A method for in situ cryopreservation of adherent cells

Abstract

The application discloses an in-situ freezing method for adherent cells, and the method obtains a "sandwich layer" structure by using Matrigel and gelatin before freezing; the bottom layer can prevent the middle layer of cells from falling off due to temperature change and prevent the cells from being frozen; the uppermost layer is used to protect the middle layer of cells from being frozen, and can be removed well during rewarming, thereby facilitating use and subculture operation. The method does not need to digest cells and collect cells, and avoids damage to cells caused by operations such as digestion and blowing before freezing; the method solves the problems of limitation of adherent materials and falling off of adherent cells caused by non-affinity materials in the in-situ freezing of adherent cells; after thawing and recovery, the cells do not need to be re-inoculated and subcultured for a long time to recover the cell state, and the cells can be used after thawing, thereby shortening the culture time of the cells after recovery; the survival rate of the adherent cells after recovery can reach 73.32%, the survival rate is good, the adherent state after recovery is good, there is no obvious falling off, and the cells have normal morphology.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a method for in situ cryopreservation of adherent cells. Background Technology

[0002] Cell cryopreservation is commonly used to preserve seed cells for immediate use; it avoids genetic variations and morphological changes caused by repeated passages during long-term cell culture; furthermore, cryopreservation reduces the cost of maintaining cell condition. Currently, most methods for cryopreserving adherent cells involve suspension cryopreservation. This involves detaching cells from the adherent surface, centrifuging to collect them, suspending them in a prepared cryopreservation solution (usually a mixture of complete culture medium, FBS, and 10% DMSO), aliquoting them into sterile cryovials, and incubating at 4°C for 30 minutes, -20°C for 30 minutes, and -80°C overnight. The cells are then stored in liquid nitrogen for long-term preservation. When needed, the cells are thawed, inoculated into culture dishes or other containers, and allowed to adhere and grow. They are passaged 2-3 times to restore their cell condition before use. This cryopreservation method is labor-intensive, resource-intensive, and time-consuming.

[0003] This invention develops a method that enables adherent cells to be cryopreserved directly without digestion. The method is simple and easy to operate, solves the problems of adherent cells detaching in sheets and cells being damaged by freezing during in situ cryopreservation, and shortens the culture time from cell thawing to restoration to the original state, thereby reducing the cost of cryopreserved cell thawing and culture. Summary of the Invention

[0004] The first aspect of this invention aims to provide a method for in situ cell cryopreservation; this method is based on the phase transition principle of gels;

[0005] High-temperature shrinkage thermosensitive hydrogel: When the temperature is below the phase transition temperature, the polymer is soluble in water to form a free-flowing liquid. When the temperature rises above the phase transition temperature, the aqueous solution of the polymer undergoes a phase transition to form a non-chemically cross-linked gel.

[0006] Low-temperature shrinkage thermosensitive hydrogel: When the temperature is above the phase transition temperature, the polymer can dissolve in water to form a free-flowing liquid. When the temperature drops below the phase transition temperature, the aqueous solution of the polymer undergoes a phase transition to form a non-chemically cross-linked gel.

[0007] The above process is reversible, and the concentration level also affects the phase transition temperature.

[0008] A second aspect of the present invention is to provide a resuscitation method.

[0009] The technical solution adopted in this invention is:

[0010] A first aspect of the present invention provides a method for in situ cell cryopreservation, comprising the following steps:

[0011] S1: The adhesion surface of the culture carrier is coated with a high-temperature shrinkage thermosensitive hydrogel.

[0012] S2: Inoculate cells on the adhesion surface of the culture carrier;

[0013] S3: After encapsulating the adherent cells with a low-temperature shrinkage thermosensitive hydrogel, pre-cool them below their sol temperature.

[0014] S4: After covering the cells with cryopreservation solution, freeze the cells.

