundamental and inherent problem in the technical field of live cell freezing, which is the de novo ice crystal formation that occurs during the process of freezing unfrozen living cells, but rather strictly to keep cells alive. It wasn't even a literature that studied things. In other words, the fact that the modified application of mechanical principles, such as those described in US Pat. .

The present disclosure includes the following embodiments.
[1]
A method for freezing an unfrozen sample containing living cells while keeping the living cells alive, the method comprising:
The unfrozen sample, which includes a freezing solution containing a cryoprotectant and the living cells immersed in the freezing solution, is placed in an electric field generated from an electrode to which an alternating voltage is applied, while applying a conduction current. cooling the unfrozen sample to a frozen state;
The frequency of the alternating current voltage is 10 to 200 Hz,
The electric field is generated continuously without interruption while the sample changes from an unfrozen state to a frozen state.
Method.
[2]
The cooling means placing the unfrozen sample in the freezing chamber of a freezer, which has a freezing chamber at a constant temperature lower than the freezing temperature of the freezing solution, and an electrode installed in the freezing chamber and to which the AC voltage is applied. The method described in [1], which is carried out by placing .
[3]
The method according to [1] or [2], wherein the cryoprotectant comprises dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or a combination of any of these.
[4]
The method according to [3], wherein the concentration of the dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or any combination thereof in the freezing solution is 5 to 30% (w/v). .
[5]
The method according to any one of [1] to [4], wherein the magnitude of the conduction current flowing through each sample during cooling of the unfrozen sample to a frozen state does not exceed 0.5 μA.
[6]
The method according to any one of [1] to [5], further comprising storing the frozen sample at a temperature of -80°C or lower after the sample is frozen.
[7]
Storing the frozen sample at a temperature below -80°C includes transferring the frozen sample into a tank containing liquid nitrogen, and storing the frozen sample in the tank containing liquid nitrogen. The method according to [6], which comprises storing while being exposed to nitrogen or its vapor.
[8]
The method according to any one of [1] to [7], wherein the living cells are induced pluripotent stem cells, embryonic stem cells, or differentiated cells derived from any of them.
[9]
A method for forming spheroids, the method comprising freezing living cells having spheroid-forming ability by the method according to any one of [1] to [8], and thawing the frozen living cells. and culturing the thawed living cells to form spheroids.
[10]
A live cell freezing system for freezing an unfrozen cell sample containing living cells while keeping the living cells alive, the system comprising:
comprising a transformer, a freezing chamber, an electrode electrically connected to the transformer and installed in the freezing chamber, and the cell sample placed in the freezing chamber,
The cell sample includes a freezing solution containing a cryoprotectant and living cells immersed in the freezing solution,
the freezing chamber has a temperature lower than the freezing temperature of the freezing solution;
An electric field is generated in the freezing chamber from the electrode by an alternating voltage applied by the transformer, and a conduction current flows through the cell sample;
The frequency of the alternating current voltage is 10 to 200 Hz,
The transformer is configured to continuously generate the electric field without interruption while the cell sample changes from an unfrozen state to a frozen state.
Live cell freezing system.
[11]
The system of [10], wherein the cryoprotectant comprises dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or a combination of any of these.
[12]
The system according to [11], wherein the concentration of the dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or any combination thereof in the freezing solution is 5-30% (w/v). .
[13]
The system according to any one of [10] to [12], wherein the magnitude of the conduction current flowing through each sample does not exceed 0.5 μA.
[14]
The system according to any one of [10] to [13], wherein the living cells are induced pluripotent stem cells, embryonic stem cells, or differentiated cells derived from any of them.

FIG. 1 is a perspective view showing a schematic configuration of an example of a freezer. FIG. 2 is a sectional view showing a schematic configuration of an example of a freezer. FIG. 3 shows an example of an analysis of the current density distribution in the freezing chamber of a freezer in which an electrode plate was installed and a sample was placed. Figure 4 shows a comparison of the ability of iPS-derived neurons subjected to freezing and liquid nitrogen storage ( _LN2 storage) under different conditions to form spheroids after thawing. (1) to (3) show different freezing conditions (the same applies to Figure 5). FIG. 5 shows a comparison of cell numbers when iPS cells that have been frozen under different conditions are grown after thawing. Figure 6 shows a comparison of cell numbers when various types of cells are grown after freezing and thawing. (a) and (b) are floating cells, and (c) and (d) are adherent cells. (A) to (F) show different freezing conditions (the same applies to Figures 7 and 8). FIG. 7 shows the results of the experiment of FIG. 6 by changing the freezing solution to a DMSO-free composition. (a) and (b) use a cryoprotectant based on propylene glycol, and (c) and (d) use a cryoprotectant based on poly-L-lysine. FIG. 8(a) shows anti-βIII tubulin staining (top) and diamidino-2-phenylindole (DAPI) staining (bottom) of nervous system spheroids cultured for 3 days after freezing and thawing. (b) and (c) show the results of quantifying neurite outgrowth in spheroids derived from 1231A3 cells and 201B7-Ff cells, respectively.

In one aspect, the present disclosure provides a method for freezing an unfrozen sample containing living cells while keeping the living cells alive. The method consists of placing an unfrozen sample containing a freezing solution containing a cryoprotectant and living cells immersed in the freezing solution in an electric field generated by an electrode to which an alternating current (AC) voltage is applied. It involves freezing an unfrozen sample, that is, bringing it to a frozen state, by cooling while applying a conduction current. Normally, when an unfrozen cell is frozen, ice crystals form and develop inside and outside the cell, which are primarily responsible for the cell's viability, proliferation ability, and other cell-specific biological functions. A loss of properties occurs. By freezing using the method of this embodiment, the loss can be significantly suppressed. That is, living cells frozen by the method of the present embodiment can maintain viability at a high rate even after thawing, and can maintain substantially the same or similar characteristics as the cells before freezing.

