CN113907067B

CN113907067B - Ice crystal-free low-temperature storage method and recovery method in supercooled state

Info

Publication number: CN113907067B
Application number: CN202111519170.7A
Authority: CN
Inventors: 高毅; 李阳; 黄楚颖
Original assignee: Guangdong Qianhui Biotechnology Co ltd
Current assignee: Guangdong Qianhui Biotechnology Co ltd
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-04-29
Anticipated expiration: 2041-12-14
Also published as: CN113907067A

Abstract

The invention discloses an ice crystal-free low-temperature storage method and a recovery method in a supercooled state, and relates to the technical field of low-temperature medicine. The ice crystal-free low-temperature preservation method in the supercooled state comprises the following steps: adding oil substances into the freezing protection solution in which the cells or tissues are suspended, completely covering the oil substances on the surface of the freezing protection solution to form a sample, and preserving the sample at low temperature. The freezing protection solution can be inhibited from freezing at the temperature of-20-0 ℃, and the ice crystal freezing protection solution has obvious effects of inhibiting the formation of ice crystals and preventing the growth of the ice crystals, so that the tissue cell bank and the like can avoid using the freezing protection solution with cytotoxicity such as DMSO and the like, and the safe and nontoxic biological sample bank can be constructed more favorably. The tissue cells can be stored at a lower temperature for a longer time (> 2 days) at a low temperature, ischemia-reperfusion injury is relieved, the survival rate of the tissue cells after resuscitation is higher, and the original biological characteristics can be maintained.

Description

Ice crystal-free low-temperature storage method and recovery method in supercooled state

Technical Field

The invention relates to the technical field of low-temperature medicine, in particular to an ice crystal-free low-temperature storage method and a recovery method in a supercooled state.

Background

More than ten million patients with severe hepatitis are newly added in China every year, wherein the number of patients treated by liver transplantation operation is less than 1%, and the treatment requirements of the patients are urgently needed to be met clinically. Bioartificial liver has been proven by clinical practice as an effective treatment alternative to organs. In fact, with the clinical massive application of bioartificial livers, it is necessary to establish a "cell factory" for standardized evaluation of hepatocyte expansion, function and safety under sterile conditions by a highly experienced team, and to establish a bioartificial hepatocyte bank similar to a blood bank to meet clinical needs.

The low-temperature preservation of the cells is to limit the activity of cellular enzymes at low temperature, slow down the biochemical reaction process, inhibit the metabolism of organisms, reduce energy consumption, gradually reach the state of low energy consumption and prolong the preservation time of the cells.

About 0-4 ℃ is a solution for short-term storage of tissues, organs and cells, but the problems of ischemia-reperfusion injury and short storage time still exist objectively. Although the deep cryopreservation can preserve the cells for a long time, the formation of ice crystals in the cells, high concentration of cytotoxic preservative, complicated operation procedures and the like limit clinical application. In the metastable state that the supercooling state of the cell suspension is lower than the equilibrium freezing point but is not frozen, and under the condition of no special condition intervention, slight temperature change can trigger the formation of ice crystals in cells and the risk of freezing to cause massive cell death.

Although the tissue and cell are stored at the normal temperature of 0-4 ℃ at low temperature, ice crystals cannot be formed to cause damage, the tissue and cell still have ischemia reperfusion injury, the storage time is short, the longest time is not more than 3-5 days, and the vitality of the recovered tissue and cell is poor and does not meet the requirement of a hepatocyte bank for biological labor.

The traditional slow gradient freezing needs slow cooling to avoid cell membrane rupture and ice crystal formation caused by sudden change of osmotic pressure inside and outside cells, so a programmed cooling system is needed, and the equipment cost and the time and labor cost are increased.

The vitrification cryopreservation technology requires rapid cooling, and if the amount of the preserved cells is too large, the density is too high or the tissue volume is large, the cells can be damaged by heat stress generated by uneven heat diffusion in the cooling process, so the vitrification cryopreservation is only suitable for a small amount of cells with low density and small volume tissues. In addition, vitrification cryopreservation requires high concentrations of cryopreservative protecting agents, most commonly dimethyl sulfoxide (DMSO), which is itself cytotoxic and can affect cell differentiation.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for storing ice crystal-free low temperature in a supercooled state and a method for recovering ice crystal-free low temperature in the supercooled state.

