A cell freezing medium for organoid cryopreservation and its preparation method and application

Abstract

The application belongs to the technical field of cell cryopreservation solution, and particularly relates to a cell cryopreservation solution for organoid cryopreservation, a preparation method and application thereof. The cell cryopreservation solution for organoid cryopreservation provided by the application at least comprises lomerizine and D-sorbitol. The cell cryopreservation solution provided by the application has a high cell recovery rate after organoid cryopreservation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell cryopreservation solution, and particularly relates to a cell cryopreservation solution for organoid cryopreservation, a preparation method and application thereof. BACKGROUND

[0002] An organoid is a three-dimensional (3D) cell culture, which is cultivated in vitro and includes a self-renewing stem cell population that can differentiate into multiple organ-specific cell types, has similar spatial organization to the corresponding organ, and can reproduce part of the function of the corresponding organ, thereby providing a highly physiologically relevant system. Therefore, as an important emerging frontier technology, it can help disease research and treatment, and currently plays an increasingly important role in the fields of biology and medicine.

[0003] The most common cryoprotectant for organoid cryopreservation is dimethyl sulfoxide (DMSO) or high-concentration glycerol. DMSO is considered to be the best protective agent, but at certain concentrations it is toxic to cells and can have harmful effects on the physiological functions and activities of cells. Compared with DMSO, glycerol is more friendly to cells, but as a cryoprotective agent, it has a poor effect. Apoptosis easily occurs during the cryopreservation process, resulting in failure of subsequent recovery culture. In recent years, new cryoprotective agents such as D-sorbitol have been used in the low-temperature cryopreservation of cells. D-sorbitol is a sugar-containing polyol that can protect cells by stabilizing cell membranes at low temperatures. However, there are still many problems when it is used alone, for example, D-sorbitol cannot protect cells from apoptosis caused by oxidative stress.

[0004] Patent CN115251045A discloses a cell cryopreservation solution containing an aqueous inorganic salt solution, DMSO, polyvinyl alcohol, polyethylene glycol, vitamins, and hydroxyethyl starch. The cell cryopreservation solution can be stored at 2-8℃, does not need to be thawed in advance when used, can be used immediately, greatly improves the convenience of operation, and has excellent cell viability after cell recovery during cryopreservation of cells. However, this patent cannot ensure that DMSO is completely removed during the thawing and washing process, which may have a certain toxic effect on cells.

[0005] Calcium is an important mediator of cell necrosis in the process of irreversible cell damage or cell death. During the process of cell cryopreservation, due to the sharp drop in temperature, unbalanced free radicals are formed, which increase the concentration of calcium ions in cells and promote apoptosis. Lemildipine is a dihydropyridine calcium antagonist that can bind to the DHP binding site of the cell membrane potential-dependent calcium channel and inhibit the influx of calcium ions. Previous studies have shown that lemildipine can protect sperm during the process of sperm cryopreservation. Therefore, as an additive, lemildipine can improve the effect of low-temperature preservation of cells by inhibiting calcium ion channels, and has great potential.

[0006] Therefore, the existing cryopreservation solution technology has the following problems:

[0007] 1) The organoids in the prior art are usually prone to apoptosis during the freezing process, and the success rate is low after resuscitation culture.

[0008] 2) The prior art needs to go through a conventional "slow freezing" process, which is time-consuming.

[0009] 3) The existing cryopreservation solution contains components such as glycerol, DMSO, fetal bovine serum and human serum albumin solution, which has the problems of cytotoxicity, uncertain composition, possible introduction of bacterial and viral contamination sources. SUMMARY

[0010] To solve the problems of the prior art, the present application provides a cell cryopreservation solution for organoid freezing, and a preparation method and application thereof.

[0011] The technical solution provided by the present application is as follows:

[0012] A cell cryopreservation solution for organoid freezing, at least comprising lomerizine and D-sorbitol, the molar ratio of which is (0.9-1.1) x 10 -6 :(0.5-1.0).