The term "freezing solution" as used in this disclosure refers to a solution for freezing cells by immersing them in the solution, and the term itself does not indicate whether the solution is frozen, unfrozen, or frozen. It does not indicate that it is in the process of being done. It will be appreciated by those skilled in the art that a typical freezing solution is an aqueous liquid, and usually at least half of the weight of the freezing solution will be comprised of water. The freezing solution of this embodiment includes a cryoprotectant. Cryoprotectants are well known to those skilled in the art of live cell freezing, and a wide variety of types exist. Cryoprotectant types and concentrations used in conventional slow freezing methods can be used in this embodiment. Cryoprotectants, when dissolved in water, provide the ability to suppress cell damage associated with ice crystal formation. Examples of preferred cryoprotectants in this embodiment include dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, or a combination of any of these. These are all water-soluble molecules that inhibit the damage associated with ice crystal formation by a common mechanism: they penetrate cells and displace intracellular water molecules. Dimethyl sulfoxide (DMSO) is the most widely used cryoprotectant and may be particularly preferably used in this embodiment. Another example of a cryoprotectant preferred in this embodiment is polyampholytes (see Communications Materials (2021) 2:15). The polyampholyte may be, for example, carboxylated poly-L-lysine, which, as known to those skilled in the art, is ε-poly-L-lysine in which some of the amino groups are carboxylated. Amphoteric polymer electrolysis differs from the above-mentioned DMSO and the like in that it does not penetrate into cells, but it can also be used in this embodiment, and it can also be used in combination with the other cryoprotectants mentioned above.

In some embodiments, the concentration of dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or any combination thereof in the freezing solution is, for example, 5-30% (w/v); It can be 7-25%, 8-20%, or 9-15%. "% (w/v)", as commonly understood by those skilled in the art, represents a weight/volume concentration and is a percentage indicating how many grams of the target component are dissolved per 100 mL of aqueous solution. In one embodiment, the freezing solution contains 8-20% or 9-15% (w/v) dimethyl sulfoxide and may contain no other cryoprotectants.

As known to those skilled in the art, the freezing solution may contain other cryoprotectants and components selected from other cryoprotectants. Examples of such ingredients include sugars (e.g. glucose, trehalose, etc.), proteins (e.g. serum albumin such as bovine serum albumin or human serum albumin), polymers (e.g. cellulose derivatives, hydroxyethyl starch, dextrans, polyethylene glycols, -L-lysine, etc.), buffers (eg, HEPES, phosphates, carbonates, etc.), serum, and components of cell culture media.

When cells are "immersed" in a solution, they come into contact with the solution and components in the solution (e.g. cryoprotectants such as DMSO) penetrate into the cells. Immersion of cells can be accomplished by suspending, dispersing, or otherwise encapsulating cells or groups of cells in a solution.

A living cell may be a eukaryotic cell or a prokaryotic cell, and a eukaryotic cell may be, for example, an animal cell or a plant cell. The living cells are preferably animal cells, and may for example be insect cells, avian cells, fish cells or mammalian cells. Mammalian cells can be, for example, rodent cells (eg, mouse, rat, or hamster), artiodactyl cells (eg, bovine, porcine, ovine, goat, etc.), or primate cells (eg, monkey or human). Living cells can be, for example, stem cells, immortalized cells, or cancer cells. In one preferred embodiment, the living cells are induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, or differentiated cells derived from either. In one preferred embodiment, the living cell is a sperm, an egg, or a fertilized egg of an animal (especially a mammal such as a human). In some embodiments, the living cells are derived from or are derived from cell type(s) of interest that have been separated from other cell types with which they were associated in living tissue and maintained in culture (e.g. , genetically engineered, and/or differentiated) cells.

The cell concentration in the freezing solution can be appropriately selected by those skilled in the art based on common knowledge, and may vary depending on the type of cell. In a typical example, the cell concentration may be 10 ⁴ to 10 ⁷ cells per mL of freezing solution, such as 1×10 ⁶ to 5×10 ⁶ cells/mL. In some embodiments, the cell concentration can be between 10 ⁴ and 10 ⁷ cells per mL of sample, such as between 1×10 ⁶ and 5×10 ⁶ cells/mL. The amount of freezing solution in each sample frozen in this embodiment, or the total amount of each sample, can be, for example, but not limited to, 0.05-10 mL, 0.1-5 mL, or 0.2-1 mL. Those skilled in the art will appreciate that samples of living cells are typically frozen in containers called cryotubes, cryovials, or glass ampoule tubes. In this embodiment, the shape and material of the sample container suitable for freezing while applying an electric current can be appropriately determined by those skilled in the art, and may be a commonly used cryotube, cryovial, or glass ampoule tube, for example. Commercially available cryotubes, cryovials, or glass ampoule tubes may be suitably used. Most typically, a container containing a sample for cell freezing has a substantially cylindrical or tubular shape, and such a shape is preferred because it can promote uniform conduction of heat and current in the sample. Examples of suitable materials for the container include, but are not limited to, polypropylene, polyethylene, and glass.