The invention is realized by the following steps:

in the first aspect, the invention provides a method for preserving ice crystals at a low temperature in a supercooled state, which comprises the steps of adding oil substances into a freezing protection solution in which cells or tissues are suspended, completely covering the oil substances on the surface of the freezing protection solution to form a sample, and preserving the sample at a low temperature; the oil substance comprises at least one of light chain mineral oil and olive oil; the freezing protection solution comprises a DMEM medium or UW solution.

In alternative embodiments, the cell density in the cryoprotectant solution is greater than 1 × 10⁶~5×10⁶/ml。

In an alternative embodiment, the cryoprotectant solution further comprises polyethylene glycol and 3-O-methyl-d-glucose, wherein the concentration of the polyethylene glycol in the cryoprotectant solution is 3-7% by volume, and the concentration of the 3-O-methyl-d-glucose in the cryoprotectant solution is 0.1-0.5 mol/L;

preferably, the volume percentage concentration of the polyethylene glycol in the cryoprotectant solution is 4-6%, and the concentration of the 3-O-methyl-d glucose in the cryoprotectant solution is 0.1-0.3 mol/L;

preferably, the cryoprotectant solution does not contain DMSO.

In an alternative embodiment, cryo-preserving the sample comprises: placing the sample in a precooling temperature of-20-0 ℃ for supercooling state low-temperature storage;

preferably, before the sample is preserved in the supercooled state at low temperature, the sample is pre-cooled to 3-5 ℃ for temperature equilibration for 10-20 min.

In alternative embodiments, the cell or tissue is selected from a human or non-human mammal.

In a second aspect, the present invention provides a method for cryogenically recovering ice crystals in a supercooled state, which comprises recovering a sample stored by the cryogenically preserving method for ice crystals in a supercooled state according to any one of the preceding embodiments.

In an alternative embodiment, resuscitating the sample comprises: and (2) putting the sample at the temperature of 3-5 ℃ for rewarming for 8-12min, adding a complete culture medium preheated to room temperature below the oil liquid level by using a suction pipe, then physically absorbing the oil substances on the freezing protection liquid level, mixing uniformly, centrifuging, removing supernatant, adding a room-temperature high-sugar complete culture medium, and performing resuscitation culture for 20-30 h.

In an alternative embodiment, the oil substance is physically aspirated by capillary action of a pasteur pipette;

preferably, the centrifugation speed is 800-;

preferably, the complete medium is DMEM complete medium.

The invention has the following beneficial effects:

compared with the conventional clinical tissue and cell bank low-temperature storage mode, the ice crystal-free low-temperature storage method in the supercooled state can inhibit the freezing of the freezing protection solution at-20-0 ℃, has obvious effects of inhibiting the formation of ice crystals and preventing the growth of the ice crystals, can avoid using the freezing protection solution with cytotoxicity such as DMSO (dimethyl sulfoxide) and the like in a tissue cell bank and the like, and is more favorable for constructing a safe and nontoxic biological sample bank. Compared with the conventional normal-temperature storage and short-term storage time of 4 ℃, the ice crystal-free low-temperature storage method in the supercooled state can be used for carrying out long-time (> 2 days) low-temperature storage on tissue cells at lower temperature, so that ischemia-reperfusion injury is relieved, the survival rate of the recovered tissue cells is higher, and the original biological characteristics can be maintained. Compared with the vitrification cryopreservation technology applied to the field of assisted reproduction at present, the cryo-preservation method without ice crystals in the supercooled state has the advantages that the volume limit of the tissue is small, the density and the cell quantity of the preserved cells are larger, and the cryo-preservation method can be carried out without complex equipment and devices.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic view of the samples of example 1 and comparative example 1 in a state in which the containers are upright after being frozen in a refrigerator at constant-20 ℃ for 7 days, respectively;

FIG. 2 is a schematic view showing the samples of example 1 and comparative example 1 in a state where the container is tilted after being frozen in a refrigerator at constant-20 ℃ for 7 days, respectively;

fig. 3 is a schematic diagram showing the proliferation tendency and activity of HepG2 viable cells after freeze preservation and resuscitation of different treatment groups in the second experimental example provided by the present application, wherein a Control group, a UW + oil seal group and a UW + PEG +3-OMG + oil seal group are sequentially arranged from left to right.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The embodiment of the invention provides an ice crystal-free low-temperature storage method in a supercooled state, which comprises the following steps: adding oil substances into the freezing protection solution in which the cells or tissues are suspended, completely covering the oil substances on the surface of the freezing protection solution to form a sample, and preserving the sample at low temperature.

