Cell cryopreservation liquid, application thereof and cell cryopreservation method

Abstract

The invention belongs to the technical field of cell cryopreservation, and particularly relates to a cell cryopreservation solution, application thereof and a cell cryopreservation method. A death inhibitor and a solvent are selected for specific cells, and the cell death process related to cytolytic organelle membrane damage is intervened from the molecular level, specifically, the solvent is used for reducing ice crystal formation inside and outside the cells, the membrane protection effect is exerted to achieve the high resuscitation motility rate, a programmed death pathway is further blocked in a targeted mode through the death inhibitor, and the cell death effect is improved. The programmed death after cell resuscitation is relieved, the delayed death rate of the resuscitated cells is reduced, the long-term cell fusion degree of the resuscitated cells is improved, and the long-term survival rate of the resuscitated cells is improved.

Description

TECHNICAL FIELD

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

[0002] Cell cryopreservation technology is a key link to realize long-term preservation and function maintenance of cells. At present, cell cryopreservation technology mainly relies on programmed cooling (such as gradually reducing the temperature to-80℃ and then transferring to liquid nitrogen) and optimization of cryopreservation solution components (such as adding permeable protective agents dimethyl sulfoxide, glycerol, etc.) to reduce the mechanical damage of ice crystal formation to cells and maintain the stability of cell membranes and organelles. These methods have made significant progress in improving the survival rate at the initial stage of cell recovery and have become a standardized process in laboratory and industrial applications.

[0003] However, the cryopreservation-recovery process may cause abnormal structure or function of organelle membranes through physical stress or chemical stress, such as ice crystal formation, sharp temperature change, osmotic pressure fluctuation, ion concentration gradient change. Mitochondrial membrane potential imbalance may start the intrinsic apoptosis pathway by releasing cytochrome C and other apoptosis factors; once the membrane integrity of lysosomes, as the “digestion center” and “stress sensor” of cells, is destroyed, a large amount of acid hydrolase will leak into the cytoplasm, directly or indirectly activating various programmed death pathways, including apoptosis, pyroptosis and necroptosis. Such death mechanisms are usually triggered during the recovery period (such as within 24h) after recovery, thereby significantly reducing the long-term survival rate and functional activity of cells. The design of existing cryopreservation solutions focuses on protecting cell membranes, and insufficient attention is paid to programmed death caused by organelle damage after recovery. Many kinds of cells show high initial survival rate after recovery, but significant secondary death occurs within the subsequent 24h. SUMMARY

[0004] The purpose of the present application is to provide a cell cryopreservation solution, application thereof and a cell cryopreservation method, which specifically inhibit programmed death triggered by cryodamage after cryopreservation and recovery, and improve the long-term survival rate of cells after recovery.

[0005] The present application provides a cell cryopreservation solution, which comprises one or more of an induced pluripotent stem cell cryopreservation solution, a natural killer cell cryopreservation solution and a mesenchymal stromal cell cryopreservation solution, The induced pluripotent stem cell cryopreservation solution comprises a first death inhibitor and a first solvent; the first death inhibitor is one or more of Y27632, chloroquine and SRI-011381; The natural killer cell cryopreservation solution comprises a second death inhibitor and a second solvent; the second death inhibitor is one or more of necrostatin-1, Z-VAD-fmk and disulfiram; The mesenchymal stromal cell cryopreservation solution comprises a third death inhibitor and a third solvent; the third death inhibitor is one or more of ferrostatin-1, Y27632 and β-mercaptoethanol; The first, second and third solvents respectively comprise the following volume percentage of components: 20-80% colloid solution, 5-20% energy substance, 5-60% high osmotic solution and 5-15% osmoprotectant.

[0006] Preferably, the concentration of Y27632 in the induced pluripotent stem cell cryopreservation solution is 5-20 μM; The concentration of chloroquine in the induced pluripotent stem cell cryopreservation solution is 25-200 μM; The concentration of SRI-011381 in the induced pluripotent stem cell cryopreservation solution is 10-200 μM.

[0007] Preferably, the concentration of necrostatin-1 in the natural killer cell cryopreservation solution is 10-100 μM; The concentration of Z-VAD-fmk in the natural killer cell cryopreservation solution is 10-100 μM; The concentration of disulfiram in the natural killer cell cryopreservation solution is 0.1-25 μM.