[0015] Preferably, the above-mentioned high-temperature shrinkage thermosensitive hydrogel is solid above 30°C and liquid below 10°C. Preferably, the above-mentioned high-temperature shrinkage thermosensitive hydrogel includes Matrigel matrix gel.

[0016] Preferably, the low-temperature shrinkage thermosensitive hydrogel is solid below 10°C and liquid above 30°C. Preferably, the low-temperature shrinkage thermosensitive hydrogel includes gelatin.

[0017] Preferably, the Matrigel matrix adhesive described in step S1 above needs to be diluted with a culture medium before coating, with a dilution ratio of 1:1 to 100.

[0018] Preferably, the culture medium used for dilution is a serum-free culture medium.

[0019] Preferably, the coating process in step S1 specifically includes coating the Matrigel matrix adhesive below the solidification temperature, and then placing it above the solidification temperature until the Matrigel matrix adhesive solidifies.

[0020] Preferably, the solidification temperature is 10-37°C.

[0021] Preferably, the mass fraction of the gelatin is 1-10%.

[0022] Preferably, the precooling conditions are a temperature below 10°C and a time of 1-10 minutes.

[0023] Preferably, the low-temperature shrinkage thermosensitive hydrogel in step S3 contains an antifreeze agent; or, after pre-cooling, a culture medium containing an antifreeze agent is added for incubation, and then the culture medium is removed.

[0024] Preferably, the incubation conditions are 10–20°C for 20–60 minutes.

[0025] Preferably, the antifreeze component includes at least one of trehalose, hydroxyethyl starch, antifreeze protein, polyol, glucose, and sucrose.

[0026] Preferably, the antifreeze protein includes at least one of antifreeze glycoprotein, antifreeze protein I, antifreeze protein II, antifreeze protein III, and antifreeze protein IV.

[0027] Preferably, the polyols mentioned above include at least one of glycerol, sorbitol, mannitol, etc.

[0028] Preferably, the antifreeze agent in step S3 is trehalose with a concentration of 20-50 mM.

[0029] Preferably, the culture medium used for incubation is a serum-free culture medium.

[0030] Preferably, the temperature range below the sol temperature is 1 to 10°C.

[0031] Preferably, the cryopreservation solution in S4 above contains an antifreeze component.

[0032] Preferably, the cryopreservation solution includes STEMCELL CryoStor CS10.

[0033] Preferably, the antifreeze component in step S4 includes at least one of trehalose, hydroxyethyl starch, antifreeze protein, glucose, sucrose, and polyol.

[0034] Preferably, the antifreeze component in step S4 is hydroxyethyl starch.

[0035] Preferably, the hydroxyethyl starch has a mass fraction of 3-10%.

[0036] Preferably, the cryopreservation conditions are any of the following:

[0037] a) Cool to 2-6℃ for 30-60 min, incubate at -18--22℃ for 1-4 h, and store at -75--85℃;

[0038] b) Programmatic cooling, with a cooling rate set to -(0.1-5)℃ / min, cooling to -75~-85℃ for storage.

[0039] Preferably, the storage time is 0 to 15 days.

[0040] A second aspect of the present invention provides a cell resuscitation method, comprising thawing and culturing cells frozen using the method described in the first aspect of the present invention.

[0041] Preferably, the thawing conditions are any of the following:

[0042] a) Thaw at 37–40℃ for 2–5 minutes;

[0043] b) Temperature rise: The temperature program is set to a rapid temperature rise in the range of 0 to 10°C, with a rise rate of 5 to 20°C / min and a rise time of ≤ (3 to 8) min.

[0044] Preferably, the specific steps of the resuscitation culture include: removing the frozen liquid, washing, adding serum culture medium and culturing for 1-5 hours.

[0045] Preferably, the concentration of the serum is 10-50%.