Specifically, the method comprises the following steps:

s1, selecting or preparing a proper cryoprotectant.

The cryoprotectant solution may be a conventional cell culture medium such as DMEM (preferably DMEM containing 10% fetal bovine serum) and may be UW (the University of Wisconsin solution), wherein DMSO is an optional component in the cryoprotectant solution.

Further, the cryoprotectant also comprises polyethylene glycol and 3-O-methyl-d glucose, wherein the volume percentage concentration of the polyethylene glycol in the cryoprotectant is 3-7%, and the concentration of the 3-O-methyl-d glucose in the cryoprotectant is 0.1-0.5 mol/L; among them, Polyethylene glycol (PEG) is an impermeable cryoprotectant, which cannot permeate cell membrane, maintain extracellular osmotic pressure, and prevent cell edema, and it has a special action mechanism: breaking the hydrogen bonds of the aqueous solution thereby lowering the freezing point of the water and promoting the formation of a supercooled state of the water. 3-O-methyl-d-glucose (3-OMG) is a glucose derivative which can be actively absorbed by cells, but can not be metabolized, so that the 3-O-methyl-d-glucose can be accumulated in the cells and plays a role in maintaining the morphology and the volume of the cells, and researches show that the 3-OMG is an effective cryoprotective factor of rat primary hepatocytes. In the application, the activity of cells under the low-temperature preservation condition can be further improved by adding polyethylene glycol and 3-O-methyl-d glucose into a DMEM medium or a UW liquid, and the anchorage rate of recovered cells can be improved. Preferably, the volume percentage concentration of the polyethylene glycol in the cryoprotectant solution is 4-6%, and the concentration of the 3-O-methyl-d-glucose in the cryoprotectant solution is 0.1-0.3 mol/L.

S2, obtaining cells or tissues.

The obtained cells should be changed every day, the cells are in a logarithmic growth period during cryopreservation, the state is good, the shape and the function are normal, the obtained tissues are fresh tissues, the pollution phenomenon does not occur, the warm ischemia time meets the cryopreservation requirement, the activity and the stability of the recovered tissues and cells can be greatly improved by the treatment, and the problem of direct large-scale amplification after recovery can be effectively solved. The cells or tissues herein are selected from human or non-human mammals, including but not limited to: apes, monkeys, cows, horses, rabbits, rats, etc.

S3, suspending the cells or tissues with the cryoprotectant solution, pouring into a suitable container, such as a 15ml centrifuge tube, and allowing the cell density to reach 1 × 10⁶~5×10⁶/ml。

And S4, injecting oil substances.

And injecting a proper amount of oil substances into the freezing protection liquid in which the cells or tissues are suspended, wherein the oil substances float on the liquid surface of the freezing protection liquid and gradually and completely cover the liquid surface to form a sample, and air bubbles are prevented from being mixed in the operation process. The oily substance includes, but is not limited to, at least one of light chain mineral oil and olive oil. The amount of oil used is suitably such that it completely covers the surface of the liquid, the amount is suitably increased in consideration of the bumpiness during handling and transportation, and there should be no bubble residue between the liquid and the oil after the oil is added.