[0008] Preferably, the concentration of ferrostatin-1 in the mesenchymal stromal cell cryopreservation solution is 0.05-1 μM; The concentration of Y27632 in the mesenchymal stromal cell cryopreservation solution is 5-20 μM; The concentration of β-mercaptoethanol in the mesenchymal stromal cell cryopreservation solution is 0.05-2 mM.

[0009] Preferably, the colloid solution comprises hydroxyethyl starch and / or dextran; The energy substance comprises one or more of adenosine disodium triphosphate, adenosine and glucose; The high osmotic solution comprises one or more of sorbitol, mannitol and human albumin; The osmoprotectant comprises one or more of dimethyl sulfoxide, propylene glycol and glycerol.

[0010] Preferably, the colloid solution comprises 30-90 mg / mL hydroxyethyl starch solution and / or 30-90 mg / mL dextran solution; The energy substance comprises one or more of 5-20 mg / mL adenosine disodium triphosphate, 1-10 mg / mL adenosine and 50-500 mg / mL glucose; The hypertonic solution comprises one or more of 200-300 mg / mL sorbitol, 150-250 mg / mL mannitol, and 50-200 mg / mL human albumin.

[0011] The application provides application of the cell freezing solution in one or more of the following aspects. (1) cell freezing; (2) improving cell fusion degree of the cells recovered after cell freezing; (3) reducing programmed cell death of the cells recovered after cell freezing; and (4) improving survival rate of the cells recovered after cell freezing.

[0012] Preferably, the temperature of the cell freezing is less than or equal to -80 DEG C.

[0013] Preferably, the cell fusion degree comprises cell fusion degree within 24 hours; the programmed cell death comprises programmed cell death within 24 hours; and the recovery rate comprises recovery rate within 24 hours.

[0014] The application provides a cell freezing method, comprising one or more of an induced pluripotent stem cell freezing method, a natural killer cell freezing method and a mesenchymal stromal cell freezing method. The induced pluripotent stem cell freezing method comprises the following steps: mixing the induced pluripotent stem cell freezing solution and the induced pluripotent stem cells according to 1x10 6 ~5x10 7 / mL, and freezing at a temperature of less than or equal to -80 DEG C. The natural killer cell freezing method comprises the following steps: mixing the natural killer cell freezing solution and the natural killer cells according to 1x10 7 ~5x10 8 / mL, and freezing at a temperature of less than or equal to -80 DEG C. The mesenchymal stromal cell freezing method comprises the following steps: mixing the mesenchymal stromal cell freezing solution and the mesenchymal stromal cells according to 1x10 6 ~5x10 7 / mL, and freezing at a temperature of less than or equal to -80 DEG C.

[0015] Beneficial effects: The application selects death inhibitors and solvents for specific cells, intervenes in the process of cell death related to membrane damage of organelles from a molecular level, specifically: the solvents reduce the formation of ice crystals inside and outside the cells, play a membrane protection role to achieve high recovery activity, further target the death inhibitors to block the programmed cell death pathway, reduce programmed cell death after cell recovery, reduce the rate of delayed cell death after recovery, improve long-term cell fusion degree of the cells after recovery, and improve long-term survival rate of the cells after recovery. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required to be used in the embodiments will be briefly introduced as follows.

[0017] Figure 1 The cell fusion degree within 24 hours after the induced pluripotent stem cell cryopreservation for 2 weeks; Figure 2 The recovery rate 24 hours after the natural killer cell cryopreservation for 2 weeks; Figure 3 The cell fusion degree within 24 hours after the mesenchymal stromal cell cryopreservation for 2 weeks; Among them, Indicates P <0.05; Indicates P <0.001; Indicates P <0.0001. DETAILED DESCRIPTION

[0018] The present application provides a cell cryopreservation solution, including one or more of induced pluripotent stem cell cryopreservation solution, natural killer cell cryopreservation solution and mesenchymal stromal cell cryopreservation solution, The induced pluripotent stem cell cryopreservation solution includes a first death inhibitor and a first solvent; the first death inhibitor is one or more of Y27632, chloroquine and SRI-011381; The natural killer cell cryopreservation solution includes a second death inhibitor and a second solvent; the second death inhibitor is one or more of necrostatin-1, Z-VAD-fmk and disulfiram; The mesenchymal stromal cell cryopreservation solution includes a third death inhibitor and a third solvent; the third death inhibitor is one or more of ferrostatin-1, Y27632 and β-mercaptoethanol; The first solvent, the second solvent and the third solvent respectively include the following components with volume percentage: 20-80% colloid solution, 5-20% energy substance, 5-60% high osmotic solution and 5-15% osmotic protective agent.