[0046] The beneficial effects of this invention are:

[0047] This invention develops an in situ cryopreservation method for adherent cells. This method involves treating the cells on the carrier surface with a gel before cryopreservation, resulting in a "sandwich" structure, followed by cryopreservation. The bottom layer of the carrier surface is a matrix gel that gels at 37°C and remains liquid at low temperatures (high-temperature shrinkage type), providing a cryopreservation substrate for the middle layer of cells and preventing them from detaching due to temperature changes. The second bottom layer also provides a protective layer for the middle layer of cells, preventing frostbite. The top layer is a low-temperature gel that remains liquid at 37°C (low-temperature shrinkage type), which can be coated with cryoprotectants to protect the middle layer of cells from frostbite and can be easily removed during thawing, facilitating use and passage.

[0048] This method eliminates the need for cell digestion and collection, avoiding damage caused by pre-freezing digestion and pipetting. It expands the cryopreservation method beyond simple suspension-dispersion cryopreservation, solving the problems of limited adherent materials and cell detachment due to non-affinity materials in in situ cryopreservation. Adding antifreeze components during cryopreservation mitigates severe frost damage associated with in situ cryopreservation. After thawing, cells do not require re-inoculation or long-term passage culture to restore their state; they are ready to use immediately after thawing, shortening the post-thawing culture time. The viability of thawed adherent cells reaches 73.32%, demonstrating good survival rates and excellent adherence with no significant detachment and normal morphology. This method can provide a reference for the short- to medium-term preservation of cell-containing (especially adherent) bioartificial organs, reducing preservation costs. Attached Figure Description

[0049] Figure 1 This is a diagram showing the cell state after resuscitation in Example 1.

[0050] Figure 2 This is a diagram showing the cell state after resuscitation in Example 2.

[0051] Figure 3 This is a diagram showing the state of cells after resuscitation in Comparative Example 1.

[0052] Figure 4This is a graph showing the state of cells after resuscitation in Comparative Example 2.

[0053] Figure 5 This is a graph showing the state of cells after resuscitation in Comparative Example 3. Detailed Implementation

[0054] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0055] The basal culture medium was DMEM / F12, brand name Gibco, purchased from Guangzhou Aisha, and its composition is shown in Table 1; the complete culture medium was the above basal culture medium with added fetal bovine serum.

[0056] Table 1

[0057] form liquid concentration 1X Specification 500mL pH 7.2～7.4 L-Alanyl-L-Glutamine 2.5mM <![CDATA[NaHCO3]]> 1200mg / L D-glucose 3151mg / L Sodium pyruvate 0.5mM HEPES buffer 15mM Phenol red indicator 8.1 mg / L

[0058] This invention provides a method for direct in-situ cryopreservation of adherent cells without digestion, particularly suitable for in-situ cryopreservation of cells on adhesive surfaces with significant differences in glass transition temperature, comprising the following steps:

[0059] 1. Adhesion surface treatment

[0060] Before seeding cells onto the adhesion surface of the carrier (which may be made of metal, metal oxide, polyurethane, etc.), the carrier surface needs to be treated.

[0061] a) Coat the adhesion surface with a high-temperature shrinkage thermosensitive hydrogel (such as matrix gel (8-13 mg / ml)). Mix and dilute the matrix gel and serum-free culture medium at a volume ratio of 1:1-100, and then coat the adhesion surface (while the matrix gel is still relatively cold and before it solidifies; the solidification temperature of the matrix gel is generally 10-20℃ (the temperature can be appropriately increased to speed up the solidification rate depending on the concentration and composition). After coating the carrier adhesion surface, let it stand at 37℃ for 20-60 minutes until the matrix gel slightly solidifies.

[0062] 2. Cell adhesion culture

[0063] After the cells have reached 70%-100% confluence, pre-freezing treatment and cryopreservation are carried out.

[0064] 3. Pre-freezing treatment

[0065] Use gels to encapsulate the carrier and adhere to the cells on the surface. Select gels that gel at low temperatures and can become liquid at 37°C (low-temperature shrinkage thermosensitive hydrogels), such as gelatin.