It has been found by the inventors that homogeneous ice crystal formation generally occurs at lower temperatures (≦ -40℃) and that the ice nucleation mechanism is a random movement of water molecules within the pure water. Above this temperature, however, the solid-liquid phase boundary in the impurity provides a priority for ice nucleation, resulting in the formation of heterogeneous ice crystals. Limiting the random motion of water molecules is difficult to achieve, and therefore, in creating a supercooled environment, the present application focuses on reducing heterogeneous ice crystals that form in the presence of solid phase boundaries of impurities. Under pure water conditions, interference from impurities in the liquid is avoided, and the gas/liquid plane becomes the theoretical primary site for ice crystal nucleus formation. According to the method, on the basis of the cell freezing protective solution, appropriate oil substances (including but not limited to light chain mineral oil and olive oil) which are insoluble in water are added, the oil substances completely cover the liquid surface, air and the liquid surface of the freezing protective solution are isolated, heterogeneous ice crystal nucleation sites of the gas/protective solution plane are eliminated, at the moment, under the environment of-20-0 ℃, an ice crystal forming mechanism of the cell freezing protective solution tends to homogeneous crystallization, the heterogeneous crystallization mechanism is inhibited, the freezing frequency of the cell freezing protective solution is greatly reduced, damage to cells caused by formation of ice crystals is avoided, meanwhile, the use concentration (such as DMSO) of toxic substances in the freezing protective solution can be reduced, and in addition, in the cell recovery process, the oil substances can be removed through simple physical adsorption. The method inhibits the freezing of the freezing protection solution, makes low-temperature preservation of tissues and cells at-20-0 ℃ possible, can obviously improve the survival rate of the tissues and cells, guarantees the quality of the recovered tissues and cells, and is suitable for constructing tissue cell instant libraries in short and medium periods, such as bioartificial hepatocyte libraries and the like.

And S5, storing at low temperature.

And (3) placing the sample at the temperature of precooling to 3-5 ℃ for temperature balancing for 10-20min, and then quickly placing the sample at the temperature of precooling to-20-0 ℃ for supercooling state low-temperature storage. The temperature balance can prevent the sample from generating huge temperature difference in a short time, and is favorable for protecting cells or tissues in the sample.

In addition, the invention also provides a low-temperature recovery method for ice crystal-free samples in a supercooled state, which comprises the step of recovering the samples stored by the low-temperature storage method for ice crystal-free samples in the supercooled state.

Specifically, the sample is placed at the temperature of 3-5 ℃ for re-warming for 8-12min, DMEM complete culture medium preheated to room temperature is added below the oil liquid surface by using a suction pipe, then oil substances on the freezing protection liquid surface are physically sucked by adopting the capillary action of a Pasteur suction pipe, centrifugation is carried out after uniform mixing (the centrifugation speed is 800-1200 rpm, the centrifugation time is 2-5 min), the supernatant is discarded, and then room-temperature high-sugar DMEM complete culture medium is added for recovery culture for 20-30 h.

The recovery method is simple to operate, the quality of the recovered cells or tissues is guaranteed, the survival rate of the recovered cells or tissues is remarkably improved, and the recovery method is suitable for building a short-term and medium-term tissue cell bank, such as a bioartificial hepatocyte bank.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment provides a method for storing ice crystal-free low temperature in a supercooled state, which comprises the following steps:

s1, selecting UW liquid as the freezing protection liquid.

S2, obtaining human cells and tissues.

S3, suspending human cells and tissues by using a frozen protective solution precooled to 4 ℃, injecting the human cells and tissues into a 15ml centrifuge tube, and enabling the cell density to reach 1 × 10⁶/ml。

And S4, injecting a proper amount of light chain mineral oil, wherein the light chain mineral oil floats on the liquid surface of the freezing protection liquid and gradually and completely covers the liquid surface to form a sample, and air bubbles are prevented from being mixed in the operation process.

And S5, placing the sample at the temperature of pre-cooling to 4 ℃ for temperature balancing for 15min, and then rapidly placing the sample at the temperature of pre-cooling to-16 ℃ for cold-storage in a supercooled state for 24 h.

Example 2

s1, selecting DMEM medium containing 10% fetal calf serum as a freezing protection solution.

S2, obtaining human cells and tissues.

S3, suspending human cells and tissues with a cryoprotectant solution, injecting into a 15ml centrifuge tube, and making the cell density to 5 × 10⁶/ml。

And S4, injecting a proper amount of olive oil, wherein the olive oil floats on the liquid level of the freezing protection liquid and gradually and completely covers the liquid level to form a sample, and air bubbles are prevented from being mixed in the operation process.