In this disclosure, the term "unfrozen" refers to an unfrozen state, typically a flowable liquid state. In the method of this embodiment, a sample comprising a freezing solution and live cells is cooled starting from an unfrozen state to a frozen state, and the sample is exposed to an electrical current during the process (e.g., for at least 30 minutes). A voltage is applied (ie, the sample is placed in an electric field) and a conduction current is caused to flow. This means that the sample starts at a temperature above the freezing temperature of the freezing solution (e.g., at least 5°C above, at least 10°C above, or at least 15°C above) and starts at a temperature below the freezing temperature of the freezing solution (for example, at least 5° C. lower, at least 10° C. lower, or at least 15° C. lower) during which said alternating voltage is applied. The freezing temperature of a freezing solution may vary depending on the composition of the liquid and can be readily determined by one skilled in the art. The solidification temperature and conductivity of the sample are primarily governed by the composition of the freezing solution, and the influence of suspended cells can be relatively limited. The point is that a frozen state is achieved in the freezer starting from an unfrozen state brought in from outside the freezer, and it is usually not necessary to accurately specify the freezing temperature of each freezing solution or sample. For example, from a temperature in the range 25°C to -4°C or 20°C to 0°C (above the freezing temperature of the freezing solution) to a temperature in the range -15°C to -80°C or -20°C to -50°C. The sample is cooled to a temperature (lower than the solidification temperature of the freezing solution), and the alternating current voltage is applied to the sample at least during that time. In this embodiment, the electric field is continuously generated without interruption while the sample changes from an unfrozen state to a frozen state. Therefore, the alternating current voltage may be continuously applied and the conduction current may continue to flow. The electric field may occur uninterruptedly before, during, and after freezing the sample; for example, a frozen sample may be placed in the electric field until removed from the freezer.

The conduction current flowing through the sample during the freezing process in this embodiment may typically be between 0.1 and 500 nA, such as between 0.2 and 200 nA, between 0.5 and 100 nA, or between 1 and 50 nA. Preferably, the magnitude of the conducted current flowing through each sample during cooling of an unfrozen sample to a frozen state does not exceed 0.5 μA (500 nA), and preferably does not exceed 0.4 μA or 0.1 μA. . Although there may inevitably be slight fluctuations in the current value due to different locations within the sample and/or over time, the current value of a sample in the present disclosure is interpreted as an average value for that sample. Conducted currents greater than 0.5 μA may maintain high cell viability after thawing, but may result in inhibition, variability, or other unnatural effects on the biological function of the cells. Generally, in the field of live cell freezing, the goal is to maintain the same viability and function as the cell before freezing, but the term "unnatural effects" here refers to This includes functional differences that arise due to the freezing and thawing process compared to cells that did not.

The magnitude of the applied voltage that generates an electric field within the freezer space and/or the magnitude of the voltage within the freezer space so that a conduction current within these ranges flows through the sample or freezing solution when an unfrozen sample is cooled to freezing. The position of the sample can be adjusted. The appropriate applied voltage magnitude and sample position depending on the freezer can be tested in advance using a simulated sample of composition corresponding to the sample or freezing solution. Specifically, passing a conduction current by applying an alternating current voltage to a sample refers to applying an alternating current voltage (e.g., 0.1 to 100 kV or 0.5 to 10 kV in effective value) on an electrode to generate an electric field. This can be achieved by placing the sample near it. Preferably, the sample is placed such that the freezing solution is within 20 cm, within 10 cm, or within 5 cm of the electrode.

The specific structure of a freezer capable of passing such conduction current through a sample can be appropriately determined by one skilled in the art based on the present disclosure and the information provided in Patent Document 7. The conduction current and corresponding applied voltage flowing to a sample placed at a given position within the freezing chamber for a freezer of given dimensions with electrodes within the freezing chamber can be measured by one skilled in the art (e.g., when actually freezing It can also be calculated by simulation (using a simulated sample of the corresponding composition rather than the true sample being attempted). The current density J is expressed by the formula J=σE, where E is the electric field strength and σ is the conductivity. Therefore, the current density of the sample, and hence the current value, can be calculated from the electric field strength (potential gradient) generated by the electrode to which a voltage is applied. As illustrated in Figure 3, a given region within a sample located parallel to the electrode plane can have a generally uniform current density, and by multiplying the current density by the area of that region, the current You can find the value. Although there may be small variations in the current density within the sample tube, it is possible to clearly specify the conditions under which the average current flowing through the entire sample (or the bulk of the sample) falls within a specific range, for example, as described above. The current density of the sample can be varied, for example, between 2 and 500 μA/m ² . The alternating current is preferably a sinusoidal alternating current, but is not limited thereto. The frequency of the alternating current in this embodiment is suitably lower than 500 Hz, preferably 10 Hz to 200 Hz or 20 Hz to 100 Hz, and the frequency of commercial power supply, 50 Hz or 60 Hz, is also preferably used. can. Increasing the frequency of the alternating voltage beyond these ranges may have unnatural or adverse effects on the biological functions of living cells.

In this embodiment, cooling and freezing an unfrozen sample includes, for example, a freezing chamber with a temperature lower than the solidification temperature of the freezing solution, and an electrode installed in the freezing chamber and to which the above-mentioned AC voltage is applied. This can be done by placing an unfrozen sample in the freezing chamber of a freezer. The temperature of this freezer compartment may be constant. In other words, you can store unfrozen samples in a freezer at a constant temperature (for example, -15°C to -80°C or -20°C to -50°C) without having to particularly control or slow down the cooling rate like in a program freezer. The freezing method of the present embodiment can be achieved by simply leaving it in the water. The "constant temperature" freezer compartment here means a freezer compartment that provides a stable temperature within ±1°C of the set temperature when closed. This can be a major advantage in terms of procedural simplification and cost reduction compared to programmed freezers. However, the slowing of the cooling rate by a programmed freezer or other means may be combined with the application of current according to the method of the present invention, even if no current is applied in terms of preserving the biological properties of the cells. Additional benefits may result from applying an electric current compared to the case (i.e., slow freezing alone). The method of this embodiment is a technology different from the methods described in Patent Documents 1 to 4. For example, the freezer used in the method of the present embodiment does not require a coil or magnet to generate a fluctuating (alternating current) or static magnetic field around or within the wall of the freezing chamber, as in Patent Documents 1-4. In one embodiment, cooling the sample does not substantially involve application of a magnetic field. Substantially not involving the application of a magnetic field means that the application of a coil or magnet to the sample does not involve the use of coils, magnets, etc., except for the minute magnetic field that may unavoidably exist when the sample is placed in an electric field generated from an electrode to which an alternating voltage is applied. This means that there is no intentional application of a magnetic field.