[0013] Based on the above technical solution:

[0014] The role of D-sorbitol is to stabilize the cell membrane at low temperature; the role of lomerizine is to protect cells from oxidative stress damage; lomerizine makes up for the function of D-sorbitol that cannot protect cells from oxidative stress, thereby improving the effect of cell cryopreservation and significantly improving the survival rate of cells.

[0015] Lomerizine has the molecular formula C 20 H 22 Cl2N2O6. It is a calcium antagonist, also called calcium channel blocker, which mainly blocks the calcium ion channel on the cell membrane of myocardial and vascular smooth muscle cells, inhibits the influx of extracellular calcium ions, and causes functional changes in cardiovascular tissues and organs. The drug is generally used for hypertension, coronary heart disease and arrhythmia.

[0016] In the present application, it is combined with the DHP binding site of the cell membrane membrane potential dependent calcium channel to inhibit the influx of calcium ions, thereby avoiding irreversible cell damage or cell death.

[0017] Specifically, it further comprises a phosphate buffer, wherein:

[0018] The molar concentration of lomerizine is 0.5-1.1 μM, preferably 0.9-1.1 μM, and more preferably 1 μM.

[0019] The molar concentration of D-sorbitol is 0.5-1.0 mol / L.

[0020] The pH of the phosphate buffer is 7.3-7.5.

[0021] Specifically, it further comprises vitamin C, and the concentration of vitamin C is 0.09-0.11 g / mL.

[0022] The application further provides a preparation method of the cell cryopreservation solution for organoid cryopreservation, comprising the following steps: mixing the components according to the amount of the formula, and obtaining the cell cryopreservation solution.

[0023] For example, the cell cryopreservation solution is obtained by mixing felodipine and D-sorbitol, and then adding and stirring the phosphate buffer and the vitamin.

[0024] The application further provides an application of the cell cryopreservation solution for organoid cryopreservation, which is used as a cell cryopreservation solution for organoid cryopreservation.

[0025] A specific use method comprises the following steps:

[0026] 1) Cryopreservation of organoids: centrifuge 800 g of the organoid suspension to be cryopreserved for 5 minutes, remove the supernatant, and directly resuspend the cell precipitate after centrifugation with the cryopreservation solution to obtain a cryopreservation density of 1-10 x 10 6 The container containing the organoids to be cryopreserved and the cell cryopreservation solution is immersed in a -196℃ liquid nitrogen tank, so that the organoids are preserved.

[0027] 2) Resuscitation of organoids: take the container containing the organoids to be cryopreserved and the cell cryopreservation solution out of the liquid nitrogen, and perform water bath at 37℃ until the cells are thawed, to obtain the organoids after thawing and resuscitation, then add PBS, centrifuge at 800 g for 5 minutes to remove the supernatant, and then wash once with PBS, and resuspend the washed organoids with an organoid culture medium, to obtain the organoids after thawing, removing the cryopreservation solution, and resuscitation.

[0028] In the above technical solution:

[0029] D-sorbitol can reduce the number and size of ice crystals formed when the cells are cryopreserved. Therefore, by using the cell cryopreservation solution of the application, the cells and the cell cryopreservation solution can be mixed, and then the complex "slow freezing" process of conventional pre-programmed cooling and then transferring to liquid nitrogen can be omitted, so that the cells can be directly placed in liquid nitrogen for preservation, and a high cell survival rate can be maintained.

[0030] Specifically, the organoids are lung cancer organoids, breast cancer organoids, esophageal cancer organoids, pancreatic cancer organoids or gastric cancer organoids of mice. The mice are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the corresponding tumors of the mice are constructed by chemical induction or genetic engineering method, and the corresponding tumor tissues of the mice are taken for organoid culture.

[0031] Specifically, the organoids are normal mouse liver organoids, lung organoids or small intestine organoids. The mice are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the corresponding tissues and organs of the mice are taken for organoid culture.