And S5, placing the sample at the temperature of pre-cooling to 4 ℃ for temperature balancing for 20min, and then rapidly placing the sample at the temperature of pre-cooling to-16 ℃ for supercooling state low-temperature preservation.

S6, re-warming the sample at 4 ℃ for 10min, physically absorbing oil substances on the freezing protection liquid surface by adopting the capillary action of a pasteur pipette, adding a cell complete culture medium, uniformly mixing, centrifuging at the speed of 300g/5min, re-suspending by using the cell complete culture medium, and carrying out cell culture.

Example 3

This example provides a method for cryogenically preserving ice-free crystals in a supercooled state, which is substantially the same as example 1 except that the cryoprotectant solution in this application comprises UW solution, 5% PEG and 0.2 mol/L3-OMG.

Example 4

This example provides a method for cryogenically preserving ice-free crystals in a supercooled state, which is substantially the same as example 1 except that the cryoprotectant solution in this application comprises UW solution, 3% PEG, and 0.5 mol/L3-OMG.

Example 5

This example provides cryo-preservation in a supercooled state without ice crystals, which is essentially the same as example 1 except that the cryoprotectant solution in this application comprises UW solution, 7% PEG and 0.1 mol/L3-OMG.

Comparative example 1

The present comparative example provides a conventional cryopreservation method comprising the steps of:

s1, selecting UW liquid as the freezing protection liquid.

S2, obtaining human cells and tissues.

S3, human cells,Suspending the tissue with a cryoprotectant pre-cooled to 4 deg.C, injecting into a 15ml centrifuge tube, and allowing the cell density to reach 1 × 10⁶/ml。

S4, storing the frozen protective solution suspending human cells and tissues in a refrigerator at constant-20 ℃.

Experimental example 1

The samples obtained in example 1 and comparative example 1 were frozen constantly in a refrigerator at-20 ℃ for 7 days. After 7 days, the colors of example 1 and comparative example 1 were observed vertically (see fig. 1), and then the state of example 1 and comparative example 1 was observed obliquely (see fig. 2).

As shown in fig. 1, the left side of the experimental group provided in example 1 contains 1ml of UW solution, and 1ml of light chain mineral oil is added above the solution to completely cover the surface of the UW solution, so that the UW solution is slightly transparent and has the same color as the normal temperature UW solution; the right side is a control group provided in comparative example 1, which contains 1ml of UW solution, and no other substances are added, and it is seen that it is white and has a color different from that of the normal temperature UW solution.

As shown in fig. 2, the experimental group provided in example 1 is shown below, and the UW liquid in the inclined container is in a flowing state, has the same color as the UW liquid at normal temperature, and is obviously not frozen; the upper panel is the control provided in comparative example 1, and the inclined container did not flow, and was not consistent in color with the UW solution at room temperature, and was significantly frozen.

Experimental example two

First, experiment grouping

Example 1, example 3 and comparative example 1 were grouped separately:

control group: -16 ℃ + UW liquid (i.e. comparative example 1);

UW + oil seal set: -16 ℃ + UW liquid + oil seal treatment (i.e. example 1);

UW + PEG +3-OMG + oil seal group: 16 ℃ C. + UW liquid + 5% PEG + 0.2 mol/L3-OMG + oil seal treatment (i.e., example 3).

Second, the experimental procedure

1) Taking 182cm which is 80% full of the bottom of the bottle and is completely attached to the wall²And (3) removing the culture solution from the cell culture bottle, adding a proper amount of PBS (phosphate buffer solution), slowly shaking, then removing, and washing for 3-4 times.

2) After discarding the PBS solution, 3mL of 25% trypsin solution was added, the mixture was gently shaken to spread over the bottom of the flask and placed at 37 ℃ in 5% CO₂Digesting in an incubator for 3 minutes, judging the digestion degree of cells under an inverted microscope, and stopping digestion immediately if the edges of the cells become blunt and round, the cytoplasm of the cells retract and the intercellular space widens.