[0019] As an implementation form, the volume percentage of the colloidal solution in the first solvent, the second solvent and the third solvent is 30-60%; as another implementation form, the volume percentage of the colloidal solution in the first solvent, the second solvent and the third solvent is 30-50%. As an implementation form, the volume percentage of the colloidal solution in the first solvent is 50%, specifically hydroxyethyl starch solution. As an implementation form, the volume percentage of the colloidal solution in the second solvent is 30%, specifically hydroxyethyl starch solution. As an implementation form, the volume percentage of the colloidal solution in the third solvent is 30%, specifically dextran solution.

[0020] As an implementation form, the colloidal solution of the present application comprises hydroxyethyl starch and / or dextran. As an implementation form, the hydroxyethyl starch of the present application is 30-90 mg / mL hydroxyethyl starch solution; as another implementation form, the hydroxyethyl starch of the present application is 60 mg / mL hydroxyethyl starch solution. As an implementation form, the dextran of the present application is 30-90 mg / mL dextran; as another implementation form, the dextran solution of the present application is 60 mg / mL dextran solution. The present application uses colloidal solution, which has the effect of slowing down cell sedimentation aggregation, and the present application selects hydroxyethyl starch solution and / or dextran solution, which can also stabilize cell membrane structure and provide non-permeable protection compared to other colloidal solutions.

[0021] As an implementation form, the volume percentage of the energy substance in the first solvent, the second solvent and the third solvent of the present application is 5%-17.5%. As an implementation form, the energy substance of the present application comprises one or more of adenosine disodium triphosphate, adenosine and glucose. As an implementation form, the volume percentage of the energy substance in the first solvent of the present application is 5%, specifically glucose. As an implementation form, the volume percentage of the energy substance in the second solvent of the present application is 5%, specifically adenosine disodium triphosphate and glucose, and the volume ratio of adenosine disodium triphosphate to glucose is 1:1. As an implementation form, the volume percentage of the energy substance in the third solvent of the present application is 17.5%, specifically adenosine and glucose, and the volume ratio of adenosine to glucose is 7.5:10.

[0022] As an embodiment, the adenosine triphosphate disodium of the present application is a 5-20 mg / mL adenosine triphosphate disodium solution; as another embodiment, the adenosine triphosphate disodium of the present application is a 10 mg / mL adenosine triphosphate disodium solution. As an embodiment, the adenosine of the present application is a 1-10 mg / mL adenosine solution; as another embodiment, the adenosine of the present application is a 3 mg / mL adenosine solution. As an embodiment, the glucose of the present application is a 50-500 mg / mL glucose solution; as another embodiment, the glucose of the present application is a 50 mg / mL glucose solution. The present application utilizes energy substances, has the effect of providing energy for cell basal metabolism, and the present application selects one or more of adenosine triphosphate disodium, adenosine and glucose, which provides energy more directly and quickly compared to other energy substances.

[0023] As an embodiment, the volume percentage content of the hypertonic solution in the first solvent, the second solvent and the third solvent of the present application is 10-40%; as another embodiment, the volume percentage content of the hypertonic solution in the first solvent, the second solvent and the third solvent of the present application is 35%. As an embodiment, the hypertonic solution of the present application includes one or more of sorbitol, mannitol and human albumin. As an embodiment, the volume percentage content of the hypertonic solution in the first solvent of the present application is 35%, specifically sorbitol and human albumin, and the volume ratio of sorbitol to human albumin is 2.5:1. As an embodiment, the volume percentage content of the hypertonic solution in the second solvent of the present application is 10%, specifically sorbitol and mannitol, and the volume ratio of sorbitol to mannitol is 1:1. As an embodiment, the volume percentage content of the hypertonic solution in the third solvent of the present application is 40%, specifically mannitol and human albumin, and the volume ratio of mannitol to human albumin is 1:3.