Here, an example of a freezer to which the method of this embodiment can be applied will be described. FIG. 1 is a perspective view of the freezer 1, and FIG. 2 is a sectional view of the freezer 1 taken along section II-II in FIG. The main body 2 of the freezer 1 is formed with a freezing chamber 3 that opens on the top surface, and the freezing chamber 3 can be opened and closed by a door 6 attached to the main body 2 with a hinge 7. The freezing compartment 3 is surrounded by a heat insulating material 8 provided on the main body 2 and the door 6, and a heat pump including a cooling plate 12 inside the freezing compartment 3, an external radiator 13, and a compressor 11 is operated in a refrigeration cycle. It is cooled by this. The main body 2 is provided with a control module 16, and the control module 16 can drive the heat pump according to the operation of the control panel 5 and control the temperature of the freezing compartment 3. A metal shelf 4 supported by an insulator is provided inside the freezer compartment 3 (shelf 4 is insulated from the inner wall of the freezer compartment 3), and the shelf 4 is one of the terminals on the secondary side of the transformer 15. is connected and high voltage is applied. That is, in this example, the shelf 4 serves as an electrode. As a result, an alternating electric field is generated in the freezing chamber 3, and a minute current flows through the sample placed on the shelf 4 in accordance with the conductivity of the sample. This current (or applied voltage) can also be controlled through the control panel 5. In this example, one shelf 4 is provided in the freezer compartment 3, but a plurality of shelves (electrodes) may be provided.

In some embodiments, the method may further include storing the frozen sample at a temperature of -80°C or lower or -150°C or lower after the sample is frozen. This can be accomplished, for example, by placing the frozen sample in a deep freezer at these temperatures. Alternatively, the method includes, after the sample has been frozen, transferring the frozen sample (i.e., the sample that remains frozen) into a tank containing liquid nitrogen; may also include storage while exposed to liquid nitrogen or its vapor. In this way, long-term storage of frozen cell samples using deep freezers or liquid nitrogen is a routine practice in laboratories, medical institutions, and cell banks. The form can be combined after freezing method. It has been found that the biological properties of living cells frozen by the method of this embodiment are not substantially affected by the length of subsequent storage in a deep freezer or in liquid nitrogen.

The methods of the present disclosure find particular utility in freezing living cells for the purpose of forming spheroids. Accordingly, in one aspect, the present disclosure provides a method of forming spheroids. A spheroid is a cell aggregate having a three-dimensional (usually approximately spherical) structure formed by an in vitro culture process from a single cell. The term "single cell" in this disclosure refers to a group of cells that are not associated and are dissociated into individual cells. Spheroids can provide conditions closer to in vivo cells than single cells and two-dimensional cultured cells. Furthermore, spheroids can be further induced to differentiate and develop into organoids with organ-like properties. Therefore, spheroids are highly useful in medicine, drug discovery, and the like. There is a strong need to cryopreserve a group of cells that have the ability to form spheroids and allow them to form or continue to form spheroids after thawing. However, the problem with conventional freezing methods is that the ability to form three-dimensional cell aggregates is significantly lost. That is, in many cases, cells that have undergone freezing and thawing significantly lose their ability to form three-dimensional cell aggregates. It has been found that the freezing method of this embodiment is excellent in preserving biological properties and can also preserve three-dimensional cell agglomeration ability. That is, the living cells frozen by the embodiment of the freezing method described above may be living cells that have the ability to form spheroids.

A method of forming spheroids according to one aspect of the present disclosure includes freezing living cells capable of forming spheroids by the method of the embodiment described above, and then thawing the frozen living cells, and thawing the living cells. and culturing living cells to form spheroids. Cells capable of forming spheroids are known to those skilled in the art. Spheroids are generally formed by three-dimensional self-association of cell groups while adhering to each other. It is known to those skilled in the art that spheroids can be formed by a number of different methods, such as the hanging drop method. In the simplest method, spheroids are formed by culturing single cells in U-bottom (round-bottom) wells. Low attachment U-bottom well plates suitable for spheroid formation applications are known to those skilled in the art and are commercially available in large numbers.

Normally, spheroids are passaged by once dissociating forming or expanding spheroids into single cells, and then restarting spheroid formation from a smaller number of cells. In other words, cells with inherent spheroid-forming ability have the same ability to self-associate and expand, whether they are in a single cell state or as part of a forming spheroid. I can understand that you are doing it. In this embodiment, the living cells to be frozen may be in a single cell state or may be in a state in which a spheroid is being formed. "Forming a spheroid" can mean starting spheroid formation anew from a single cell and/or continuing the formation of a spheroid in progress. Thawed living cells can be cultured to form spheroids immediately, ie, without passage or differentiation steps.

Suitable methods for thawing living cells are known to those skilled in the art. It is usually preferable to rapidly thaw frozen cell samples at 37°C.

In another aspect, the present disclosure provides a live cell freezing system for freezing an unfrozen cell sample containing living cells while keeping the living cells alive. The live cell freezing system of this embodiment can be used, for example, to implement the embodiment of the freezing method described above. The freezing method embodiments and live cell freezing system embodiments described above have common elements, and the description provided with respect to the freezing method embodiments also applies to the live cell freezing system embodiments. It should be understood that the same can be said for a given term, and vice versa.