[0032] The lemiripril and D-sorbitol in the application have good compatibility with cells and no toxic effect. Compared with the traditional glycerol and DMSO used as cell cryopreservation solution, the application does not need to use glycerol with different concentrations for gradient elution when thawing. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Figure 6 is the effect of different lemiripril concentrations on the recovery activity of breast cancer organoids after cryopreservation.

[0034] Figure 2 Figure 7 is the recovery activity of breast cancer organoids after thawing and recovery after D-sorbitol replacement.

[0035] Figure 3 Figure 8 is a comparison chart of the morphology of the recovered breast cancer organoids after 5 days of culture (left: the method of Example 1; right: the method of Comparative Example 1). DETAILED DESCRIPTION

[0036] The principles and characteristics of the application are described below, and the examples are only used to explain the application and not to limit the scope of the application.

[0037] Example 1:

[0038] A cell cryopreservation solution is composed of lemiripril, D-sorbitol, phosphate buffer and vitamin C; wherein the concentration of lemiripril is 1 μM, the molar concentration of D-sorbitol is 0.5 mol / L, the pH of the phosphate buffer is 7.4, the molar concentration of the phosphate buffer is 0.01 mol / L, and the concentration of vitamin C is 0.1 g / mL.

[0039] The use method of the above-mentioned cell cryopreservation solution is as follows:

[0040] 1) Cell cryopreservation: 1×10 6 breast cancer organoids are added to a cryopreservation tube containing 1 mL of cell cryopreservation solution, and the cryopreservation tube containing the above-mentioned cells to be cryopreserved and cell cryopreservation solution is immersed in-196℃ liquid nitrogen for organoid preservation.

[0041] 2) Cell thawing: After 2 weeks of storage, the cryopreserved organoids and cell cryopreservation solution were taken out of the liquid nitrogen and heated in a 37°C water bath for about 1 min until the organoids were thawed and recovered. The thawed and recovered organoids were then centrifuged at 800g for 5 min with 10 mL PBS to remove the supernatant, and then the organoids were washed twice with 10 mL of normal saline to remove the supernatant, obtaining the washed cells. The washed cells were resuspended with 1 mL of organoid culture medium, and the thawed and recovered organoids were obtained. The entire recovery process took 15 min.

[0042] Example 2

[0043] This example 2 is basically the same as example 1, the difference is that the concentration of lomeridipine and the concentration of D-sorbitol in the cell cryopreservation solution are different; as shown in the following table 1:

[0044] Table 1: Effect of different concentrations of lomeridipine and D-sorbitol on the recovery activity of breast cancer organoids after cryopreservation

[0045]

[0046] Figure 1 The results of AO / PI staining of breast cancer organoids after recovery, from left to right, are D-sorbitol with a molar concentration of 0.5 mol / L, lomeridipine concentrations of 0, 0.5 and 1 μM. Red represents dead cells, and green represents live cells.

[0047] From the results of table 1 and Figure 1 It can be seen from the results that when the concentration of lomeridipine is 0-1 μM and the concentration of D-sorbitol is 0.5-1.0 M, the recovery activity of organoids can be significantly improved. Adding lomeridipine or D-sorbitol alone cannot effectively protect cells during cryopreservation, and within this range, the higher the concentration of lomeridipine, the more obvious the effect. It shows that lomeridipine in the above-mentioned addition ratio can inhibit cell apoptosis. When the content of lomeridipine and D-sorbitol is not within the above-mentioned range, the recovery activity of organoids is greatly reduced.

[0048] Example 3

[0049] A cell cryopreservation solution is composed of lomeridipine, D-sorbitol, phosphate buffer and vitamin C; wherein the concentration of lomeridipine is 1 μM, the molar concentration of D-sorbitol is 0.5 mol / L, the final pH of the phosphate buffer is 7.4, the molar concentration is 0.01 mol / L, and the concentration of vitamin C is 0.1 g / mL.