[0066] a) Remove the cell culture medium, wash with PBS 3-5 times, add 1-10% gelatin solution, pre-freeze at 1℃-10℃ for 1-10 min to allow it to gel. After gelation, incubate with serum-free culture medium containing cryoprotectants (such as trehalose, hydroxyethyl starch, antifreeze protein, etc.) at 10-20℃ for 20-60 min to allow the cryoprotectants to penetrate into the gel network.

[0067] b) Alternatively, remove the cell culture medium, wash with PBS 3-5 times, add 1-10% gelatin solution containing gel solution antifreeze components (such as trehalose, hydroxyethyl starch, antifreeze protein, etc.), and pre-freeze at 1℃-10℃ for 2-10 minutes to allow it to shrink into a gel.

[0068] Thus, a "sandwich" structure is formed before cryopreservation. The bottom layer, on the carrier adhesion surface, is a matrix gel that gels at 37°C and is in a low-temperature liquid state, providing a cryopreservation substrate for the middle layer cells and preventing them from detaching due to temperature changes. This second layer also provides a protective layer for the cryopreservation of the middle layer cells, preventing frostbite. The top layer is a low-temperature gel-state protective gel (such as gelatin) that is liquid at 37°C, mixed with cryoprotectants (such as trehalose, hydroxyethyl starch, and antifreeze proteins) to protect the middle layer cells from frostbite. This top layer of gel can be easily removed during rewarming, facilitating use and passage.

[0069] The standard temperature for cell culture is 37°C. At this temperature, the bottom matrix gel is solid, while the top gelatin is liquid. During cryopreservation, the temperature is lowered from 37°C to -80°C. During this cooling process, the bottom matrix gel (containing the cells) changes from solid to liquid, while the top gelatin changes from liquid to solid. This phase transition between the upper and lower gel layers effectively protects the cells and reduces ice crystal damage. Upon thawing from -80°C to 37°C, the bottom matrix gel (containing the cells) changes from liquid to solid, while the top gelatin changes from solid to liquid.

[0070] 4. Cell cryopreservation

[0071] Remove the cryoprotectant from the culture medium and add cryopreservation solution. Use STEMCELL CryoStor CS10 cryopreservation solution, which can contain antifreeze ingredients (trehalose, hydroxyethyl starch, antifreeze proteins, etc.).

[0072] The cryopreservation procedure is as follows:

[0073] a) Place in 4℃ to cool down for 30-60 minutes, then transfer to -20℃ for incubation for 1-4 hours, and finally store at -80℃.

[0074] b) Alternatively, place the frozen cells directly in a gradient cooling box, set the cooling rate to -(0.1-5)℃ / min, and cool to -80℃ for storage.

[0075] Cells cryopreserved in situ can be thawed using conventional methods, or as follows.

[0076] 1. Cell thawing

[0077] a) Remove the frozen cells and immediately place them in a 37-40℃ water bath to thaw for 2-5 minutes. Do not let water get into the bottle opening to prevent contamination.

[0078] b) Alternatively, place the frozen cells in a thawing chamber and rapidly increase the temperature in the 0℃-10℃ range using a program, with a heating rate of 5-20℃ / min, and keep the entire heating process within 3-8 minutes.

[0079] 2. Resuscitation and Cultivation

[0080] Discard all cryopreservation liquid in a biosafety cabinet, wash with PBS 2-5 times to remove residual cryopreservation liquid and gel protective layer, add 3-5 mL of culture medium containing 10-50% serum, and incubate at 37°C in a 5% CO2 incubator for 1-5 hours until the cells recover their state. They can be passaged or used normally after about 12-24 hours.

[0081] Example 1

[0082] The in-situ cryopreservation method for adherent cells on the surface of an alumina sheet (AAO) is as follows:

[0083] 1. AAO adhesion surface treatment

[0084] a) Apply the AAO adhesion surface with a matrix gel diluted 1:50 (Matrigel (10 mg / ml): serum-free culture medium) by volume, and incubate at 37°C for 40 min.