In some embodiments, a live cell freezing system includes a transformer, a freezing chamber, electrodes, and a cell sample. The electrode is electrically connected to the transformer and placed inside the freezer. The shape of the electrode can be, for example, a plate (electrode plate) or a rod (e.g., single, multiple, or a plurality of rods combined to form a grid, straight rods, or non-straight, such as curved or meandering). rods), etc., but are not limited to these. Cell samples are also placed in the freezing chamber. The transformer may be placed outside the freezer. For example, it is preferable that a plate-shaped, grid-shaped, or other generally planar electrode is installed horizontally in the freezing chamber, and a cell sample container (for example, a tube) is placed on the generally planar electrode. In this way, the electrode can act as a sample shelf in the freezer. However, it is not essential that the cell sample container be in direct contact with the electrode. Holding the cell sample tube perpendicular to the generally planar electrode (eg, placing the cell sample tube upright on a horizontal electrode plate) is preferred for uniformity of current density.

Similarly to the embodiment of the freezing method described above, the cell sample in this live cell freezing system also includes a freezing solution containing a cryoprotectant and living cells immersed in the freezing solution. Cryoprotectants may include dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholytes, or combinations of any of these. The concentration of dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or any combination thereof in the freezing solution is 5-30% (w/v), 7-25%, 8-20%, Or it can be 9-15%. In one embodiment, the freezing solution contains 8-20% or 9-15% (w/v) dimethyl sulfoxide and may contain no other cryoprotectants. Examples of suitable living cells are induced pluripotent stem cell lines, embryonic stem cell lines, and differentiated cells derived from any of them, as well as cells that have spheroid-forming potential and are in the process of forming or have formed spheroids. It's okay.

The freezing chamber has a temperature lower than the freezing temperature of the freezing solution. The temperature of the freezer compartment can be, for example, between -15°C and -80°C or between -20°C and -50°C. The freezer compartment may be maintained at a constant temperature, ie, a stable temperature within ±1°C. Cell samples found in live cell freezing systems may be unfrozen or may be in a frozen state.

Electrical connection between the transformer and the electrodes is typically accomplished via electrical wires. Specifically, one of the pair of secondary terminals of the transformer is electrically connected to the electrode. The other of the pair of secondary terminals of the transformer may be open. It is understood that the transformer itself is also electrically connected to the AC power source. An alternating current (AC) voltage provided by a transformer is applied to the electrodes, creating an electric field in the freezer space that causes a conduction current to flow through the cell sample on or near the electrodes. This current is typically in the range of 0.1-500 nA, and may be 0.2-200 nA, 0.5-100 nA, or 1-50 nA. The magnitude of the conduction current flowing through each sample preferably does not exceed 0.5 μA (500 nA), and preferably does not exceed 0.4 μA or 0.1 μA. The alternating current voltage is suitably applied at a frequency below 500 Hz, preferably within the range of 10-200 Hz. The transformer is configured to continuously generate the electric field while the cell sample changes from an unfrozen state to a frozen state. In other words, for example, the transformer may be configured to continuously generate the electric field during operation of the live cell freezing system or during closure of the freezing chamber during operation. For example, if a freezing chamber has a door that can be opened and closed, when the door is opened (for example, to take a sample in or out), the electric field generation will be interrupted, but when the door is closed and the freezing chamber is closed, the electric field generation will continue. The transformer can be configured as follows.

In this specification, the numerical range indicated using the expression "A to B" (or "from A to B" or "between A and B") refers to the numerical value A written before and after "..." and B as the minimum and maximum values, respectively, and also includes ranges excluding the minimum and/or maximum values. Where more than one numerical range is stated, the upper or lower value of one stated numerical range may be combined with the upper or lower value of another stated numerical range, and no such combination Numerical ranges that may occur are also contemplated in this disclosure.

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of international application PCT/JP2022/024417, filed June 17, 2022, which is hereby incorporated by reference in its entirety.
The present disclosure further includes the following embodiments.
[1]
An unfrozen sample containing a freezing solution containing a cryoprotectant and living cells immersed in the freezing solution is cooled while passing a conduction current by placing it in an electric field generated from an electrode to which an alternating voltage is applied. and freezing the unfrozen sample.
[2]
The cooling means placing the unfrozen sample in the freezing chamber of a freezer, which has a freezing chamber at a constant temperature lower than the freezing temperature of the freezing solution, and an electrode installed in the freezing chamber and to which the AC voltage is applied. The method described in [1], which is carried out by placing .
[3]
The method according to [1] or [2], wherein the cryoprotectant comprises dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or a combination of any of these.
[4]
The method according to [3], wherein the concentration of the dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or any combination thereof in the freezing solution is 5 to 30% (w/v). .
[5]
The method according to any one of [1] to [4], further comprising storing the frozen sample at a temperature of -80°C or lower after the sample is frozen.
[6]
Storing the frozen sample at a temperature below -80°C includes transferring the frozen sample into a tank containing liquid nitrogen, and storing the frozen sample in the tank containing liquid nitrogen. The method according to [5], which comprises storing while being exposed to nitrogen or its vapor.
[7]
The method according to any one of [1] to [6], wherein the living cells are induced pluripotent stem cells, embryonic stem cells, or differentiated cells derived from any of them.
[8]
A method for forming spheroids, the method comprising: freezing living cells having spheroid-forming ability by the method according to any one of [1] to [7]; thawing the frozen living cells; and culturing the thawed living cells to form spheroids.
[9]
A live cell freezing system comprising a transformer, a freezing chamber, an electrode electrically connected to the transformer and installed in the freezing chamber, and a cell sample placed in the freezing chamber,
The cell sample includes a freezing solution containing a cryoprotectant and living cells immersed in the freezing solution,
the freezing chamber has a temperature lower than the freezing temperature of the freezing solution;
An electric field is generated in the freezing chamber from the electrode by an alternating voltage applied by the transformer, and a conduction current is flowing through the cell sample.
Live cell freezing system.
[10]
The system of [9], wherein the cryoprotectant comprises dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or a combination of any of these.
[11]
The system according to [10], wherein the concentration of the dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or any combination thereof in the freezing solution is 5-30% (w/v). .
[12]
The system according to any one of [9] to [11], wherein the living cells are induced pluripotent stem cells, embryonic stem cells, or differentiated cells derived from any of them.