[0050] The use method of the above-mentioned cell cryopreservation solution is as follows:

[0051] (1) Cell cryopreservation: 1 x 106 The lung cancer organoids, breast cancer organoids, esophageal cancer organoids, pancreatic cancer organoids, gastric cancer organoids, and normal mouse liver organoids, lung organoids, and small intestine organoids are respectively added to cryopreservation tubes containing 1 mL of cell cryopreservation solution, and the cryopreservation tubes containing the cells to be cryopreserved and the cell cryopreservation solution are immersed in liquid nitrogen at -196°C to achieve preservation of the organoids.

[0052] (2) Cell thawing: After 2 weeks of cryopreservation, the cryopreservation tube containing the organoids to be cryopreserved and the cell cryopreservation solution is removed from the liquid nitrogen, heated in a 37°C water bath for about 1 min, and the organoids are thawed and recovered until the organoids are thawed and recovered. The organoids are then centrifuged at 800g for 5 min to remove the supernatant, and the organoids are washed twice with 10 mL of physiological saline to remove the supernatant and obtain the washed cells. The washed cells are resuspended in 1 mL of organoid culture medium to obtain the thawed and recovered organoids. The entire recovery process takes 15 min. The cell survival rate (AO / PI staining) is detected using a cell counter, and the results are shown in Table 2. The cell viability is more than 80%.

[0053] Table 2:

[0054] Organoid species Organoid resuscitation viability Mouse lung cancer organoids 88.7％ Mouse breast cancer organoids 91.2％ Mouse esophageal cancer organoids 83.5％ Mouse pancreatic cancer organoids 85.2％ Mouse gastric cancer organoids 89.2％ Mouse normal liver organoids 85.7％ Mouse normal lung organoids 88.9％ Mouse normal small intestine organoids 91.5％

[0055] Comparative Example 1:

[0056] Comparative Example 1 is basically the same as Example 1, except that D-sorbitol in the cell cryopreservation solution is replaced by glycerol, dimethyl sulfoxide (DMSO), sphingoglycolipid, glycolipid, glycerophospholipid, sphingomyelin, algal protein glycolipid, sophorolipid, phospholipid, lecithin, and lipopolysaccharide (all purchased from Sigma Company). The recovery viability of the breast cancer organoids after thawing and recovery is shown in Table 1. Figure 2

[0057] Comparative Example 2:

[0058] Comparative Example 2 is basically the same as Example 1, except that the cell cryopreservation solution further contains 0.15 mol / L polyvinylpyrrolidone (Shanghai Chemical Reagent Co., Ltd. of Sinopharm Group), 0.2 mol / L hydroxyethyl starch (Shanghai Chemical Reagent Co., Ltd. of Sinopharm Group), 0.12 mol / L human blood albumin solution (Shanghai Blood Products Co., Ltd. of Sinopharm Group), 0.1 mol / L fetal bovine serum (Beijing Solabio Science and Technology Co., Ltd.), 0.1 mol / L acetamide (Shanghai Aladdin Biochemical Technology Co., Ltd.), and 0.25 mol / L DMSO (Shanghai Chemical Reagent Co., Ltd. of Sinopharm Group). The recovery viability of the organoids after thawing and recovery is shown in Table 3.

[0059] Table 3: ​

[0060] Method Cell resuscitation viability (%) Example 1 91.2％ Comparative Example 2 79.5％

[0061] The above results can show that, compared with the cell freezing solution of Example 1, the cell freezing solution of Comparative Example 2 has a decreased organoid resuscitation activity after adding some other components commonly used for cell freezing, which may be due to the addition of other components affecting the efficiency of D-sorbitol entering the cells, resulting in a decrease in the amount of D-sorbitol entering the cells, and thus a decrease in the freezing effect. And compared with the cell freezing solution of Example 1, the cell freezing solution of Comparative Example 2 adds fetal bovine serum and human blood albumin solution and other components, which has the problems of cytotoxicity, uncertain components, and possible introduction of bacterial and viral contamination sources.

[0062] Comparative Example 3:

[0063] The classical glycerol freezing method is used to freeze and thaw the organoids in this comparative example 3.