[0085] 2. Cell Culture (The following examples and comparative examples all use HK2 cells as an example)

[0086] a) After cell adhesion is complete, add complete culture medium and culture until 80% of the cells have merged.

[0087] 3. Pre-freezing treatment

[0088] a) Wash with PBS 3-5 times, then coat the cells adhering to AAO with gelatin (3% by mass, containing 35mM trehalose) and pre-cool at 4°C (pre-cool for about 3 minutes).

[0089] 4. Freezing

[0090] a) Add STEMCELL CryoStor CS10 (the cryopreservation solution contains 0.33M trehalose).

[0091] b) Place it in a gradient cooling box, set the cooling gradient to -1℃ / min, and store it at -80℃ for 7 days.

[0092] The cell resuscitation methods are as follows:

[0093] a) Remove the frozen cells and immediately place them in a 39°C water bath for 3 minutes to thaw. Do not let water get into the bottle opening to prevent contamination.

[0094] b) Discard all frozen liquid under aseptic conditions in a clean bench, wash with PBS 1-2 times to dilute and remove residual frozen liquid, add 3 mL of culture medium containing 30% serum, and incubate at 37°C and 5% CO2 for 1 hour to restore the condition.

[0095] Cell status was assessed using the CCK8 assay kit. The viability of adherent cells after resuscitation reached 61.58%, indicating high cell viability. Immediately after resuscitation, Calcein / PI double staining was performed on the adherent cells, showing good adhesion and normal morphology. Figure 1 ).

[0096] Example 2

[0097] The in-situ cryopreservation method for adherent cells on the surface of a 12-well plate is as follows:

[0098] 1.12 Orifice Plate Adhesion Surface Treatment

[0099] a) Apply the matrix gel diluted 1:50 (Matrigel: serum-free culture medium) to the adhesion surface of the culture wells, and then incubate at 37°C for 30 min in a CO2 incubator.

[0100] 2. Cell Culture

[0101] a) After cell adhesion is complete, add complete culture medium and culture until 80% of the cells have merged.

[0102] 3. Pre-freezing treatment

[0103] a) Wash with PBS 3-5 times, then coat the cells adhering to AAO with gelatin (3% by mass, containing 35mM trehalose) and pre-cool at 4°C (pre-cool for about 3 minutes).

[0104] 4. Freezing

[0105] a) Add cryopreservation solution. The cryopreservation solution selected is STEMCELL CryoStor CS10, which contains 6% (by mass) starch.

[0106] b) Place it in a gradient cooling box, set the cooling gradient to -0.5℃ / min, and store it at -80℃ for 7 days.

[0107] The cell resuscitation methods are as follows:

[0108] a) Remove the frozen cells and immediately place them in a 38.5°C water bath for 3 minutes to thaw. Do not let water get into the bottle opening to prevent contamination.

[0109] b) Discard all frozen liquid under aseptic conditions in a clean bench, wash with PBS 1-2 times to dilute and remove residual frozen liquid, add 3 mL of culture medium containing 50% serum, and incubate at 37°C and 5% CO2 for 1.5 h to restore the liquid to its original state.

[0110] Cell status was assessed using the CCK8 assay kit. The viability of adherent cells after resuscitation reached 73.32%, indicating high cell viability. Immediately after resuscitation, Calcein / PI double staining showed good adhesion, no significant detachment, and normal morphology of the adherent cells. Figure 2 ).

[0111] Comparative Example 1

[0112] The in-situ cryopreservation method for adherent cells on the surface of a 12-well plate is as follows:

[0113] 1.12 Orifice Plate Adhesion Surface Treatment

[0114] a) Apply the substrate gel diluted 1:50 (volume ratio of Matrigel substrate gel to serum-free culture medium) to the adhesion surface and incubate at 37°C for 30 min.