The following examples are merely for the purpose of illustrating typical embodiments, and the present invention is not limited to these specific embodiments.

[Example 1: Study of current conditions]
A commercially available conventional constant temperature freezer was modified to create a freezer 1 essentially as shown in FIGS. 1 and 2. A transformer was installed outside the freezing chamber of a conventional freezer, and a metal shelf (electrode) connected to one of the secondary terminals of the transformer was installed horizontally below the freezing chamber. An insulating glass support was sandwiched between the bottom of the freezer and the shelf board. The other secondary terminal of the transformer is open. This freezer (including a transformer) was connected to a commercial power source to cool the freezer compartment and apply voltage.

In addition to studying the biological effects of different freezing conditions as in the Examples described below, we analyzed the current flowing through the sample. Although it is possible to actually measure the current, here we will describe analysis of the current value by calculation.

Using Femtet (finite element method simulation software) from Murata Software Co., Ltd., the current flowing through the sample liquid was calculated by inputting the parameters corresponding to the experimental conditions, including: Freezer 1 Freezer compartment 3 Internal dimensions, dimensions of metal shelf 4 (length x width x thickness) and position within freezing chamber 3, sample tube dimensions (length, outer diameter, inner diameter) and position relative to shelf 4, amount of sample liquid and conductivity , as well as the applied voltage to shelf 4. The inner diameter of a typical sample tube is approximately 8 mm. The sample tube was held vertically against the horizontal shelf 4. Although FIG. 2 shows a grid-like shelf 4, here a single plate was used as the electrode/shelf. Although not shown in the figure, the dimensions and material of the tube rack used to hold the sample tube were also taken into consideration in the calculation.

As described in the Examples below, it has been found that freezing by placing unfrozen samples in this freezer improves the preservation of the cells' inherent biological capabilities prior to freezing. However, it was analyzed that the current flowing through the sample liquid in the sample tube at that time was within the range of 0.1 to 500 nA. FIG. 3 shows an example of a simulation result, which shows the distribution of current density in the freezing compartment 3 of the freezer 1 under one experimental condition. It should be noted that in this figure, the distribution of current density is represented by the color coding of individual data points rather than the density of data points. The following specific examples describe experiments conducted under conditions in which the current flowing through the sample liquid was approximately 1 to 50 nA on average.

[Example 2: Spheroid formation after cryopreservation of human iPS-derived differentiated cells]
This example describes an example experiment using human iPS cell lines 201B7-Ff and 1231A3. 201B7-Ff is derived from skin fibroblasts (Cell. 2007 Nov 30;131(5):861-72), and 1231A3 is derived from peripheral blood cells (J Parkinsons Dis. 2022;12(3):871-884 ) Human iPS cells. Culture of undifferentiated iPS cells to culture to induce dopaminergic neuron differentiation and culture to form spheroids was performed essentially according to the protocol described in Nat Commun. 2020 Jul 6;11(1):3369. This standard protocol involves dissociating the iPS cell line into single cells on day 0, starting differentiation culture in a 6-well plate, performing neuronal differentiation until day 13, and then dissociating the iPS cell line into single cells again. The cells are seeded in U-shaped low-attachment 96-well plates and cultured to form spheroids.

The difference between this example and the standard protocol above is that differentiated cells were frozen on the 13th day before seeding into U-shaped 96-well plates using a different method described below, and the frozen cells were placed in a liquid nitrogen tank for 1 hour. or 7 days. Thereafter, the cells were thawed by a conventional method and seeded in a U-shaped 96-well plate as described above, and culture for forming spheroids was started. Four days after the start of culture in the U-shaped 96-well plate, the presence and extent of spheroid formation was examined under a microscope.

Cell freezing was performed as follows. Differentiated cells dissociated into single cells using Accumax cell dissociation solution (Funakoshi) were placed in a cryotube at a concentration of 5 × 10 cells in ²⁰⁰ μL of freezing solution, and these unfrozen (4-10 °C) The sample tube was cooled and frozen under any of conditions 1 to 3 below, and then immediately transferred in a frozen state to a liquid nitrogen tank. As the freezing solution, the one described in the Examples of Japanese Patent No. 4385158 was used, which contains 10% DMSO as a cryoprotectant.
Condition 1) By placing an unfrozen sample tube in the -35°C freezing chamber of the freezer described in Example 1, the sample was cooled and frozen for 30 minutes while an electric current was passed through it, and then transferred to a liquid nitrogen tank (this Disclosure Freeze Law).
Condition 2) The unfrozen sample tube was cooled to -80°C and frozen in a program freezer set at a controlled cooling rate of 4°C → -80°C, -1°C/min, and then transferred to a liquid nitrogen tank.
Condition 3) A simple slow freezing container containing an unfrozen sample tube was placed in the -80°C freezing chamber of a standard deep freezer overnight to cool and freeze, and then the sample tube was transferred to a liquid nitrogen tank. .