[0024] As an embodiment, the sorbitol of the present application is a 200-300 mg / mL sorbitol solution; as another embodiment, the sorbitol of the present application is a 250 mg / mL sorbitol solution. As an embodiment, the mannitol of the present application is a 150-250 mg / mL mannitol solution; as another embodiment, the mannitol of the present application is a 200 mg / mL mannitol solution. As an embodiment, the human albumin of the present application is a 50-200 mg / mL human albumin solution; as another embodiment, the human albumin of the present application is a 200 mg / mL human albumin solution. The present application utilizes hypertonic solutions, has the effect of promoting cell dehydration and reducing intracellular ice crystal formation, and the present application selects one or more of sorbitol, mannitol and human albumin, which can also promote organelle repair after damage by inducing stress granule formation compared to other hypertonic solutions.

[0025] As an embodiment, the volume percentage of the permeation protective agent in the first solvent, the second solvent and the third solvent of the present application is 5%-12.5%. As an embodiment, the permeation protective agent in the first solvent of the present application includes one or more of dimethyl sulfoxide, propylene glycol and glycerol, and the volume percentage of the permeation protective agent is 5%, specifically glycerol. As an embodiment, the permeation protective agent in the second solvent of the present application includes one or more of dimethyl sulfoxide, propylene glycol and glycerol, and the volume percentage of the permeation protective agent is 5%, specifically dimethyl sulfoxide. As an embodiment, the permeation protective agent in the third solvent of the present application includes one or more of dimethyl sulfoxide, propylene glycol and glycerol, and the volume percentage of the permeation protective agent is 12.5%, specifically propylene glycol and glycerol, and the volume ratio of propylene glycol to glycerol is 7.5:5. The present application uses the permeation protective agent to directly penetrate into the cells to reduce the formation of ice crystals, and the present application selects one or more of dimethyl sulfoxide, propylene glycol and glycerol, which has a better effect on inhibiting intracellular ice crystal formation than other permeation protective agents.

[0026] As an embodiment, the concentration of Y27632 in the induced pluripotent stem cell cryopreservation solution of the present application is 5-20 μM; as another embodiment, the concentration of Y27632 in the induced pluripotent stem cell cryopreservation solution of the present application is 10-15 μM; as an embodiment, the concentration of Y27632 in the induced pluripotent stem cell cryopreservation solution of the present application is 12 μM. The present application uses Y27632 to reduce apoptosis.

[0027] As an embodiment, the concentration of chloroquine in the induced pluripotent stem cell cryopreservation solution of the present application is 25-200 μM; as another embodiment, the concentration of chloroquine in the induced pluripotent stem cell cryopreservation solution of the present application is 50-150 μM; as another embodiment, the concentration of chloroquine in the induced pluripotent stem cell cryopreservation solution of the present application is 100 μM. The present application uses chloroquine to reduce autophagy-like death.

[0028] As an embodiment, the concentration of SRI-011381 in the induced pluripotent stem cell cryopreservation solution of the present application is 10-200 μM; as another embodiment, the concentration of SRI-011381 in the induced pluripotent stem cell cryopreservation solution of the present application is 25-150 μM; as another embodiment, the concentration of SRI-011381 in the induced pluripotent stem cell cryopreservation solution of the present application is 50-100 μM; as another embodiment, the concentration of SRI-011381 in the induced pluripotent stem cell cryopreservation solution of the present application is 60-80 μM. The present application uses SRI-011381 to inhibit ammonia death.

[0029] As an embodiment, the first death inhibitor of the present application is a combination of Y27632, chloroquine and SRI-011381, and the concentrations of Y27632, chloroquine and SRI-011381 in the induced pluripotent stem cell cryopreservation solution are 10 μM, 25 μM and 25 μM, respectively. The present application combines Y27632, chloroquine and SRI-011381, reduces the use amount of Y27632, chloroquine and SRI-011381, and synergistically reduces the programmed death of the induced pluripotent stem cell after resuscitation, reduces the delayed mortality rate, and improves the long-term cell fusion degree and survival rate.