[0115] 2. Cell Culture

[0116] a) After cell adhesion is complete, add complete culture medium and culture until 80% of the cells have merged.

[0117] 3. Freezing

[0118] a) Add cryopreservation solution. The cryopreservation solution selected is STEMCELL CryoStor CS10, which contains 6% (by mass) starch.

[0119] b) Place it in a gradient cooling box, set the cooling gradient to -0.5℃ / min, and store it at -80℃ for 7 days.

[0120] The cell resuscitation methods are as follows:

[0121] a) Remove the frozen cells and immediately place them in a 38.5°C water bath for 3 minutes to thaw. Do not let water get into the bottle opening to prevent contamination.

[0122] b) Discard all frozen liquid under aseptic conditions in a clean bench, wash with PBS 1-2 times to dilute and remove residual frozen liquid, add 3 mL of culture medium containing 50% serum, and incubate at 37°C and 5% CO2 for 1.5 h to restore the liquid to its original state.

[0123] Cell status was assessed using the CCK8 assay kit. After thawing, the viability of adherent cells was only 33.86%. Immediately after thawing, Calcein / PI double staining of the adherent cells showed good cell adhesion with no significant detachment, but also significant frost damage and high cell death. Figure 3 ).

[0124] Comparative Example 2

[0125] The in-situ cryopreservation method for adherent cells on the AAO membrane adhesion surface is as follows:

[0126] 1. Surface treatment for AAO film adhesion

[0127] a) Apply the substrate gel diluted 1:50 (volume ratio of Matrigel substrate gel to serum-free culture medium) to the adhesion surface and incubate at 37°C for 40 min.

[0128] 2. Cell Culture

[0129] a) After cell adhesion is complete, add complete culture medium and culture until 80% of the cells have merged.

[0130] 3. Pre-freezing treatment

[0131] a) Wash cells 3-5 times with PBS. Dilute the matrix gel 1:5 (Matrigel: serum-free medium) by volume, and add trehalose (working concentration 35mM). While the matrix gel mixture is still cold, coat the cells and incubate at 37°C for 30 minutes to allow it to solidify.

[0132] 4. Freezing

[0133] a) Add cryopreservation solution. The cryopreservation solution selected is STEMCELL CryoStor CS10, which contains 0.33M trehalose.

[0134] b) Place it in a gradient cooling box, set the cooling gradient to -1℃ / min, and store it at -80℃ for 7 days.

[0135] The cell resuscitation methods are as follows:

[0136] a) Remove the frozen cells and immediately place them in a 39°C water bath for 3 minutes to thaw. Do not let water get into the bottle opening to prevent contamination.

[0137] b) Discard all frozen liquid under aseptic conditions in a clean bench, and wash with PBS 1-2 times to remove residual frozen liquid.

[0138] c) Incubate at 4°C for 10 min to remove the upper layer of Matrigel matrix gel.

[0139] d) Add 3 mL of culture medium containing 30% serum and incubate at 37°C in a 5% CO2 incubator for 1 h to restore the condition.

[0140] Cell status was assessed using the CCK8 assay kit; the viability of adherent cells after resuscitation was only 15%. Further staining with Calcein / PI double staining after resuscitation revealed significant cell detachment and substantial cell death. Figure 4 ).

[0141] Comparative Example 3

[0142] The in-situ cryopreservation method for adherent cells on the AAO membrane adhesion surface is as follows:

[0143] 1. AAO adhesion surface treatment

[0144] a) Use a 1:50 (Matrigel: serum-free culture medium) volume ratio to coat the AAO adhesion surface, and then incubate at 37°C for 40 min in a CO2 incubator.

[0145] 2. Cell Culture

[0146] a) After cell adhesion is complete, add complete culture medium and culture until 80% of the cells have merged.