The program freezer is commercially available from Company X. The container for simple slow freezing is commercially available from Company Y. Containers for slow freezing are constructed from materials with a specific thermal conductivity, allowing them to be cooled at controlled cooling rates similar to those obtained in programmable freezers by simply leaving the container containing the sample in a standard deep freezer. This is a cell freezing container that can approximate

The results are shown in Figure 4. The numbers (1) to (3) shown in Figure 4 correspond to the freezing conditions 1 to 3 above, and each three representative wells with the typical appearance of the majority of wells under each condition are shown. An example is shown. Cooling rate control using a programmed freezer (condition 2) is generally considered the gold standard for cell freezing, but surprisingly, cells frozen using the freezing method of the present disclosure (condition 1) do not meet conditions 2 and 3. The spheroid-forming ability was clearly superior to that of the cells that had been frozen. The spheroid-forming ability of cells frozen under condition 1 was comparable to that of cells that had not undergone freezing (no freezing). There was no significant difference in spheroid formation ability depending on whether the cryopreservation period in liquid nitrogen (LN ₂ ) was 1 day ((a), (c)) or 7 days ((b), (d)). I couldn't. Although an example in which spheroid formation of dopaminergic neuron differentiated cells was investigated has been described here, the freezing method of the present disclosure can also preserve the spheroid formation ability of other types of cells.

[Example 3: Proliferation of human iPS cells after freezing]
In this example, the same iPS cell line used in Example 2 was frozen in an undifferentiated state under conditions 1 to 3 above. The same freezing solution as in Example 2 was used, except that the cell concentration was 6.30×10 ⁵ (strain 201B7-Ff) or 8.84×10 ⁵ (strain 1231A3) in 200 μL of freezing solution. After cryopreservation in a liquid nitrogen tank for 7 days, the cells were quickly thawed by spraying with a liquid medium warmed to 37°C using a dropper, and the cells were collected by centrifugation (190x g, 3 minutes). The cells were resuspended in fresh medium and cultured in laminin-coated 6-well plates. The medium was replaced with a fresh one 1 and 3 days after the start of culture, and 5 days (201B7-Ff strain) or 4 days (1231A3 strain) after the start of culture, the cells were grown using a TC20 automatic cell counter (Bio-Rad). Cell numbers were counted.

The results are shown in Figure 5. The numbers (1) to (3) displayed in FIG. 5 correspond to the freezing conditions 1 to 3 above. n = 5 for each freezing condition for each cell line, and significant differences were tested using an unpaired t-test using statistical testing software GraphPad Prism 8 J (GraphPad Software, San Diego, CA, USA). There is. The cell proliferation ability of iPS cells frozen under condition 1 was higher than that of iPS cells frozen under conditions 2 and 3, and the difference between them was statistically significant.

[Example 4: Post-freezing expansion of various cell lines]
This example describes an experimental example to verify the generality of preserving cell proliferation ability by the freezing method of the present disclosure. Typical examples of suspension cells are the human natural killer-like cell line KHYG-1 and human lymphoid cell line THP-1, and representative adherent cells include the human hepatocellular carcinoma line HuH-1. 7 and OMVANA, a human ovary-derived cell line, were used. These are immortalized cancer cell lines. The same freezing solution as in Example 2 was used. Freezing volumes and cell densities of 4 × ¹⁰ cells in 200 μL of freezing solution (suspension cells) or 2 × ¹⁰ cells in 1 mL of freezing solution (adherent cells) were used. n=4 for each freezing condition for each cell line, and statistical tests were the same as in Example 3.

Six different freezing conditions were compared:
Condition A) By placing an unfrozen sample tube in the -35°C freezing chamber of the freezer described in Example 1, the sample was cooled and frozen for 30 minutes while passing an electric current through it, and then transferred to a liquid nitrogen tank (this Disclosure Freeze Law).
Condition B) The unfrozen sample tube was cooled and frozen by placing it in the -35°C freezing chamber of a proton freezer for 30 minutes, and then transferred to a liquid nitrogen tank.
Condition C) Cool and freeze the unfrozen sample tube to -80°C in a company X program freezer set at a controlled cooling rate of 4°C → -80°C and -1°C/min, then transfer it to a liquid nitrogen tank. did.
Condition D) Same as Condition C except that a program freezer from Company Z was used.
Condition E) Place the Y company's simple slow freezing container containing unfrozen sample tubes in the -80℃ freezing chamber of a standard deep freezer overnight to cool and freeze, then transfer the sample tubes to a liquid nitrogen tank. Moved.
Condition F) Same as Condition E except that a simple slow freezing container from Company W was used.

The proton freezer under condition B (Ryoho Freeze Systems Co., Ltd.) is disclosed in Non-Patent Document 2 (J Parkinsons Dis. 2022;12(3):871-884) and Patent Document 4 (International Publication No. 2021/100829, Japanese Patent No. 4424693). This is a freezer that cools and freezes samples while applying a static magnetic field of 1 to 200 mT, radio waves of 0.2 to 1 MHz, and cold air.

After being stored in a liquid nitrogen tank for more than a week, the cells were thawed, and the suspended cells were seeded in 24-well plates and the adherent cells were seeded in 6-well plates for culture. The number of living cells was counted using a TC20 automatic cell counter 5 days (KHYG-1), 7 days (THP-1), 4 days (HuH-7), or 3 days (OVMANA) after the start of culture. The results are shown in FIG. The symbols (A) to (F) displayed in FIG. 6 correspond to the above freezing conditions A to F. The freezing method of the present disclosure (Condition A) provides results that are superior to, or at least comparable to, conventional freezing methods in preserving the inherent capabilities of cells when freezing a variety of cells. This can be seen from these data.