[0064] Classical method: take 1x10 6 breast cancer organoid suspension, mix the organoids with the 57% (v / v) compound glycerol solution to be added at a volume ratio of 5:4 of organoids to compound glycerol solution, and then place it in a 37°C water bath for 10 min. balance, then transfer it to the program cooling instrument to -80°C, and then transfer it to liquid nitrogen for storage. When resuscitating, take it out from the liquid nitrogen, quickly put it into a 37°C water bath, and gently shake it until it is completely melted. Then add 9% (m / v) sodium chloride solution 800g to centrifuge for 10 min. Wash once, and then balance and use 9% sodium chloride solution 800g to centrifuge for 10 min. Wash 2-3 times, and then resuspend the organoids with 1 mL of organoid culture medium to obtain the thawed and resuscitated organoids.

[0065] The organoids resuscitated after thawing and removing the freezing solution of the method of Example 1 and the organoids resuscitated after thawing and removing glycerol of the method of Comparative Example 3 were cultured for 5 days, and the morphology was as shown in Figure 3 , and the resuscitation activity was as shown in Table 4.

[0066] From Figure 3 it can be seen that the organoids resuscitated after thawing and removing the freezing solution of the method of Example 1 have more organoids growing after culture, which shows that the cell freezing solution of the application has good freezing and thawing effect.

[0067] Table 4:

[0068] Method Cell resuscitation viability (%) Resuscitation time consumption Example 1 method 91.2％ 15 min Comparative Example 3 method 42.7％ 1h

[0069] From the above results, compared with the classical glycerol freezing method of the comparative example 3 method, the cell freezing solution used in the present application does not use high concentration of glycerol, so it will not cause high osmotic pressure inside the organoids, avoid cell rupture when resuscitation, the organoid survival rate after freezing and resuscitation is significantly higher than that of the classical method (comparative example 3 method), and there is no need to remove glycerol by multiple washing, the resuscitation time is significantly shortened, the inventors analyze that lemmidipine promotes the absorption of D-sorbitol by cells, and at the same time avoids the rupture of cells during resuscitation.

[0070] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A cell cryopreservation solution for organoid cryopreservation, characterized in that, It consists of the following components: lemidipine, D-sorbitol, phosphate buffer, and vitamin C. The molar concentration of lemidipine is 0.9–1.1 μM, the molar concentration of D-sorbitol is 0.5–1.0 mol / L, the final pH of the added phosphate buffer is 7.3–7.5, and the concentration of vitamin C is 0.09–0.11 g / mL.

2. A method for preparing a cell cryopreservation solution for organoid cryopreservation according to claim 1, characterized in that, Includes the following steps: Mix the ingredients according to the recipe to obtain the final product.

3. An application of the cell cryopreservation solution for organoid cryopreservation according to claim 1, characterized in that: Cell cryopreservation solution used for organoid cryopreservation.

4. The application of the cell cryopreservation solution for organoid cryopreservation according to claim 3, characterized in that: The organoids mentioned are mouse lung cancer organoids, breast cancer organoids, esophageal cancer organoids, pancreatic cancer organoids, or gastric cancer organoids.

5. The application of the cell cryopreservation solution for organoid cryopreservation according to claim 3, characterized in that: The organoids mentioned are normal mouse liver organoids, lung organoids, or small intestine organoids.

6. The application of the cell cryopreservation solution for organoid cryopreservation according to claim 3, characterized in that: Cryopreservation of organoids includes the following steps: centrifuging the suspension of organoids to be cryopreserved, removing the supernatant, and resuspending the resulting cell pellet directly in cryopreservation solution until homogeneous, with a cryopreservation density of (1–10) × 10⁻⁶. 6 The organoids can be preserved by immersing the container containing the organoids to be frozen and the cell cryopreservation solution in a liquid nitrogen tank at -196°C. The resuscitation of organoids includes the following steps: the container containing the organoids to be cryopreserved and the cell cryopreservation solution is removed from liquid nitrogen, and the cells are thawed in a 37°C water bath to obtain the thawed and resuscitated organoids. Then, the organoids are washed and resuspended in organoid culture medium to obtain the thawed and resuscitated organoids without cryopreservation solution.

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