[0030] As an embodiment, the concentration of necrostatin-1 in the natural killer cell cryopreservation solution of the present application is 10-100 μM; as another embodiment, the concentration of necrostatin-1 in the natural killer cell cryopreservation solution of the present application is 20-80 μM; as an embodiment, the concentration of necrostatin-1 in the natural killer cell cryopreservation solution of the present application is 30-60 μM; as an embodiment, the concentration of necrostatin-1 in the natural killer cell cryopreservation solution of the present application is 50 μM. The present application uses necrostatin-1 to have the effect of inhibiting necroptosis.

[0031] As an embodiment, the concentration of Z-VAD-fmk in the natural killer cell cryopreservation solution of the present application is 10-100 μM; as another embodiment, the concentration of Z-VAD-fmk in the natural killer cell cryopreservation solution of the present application is 20-80 μM; as an embodiment, the concentration of Z-VAD-fmk in the natural killer cell cryopreservation solution of the present application is 30-60 μM; as an embodiment, the concentration of Z-VAD-fmk in the natural killer cell cryopreservation solution of the present application is 50 μM. The present application uses Z-VAD-fmk to have the effect of reducing apoptosis and necroptosis.

[0032] As an embodiment, the concentration of disulfiram in the natural killer cell cryopreservation solution of the present application is 0.1-25 μM; as another embodiment, the concentration of disulfiram in the natural killer cell cryopreservation solution of the present application is 0.5-20 μM; as an embodiment, the concentration of disulfiram in the natural killer cell cryopreservation solution of the present application is 1-30 μM; as an embodiment, the concentration of disulfiram in the natural killer cell cryopreservation solution of the present application is 10-20 μM. The present application uses disulfiram to have the effect of inhibiting pyroptosis.

[0033] As an embodiment, the second death inhibitor of the present application is a combination of necrostatin-1, Z-VAD-fmk and disulfiram, and the concentrations of necrostatin-1, Z-VAD-fmk and disulfiram in the natural killer cell cryopreservation solution are 10 μM, 10 μM and 0.1 μM, respectively. The present application combines necrostatin-1, Z-VAD-fmk and disulfiram, reduces the use amount of necrostatin-1, Z-VAD-fmk and disulfiram, has synergistic effect, reduces the programmed death of natural killer cells after resuscitation, reduces the delayed mortality, and improves the long-term cell fusion degree and survival rate.

[0034] As an embodiment, the concentration of ferrostatin-1 in the mesenchymal stromal cell cryopreservation solution of the present application is 0.05-1 μM; as another embodiment, the concentration of ferrostatin-1 in the mesenchymal stromal cell cryopreservation solution of the present application is 0.08-0.8 μM; as another embodiment, the concentration of ferrostatin-1 in the mesenchymal stromal cell cryopreservation solution of the present application is 0.1-0.5 μM. The present application uses ferrostatin-1 to have the effect of inhibiting ferroptosis.

[0035] As an embodiment, the concentration of Y27632 in the mesenchymal stromal cell cryopreservation solution of the present application is 5-20 μM; as another embodiment, the concentration of Y27632 in the mesenchymal stromal cell cryopreservation solution of the present application is 8-15 μM; as another embodiment, the concentration of Y27632 in the mesenchymal stromal cell cryopreservation solution of the present application is 10-12 μM. The present application uses Y27632 to have the effect of reducing apoptosis.

[0036] As an embodiment, the concentration of β-mercaptoethanol in the mesenchymal stromal cell cryopreservation solution of the present application is 0.05-2 mM; as another embodiment, the concentration of β-mercaptoethanol in the mesenchymal stromal cell cryopreservation solution of the present application is 0.08-1 μM; as another embodiment, the concentration of β-mercaptoethanol in the mesenchymal stromal cell cryopreservation solution of the present application is 0.1-0.8 μM; as another embodiment, the concentration of β-mercaptoethanol in the mesenchymal stromal cell cryopreservation solution of the present application is 0.5-0.6 μM. The present application uses β-mercaptoethanol to have the effect of reducing disulfide death.

[0037] As an embodiment, the third death inhibitor is a combination of ferrostatin-1, Y27632 and β-mercaptoethanol, and the concentrations of ferrostatin-1, Y27632 and β-mercaptoethanol in the mesenchymal stromal cell cryopreservation solution are 10 μM, 10 μM and 0.1 μM, respectively. The present application combines ferrostatin-1, Y27632 and β-mercaptoethanol, reduces the dosage of ferrostatin-1, Y27632 and β-mercaptoethanol, and synergistically reduces the programmed death of mesenchymal stromal cells after resuscitation, reduces the delayed mortality rate, and improves the long-term cell fusion degree and survival rate.