Essentially the same experiment was repeated with different compositions of the freezing solution. The freezing solution used in the experiments shown in Figure 7a,b was DMSO-free and albumin-free, but contained 10% (w/v) propylene glycol cryoprotectant. . The symbols (A) to (E) displayed in FIG. 7 correspond to the above-mentioned freezing conditions A to E. Although the level of cryoprotection with propylene glycol was overall lower than that with DMSO (data not shown), when compared with the same freezing solution composition, the freezing method of the present disclosure is at least comparable to program freezers, etc. The conclusion remains that it can provide results superior to these conventional methods. The DMSO-free freezing solution used in the experiments shown in Figures 7c, d contained the Poly-L-lysine cryoprotectant described in Example 2 of Patent No. 5,630,979. In other words, it is a frozen polymer whose main component is an ampholyte obtained by carboxylating a portion (65 mol%) of the amino groups of ε-poly-L-lysine (by reaction with succinic anhydride). It is a protective agent. Even when this cryoprotectant was used, the freezing method (A) of the present disclosure showed significantly better results than the conventional method in terms of maintaining cell proliferation ability.

[Example 5: Neurite outgrowth ability of spheroids after cryopreservation]
In Example 2, an experiment was described in which neurally differentiated cells were frozen at a stage immediately before the start of spheroid formation, but an experiment was also conducted in which similar cryopreservation was performed after spheroid formation (corresponding to the 27th day of differentiation). Spheroids were frozen under conditions corresponding to freezing conditions A, B, C, and E described in Example 4 and stored for a period of time in liquid nitrogen. In conditions B, C, and E, which are comparative examples, there were cases in which about 30 to 80% of spheroids were lost after 3 days of culture after thawing, whereas in the freezing method example of the present disclosure, When freezing while applying an electric current according to condition A, more than 80% of spheroids were consistently recovered. Spheroids recovered after thawing and culture for 3 days were stained with anti-βIII tubulin antibody to measure the degree of neurite outgrowth. To explain in more detail, since the spheroid body is a collection of cell bodies, its overall outline is stained not only with anti-βIII tubulin antibody but also with DAPI, whereas the neurite parts lacking nuclei are not stained with DAPI. It is stained only by anti-βIII tubulin antibody (Fig. 8a). Therefore, the degree of neurite outgrowth in each spheroid can be quantified by determining the area obtained by subtracting the DAPI staining area from the anti-βIII tubulin antibody staining area as the "neurite area" in a fluorescence microscopic image.

The spheroids that underwent freezing in the example maintained the ability to extend neurites better than the spheroids that underwent freezing in the comparative example (FIGS. 8b, c). In a related experiment, when the current value flowing through each sample during freezing was increased, it was possible to obtain good cell survival rate and neurite outgrowth, but the degree of neurite outgrowth varied between samples. was observed to increase. It has been suggested that ensuring that the magnitude of the conducted current flowing through each sample during freezing does not exceed 0.5 μA may improve the reproducibility or stability of the biological state after the freeze-thaw procedure. Ta.

Claims (14)

1. A method for freezing an unfrozen sample containing living cells while keeping the living cells alive, the method comprising:
   The unfrozen sample, which includes a freezing solution containing a cryoprotectant and the living cells immersed in the freezing solution, is placed in an electric field generated from an electrode to which an alternating voltage is applied, while applying a conduction current. cooling the unfrozen sample to a frozen state;
   The frequency of the alternating current voltage is 10 to 200 Hz,
   The electric field is generated continuously without interruption while the sample changes from an unfrozen state to a frozen state.
   Method.
2. The cooling means placing the unfrozen sample in the freezing chamber of a freezer, which has a freezing chamber at a constant temperature lower than the freezing temperature of the freezing solution, and an electrode installed in the freezing chamber and to which the AC voltage is applied. 2. The method according to claim 1, which is carried out by placing.
3. 2. The method of claim 1, wherein the cryoprotectant comprises dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or a combination of any of these.
4. 4. The method of claim 3, wherein the concentration of dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or any combination thereof in the freezing solution is 5-30% (w/v). .
5. 2. The method of claim 1, wherein the magnitude of the conducted current flowing through each sample during cooling of the unfrozen sample to a frozen state does not exceed 0.5 μA.
6. The method of claim 1, further comprising storing the frozen sample at a temperature of −80° C. or lower after the sample is frozen.
7. Storing the frozen sample at a temperature below -80°C includes transferring the frozen sample into a tank containing liquid nitrogen, and storing the frozen sample in the tank containing liquid nitrogen. 7. The method of claim 6, comprising storage with exposure to nitrogen or its vapor.
8. The method according to any one of claims 1 to 7, wherein the living cells are induced pluripotent stem cells, embryonic stem cells, or differentiated cells derived from any of them.
9. A method for forming spheroids, the method comprising: freezing living cells having spheroid-forming ability by the method according to any one of claims 1 to 7; thawing the frozen living cells; and A method comprising culturing thawed living cells to form spheroids.
10. A live cell freezing system for freezing an unfrozen cell sample containing living cells while keeping the living cells alive, the system comprising:
    comprising a transformer, a freezing chamber, an electrode electrically connected to the transformer and installed in the freezing chamber, and the cell sample placed in the freezing chamber,
    The cell sample includes a freezing solution containing a cryoprotectant and living cells immersed in the freezing solution,
    the freezing chamber has a temperature lower than the freezing temperature of the freezing solution;
    An electric field is generated in the freezing chamber from the electrode by an alternating voltage applied by the transformer, and a conduction current flows through the cell sample;
    The frequency of the alternating current voltage is 10 to 200 Hz,
    The transformer is configured to continuously generate the electric field without interruption while the cell sample changes from an unfrozen state to a frozen state.
    Live cell freezing system.
11. 11. The system of claim 10, wherein the cryoprotectant comprises dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or a combination of any of these.
12. 12. The system of claim 11, wherein the concentration of the dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, polyampholyte, or any combination thereof in the freezing solution is 5-30% (w/v). .
13. 11. The system of claim 10, wherein the magnitude of the conduction current flowing through each sample does not exceed 0.5 μA.
14. The system according to any one of claims 10 to 13, wherein the living cells are induced pluripotent stem cells, embryonic stem cells, or differentiated cells derived from any of them.