[0147] 3. Pre-freezing treatment

[0148] a) Wash with PBS 3-5 times, then coat the cells adhering to AAO with gelatin (3% by mass, containing 35mM trehalose slurry) and pre-cool at 4°C (pre-cool for about 3 minutes).

[0149] 4. Freezing

[0150] a) Add cryopreservation solution. The cryopreservation solution is a self-made 50% FBS + 40% complete culture medium + 10% DMSO cryopreservation solution.

[0151] b) Place it in a gradient cooling box, set the cooling gradient to -1℃ / min, and store it at -80℃ for 7 days.

[0152] The cell resuscitation methods are as follows:

[0153] a) Remove the frozen cells and immediately place them in a 39°C water bath for 3 minutes to thaw. Do not let water get into the bottle opening to prevent contamination.

[0154] b) Discard all frozen liquid under aseptic conditions in a clean bench, wash with PBS 1-2 times to dilute and remove residual frozen liquid, add 3 mL of culture medium containing 30% serum, and incubate at 37°C and 5% CO2 for 1 hour to restore the condition.

[0155] Cell status was assessed using the CCK8 assay kit. After resuscitation, the viability of adherent cells was low, only 13.09% and 2.85%. Immediately after resuscitation, Calcein / PI double staining showed good adhesion and minimal detachment, but significant cell death. Figure 5 ).

[0156] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A method for in situ cell cryopreservation, comprising the following steps: S1: The adhesion surface of the culture medium is coated with Matrigel matrix adhesive; S2: Inoculate cells on the adhesion surface of the culture carrier; S3: After encapsulating the adherent cells with gelatin, pre-cool them below their sol temperature; S4: After covering the cells with cryopreservation solution, the cells are cryopreserved. The cryopreservation solution is STEMCELL CryoStor CS10.

2. The method according to claim 1, characterized in that, Before coating, the Matrigel matrix adhesive described in step S1 needs to be diluted with a culture medium at a ratio of 1:1 to 100.

3. The method according to claim 1 or 2, characterized in that, The coating process in step S1 specifically includes coating the Matrigel matrix below the solidification temperature, and then placing it above the solidification temperature until the Matrigel matrix solidifies.

4. The method according to claim 1, characterized in that, The gelatin has a mass fraction of 1-10%, and the pre-cooling conditions are a temperature below 10°C and a time of 1-10 minutes.

5. The method according to claim 1 or 4, characterized in that, The gelatin in step S3 contains an antifreeze agent.

6. The method according to claim 1, characterized in that, After pre-cooling, add a culture medium containing an antifreeze and incubate, then remove the culture medium; the incubation conditions are 10-20℃ for 20-60 min.

7. The method according to claim 5, characterized in that, In step S3, the antifreeze agent is trehalose with a concentration of 20–50 mM.

8. The method according to claim 1, characterized in that, The cryopreservation solution in step S4 contains an antifreeze component, which is hydroxyethyl starch with a mass fraction of 3-10%.

9. The method according to claim 1, characterized in that, The cryopreservation conditions are any of the following: a) Cool to 2-6℃ for 30-60 min, incubate at -18--22℃ for 1-4 h, and store at -75--85℃; b) Programmatic cooling, with a cooling rate set to -(0.1~5)℃ / min, cooling to -75~-85℃ for storage.

10. A cell resuscitation method, comprising thawing and culturing cells frozen using the method described in any one of claims 1 to 9.

11. The method according to claim 10, characterized in that, The thawing conditions are any of the following: a) Thaw at 37–40℃ for 2–5 minutes; b) Temperature rise: The temperature range of 0 to 10℃ is set to rapid temperature rise, with a temperature rise rate of 5 to 20℃ / min and a total temperature rise time of ≤ (3 to 8) min.

12. The method according to claim 11, characterized in that, The specific steps of the resuscitation culture include: removing the frozen liquid, washing, and adding serum culture medium for resuscitation culture.

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