3) Adding high-glucose DMEM complete culture medium containing 10% fetal calf serum for dilution to stop digestion, wherein the dilution ratio is at least 1: 5. and repeatedly blowing and beating HepG2 cells at the bottom of the flask by using a Pasteur pipette to ensure that adherent cells fall off from the bottom of the flask of the culture flask to form uniform single-cell suspension.

4) After cell counting and cell number determination, centrifugation was carried out at 1000rpm for 3 minutes, and the supernatant was discarded. The HepG2 cells were washed 2 times with PBS solution and finally centrifuged to discard the supernatant.

5) Adding pre-cooled UW solution or UW + PEG +3-OMG low-temperature protective solution into each group, and adjusting cell number to 1 × 10⁶/mL。

6) According to the method, 1mL of mineral oil is carefully added to a cell suspension by using a micro pipetting gun with a proper range, the process needs to be finely operated, bubbles are prevented from being generated between an oil liquid and an aqueous solution, the oil liquid is completely and uniformly covered on the surface of a culture solution, and then the culture solution is uniformly placed in a refrigerator at 4 ℃ for standby, and jolt is avoided in the moving process so as to prevent the continuity of the liquid level of an oil seal from being damaged.

7) The HepG2 cells are stored at low temperature in a 4 ℃ refrigerator or a-16 ℃ refrigerator for 12 hours and 24 hours, then are quickly taken out from the refrigerator and placed in the 4 ℃ refrigerator for re-heating for 10 minutes (jolting or vibration is avoided in the process), then are placed in a super clean bench, 2mL of DMEM complete culture medium preheated to room temperature is added below the oil liquid level by using a Pasteur pipette, then the mineral oil above the liquid level is sucked by using the Pasteur pipette (the mineral oil is sucked to be clean as much as possible), and are blown, uniformly mixed, centrifuged at 1000rpm for 3 minutes, and the supernatant is discarded.

8) 3mL of room temperature high-sugar DMEM complete medium was added, and the mixture was transferred to a 6-well plate and subjected to resuscitative culture for 24 hours.

9) After 24 hours of resuscitative culture, in a clean benchGently washing HepG2 cells with PBS for 3 times, adding 25% trypsin solution 0.5mL dropwise into each well of 6-well plate, shaking slightly to make it spread over the bottom of the whole culture flask, and placing at 37 deg.C and 5% CO₂Digesting in an incubator for 3 minutes, judging the digestion degree of cells under an inverted microscope, and stopping digestion immediately if the edges of the cells become blunt and round, the cytoplasm of the cells retract and the intercellular space widens. Adding high-glucose DMEM complete culture medium containing 10% fetal calf serum for dilution to stop digestion, wherein the dilution ratio is at least 1: 5. and repeatedly blowing HepG2 cells at the bottom of the bottle by using a Pasteur pipette to enable adherent cells to fall off a 6-hole plate to form uniform single-cell suspension, transferring the suspension into a 15mL centrifuge tube for labeling, centrifuging at 1000rpm for 3 minutes, and discarding the supernatant. Cell resuspension was performed by adding 1mL of high-glucose DMEM complete medium containing 10% fetal bovine serum, followed by cell counting of the cell suspension. Recovery cell adherence rate = (number of suspension cells/10)⁶×100%）

10) Centrifuging and resuspending HepG2 cells after being stored at low temperature for a period of time, after being blown evenly, using a 200 mu L micropipette to transfer 100 mu L of cell suspension into a 96-well plate and marking each group, and placing the cell suspension into a chamber with 5% CO at 37 DEG C₂Pre-culturing for 10min in an incubator, adding 10 μ L CCK8 working solution into a 96-well plate, and placing at 37 deg.C and 5% CO₂After incubation for 1 hour in the incubator, absorbance at 450nm of each group was measured using a multifunctional microplate reader. Note that no air bubbles were generated when dropping the liquid into the 96-well plate, which would affect the readings.

Thirdly, experimental results:

1) the CCK8 detection result can basically reflect the proliferation trend and the activity of the HepG2 survival cells. From fig. 3, a trend of the viability of the 3 groups of HepG2 cells over time was observed. The CCK8 detection result shows that after each group is stored at low temperature for 12 hours and 24 hours, the cell viability of each group begins to be obviously different, the cell viability of the UW + PEG +3-OMG + oil seal group is the highest, the cell viability of the Control group is the lowest next to the UW + oil seal group, and the cell viability of the other two groups has statistical significance (P is less than 0.05) compared with the cell viability of the Control group.

2) The low-temperature preservation time is 12 hours, the anchorage rate of the recovered cells of the UW + PEG +3-OMG + oil seal group is the highest and reaches 90.8 percent after the recovery culture is carried out for 24 hours, and the UW + oil seal group is 83.2 percent, so that the two groups have statistical significance (P is less than 0.05) compared with 23.3 percent of the Control group. After the low-temperature preservation time is prolonged to 24 hours, the recovery adherence rate of the UW + PEG +3-OMG + oil seal group is 80.9% at most, and the recovery adherence rate has statistical significance (P is less than 0.05) compared with 22.1% of the Control group. See table 1 for details.

TABLE 1 adhesion rate of HepG2 recovered cells

In summary, compared with the existing clinical tissue and cell bank low-temperature storage mode, the ice crystal-free low-temperature storage method in the supercooled state can inhibit the freezing of the freezing protection solution at-20 ℃ to 0 ℃, has obvious effects of inhibiting the formation of ice crystals and preventing the growth of the ice crystals, enables the tissue cell bank and the like to avoid using the freezing protection solution with cytotoxicity such as DMSO and the like, and is more beneficial to constructing a safe and nontoxic biological sample bank. Compared with the conventional normal-temperature storage and short-term storage time of 4 ℃, the ice crystal-free low-temperature storage method in the supercooled state can be used for carrying out long-time (> 2 days) low-temperature storage on tissue cells at lower temperature, so that ischemia-reperfusion injury is relieved, the survival rate of the recovered tissue cells is higher, and the original biological characteristics can be maintained. Compared with the vitrification cryopreservation technology applied to the field of assisted reproduction at present, the cryo-preservation method without ice crystals in the supercooled state has the advantages that the volume limit of the tissue is small, the density and the cell quantity of the preserved cells are larger, and the cryo-preservation method can be carried out without complex equipment and devices.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for storing ice crystal-free low temperature under a supercooled state is characterized by comprising the following steps: adding oil substances into the freezing protection solution in which cells or tissues are suspended, so that the oil substances completely cover the surface of the freezing protection solution to form a sample, and storing the sample at low temperature; the oil substance comprises at least one of light chain mineral oil and olive oil; the freezing protection solution comprises a DMEM medium or UW solution; cryo-preserving the sample comprises: and (3) placing the sample at a pre-cooling temperature of 3-5 ℃ for temperature balancing for 10-20min, and then placing the sample at a pre-cooling temperature of-20-0 ℃ for supercooling state low-temperature storage.

2. The method according to claim 1, wherein the cell density in the cryoprotectant solution is 1 x 10⁶~5×10⁶/ml。

3. The method according to claim 1, wherein the cryoprotectant solution further comprises polyethylene glycol and 3-O-methyl-d-glucose, wherein the concentration of the polyethylene glycol in the cryoprotectant solution is 3-7% by volume, the concentration of the 3-O-methyl-d-glucose in the cryoprotectant solution is 0.1-0.5mol/L, and the cryoprotectant solution does not contain DMSO.

4. The method of claim 1, wherein the cells or tissues are selected from human or non-human mammals.

5. A cryo-resuscitation method without ice crystals in a supercooled state, comprising resuscitating a sample stored by the cryo-conservation method without ice crystals in a supercooled state according to any of claims 1 to 4.

6. The cryo-recovery method without ice crystals in a supercooled state according to claim 5, wherein recovering the sample comprises: and (2) putting the sample at the temperature of 3-5 ℃ for rewarming for 8-12min, adding a complete culture medium preheated to room temperature below the oil liquid level by using a suction pipe, then physically absorbing the oil substances on the freezing protection liquid level, mixing uniformly, centrifuging, removing supernatant, adding a room-temperature high-sugar complete culture medium, and performing resuscitation culture for 20-30 h.

7. The cryo-resuscitation method for ice crystal-free under supercooled state of claim 5, wherein said oil substance is physically sucked up by capillary action of Pasteur pipette.