[0038] The CAS numbers of Y27632, chloroquine, SRI-011381, necrostatin-1, Z-VAD-fmk, disulfiram, ferrostatin-1 and β-mercaptoethanol are 146986-50-7, 54-05-7, 1629138-41-5, 4311-88-0, 187389-52-2, 97-77-8, 347174-05-4 and 60-24-2, respectively.

[0039] The present application provides the use of the cell cryopreservation solution described in the above technical solution in one or more of the following: (1) cell cryopreservation; (2) improving the cell fusion degree after resuscitation of the cell cryopreservation; (3) reducing the programmed death after resuscitation of the cell cryopreservation; (4) improving the survival rate after resuscitation of the cell cryopreservation.

[0040] As an embodiment, the temperature of the cell cryopreservation is ≤-80℃. As an embodiment, the cell fusion degree includes the cell fusion degree within 24 hours. As an embodiment, the programmed death includes the programmed death within 24 hours; and the resuscitation rate includes the resuscitation rate within 24 hours. The resuscitation effect of the cell solution of the present application after 20 years of cell cryopreservation is comparable to the resuscitation effect after 2 weeks of cell cryopreservation.

[0041] As an embodiment, the cells include, but are not limited to, one or more of induced pluripotent stem cells, natural killer cells and mesenchymal stromal cells, and the induced pluripotent stem cell cryopreservation solution, the natural killer cell cryopreservation solution and the mesenchymal stromal cell cryopreservation solution are used for the cryopreservation of induced pluripotent stem cells, natural killer cells and mesenchymal stromal cells, respectively.

[0042] The application provides a cell cryopreservation method, including one or more of an induced pluripotent stem cell cryopreservation method, a natural killer cell cryopreservation method and a mesenchymal stromal cell cryopreservation method, the induced pluripotent stem cell cryopreservation method comprising the following steps: mixing the induced pluripotent stem cell cryopreservation solution and the induced pluripotent stem cell according to a volume ratio of 1x10 6 ~5x10 7 / mL, and cryopreserving at ≤-80℃. The natural killer cell cryopreservation method comprises the following steps: mixing the natural killer cell cryopreservation solution and the natural killer cell according to a volume ratio of 1x10 7 ~5x10 8 / mL, and cryopreserving at ≤-80℃. The mesenchymal stromal cell cryopreservation method comprises the following steps: mixing the mesenchymal stromal cell cryopreservation solution and the mesenchymal stromal cell according to a volume ratio of 1x10 6 ~5x10 7 / mL, and cryopreserving at ≤-80℃.

[0043] As an embodiment, the induced pluripotent stem cell cryopreservation solution and the induced pluripotent stem cell are mixed according to a volume ratio of 1 mL: 2x10 6 individuals, and cryopreserving at ≤-80℃. As an embodiment, the natural killer cell cryopreservation solution and the natural killer cell are mixed according to a volume ratio of 1 mL: 1x10 8 individuals, and cryopreserving at ≤-80℃. As an embodiment, the mesenchymal stromal cell cryopreservation solution and the mesenchymal stromal cell are mixed according to a volume ratio of 1 mL: 1x10 7 individuals, and cryopreserving at ≤-80℃.

[0044] In order to further illustrate the application, the application provides a cell cryopreservation solution, application and cell cryopreservation method are described in detail below in combination with the drawings and examples, but they cannot be understood as limiting the protection scope of the application.

[0045] Preparation Example 1 The solvent is configured by components with the following volume percentage contents: 50% 60mg / mL hydroxyethyl starch solution, 5% 50mg / mL glucose solution, 25% 250mg / mL sorbitol solution, 10% 200mg / mL human albumin solution, 5% dimethyl sulfoxide and 5% glycerol.

[0046] Example 1 A cryopreservation solution is composed of 20μM Y27632 and the solvent of Preparation Example 1.

[0047] Example 2 A cryopreservation solution consisting of 200 μΜ chloroquine and the solvent of Preparation Example 1.

[0048] Example 3 A cryopreservation solution consisting of 200 μΜ SRI-011381 and the solvent of Preparation Example 1.

[0049] Example 4 A cryopreservation solution consisting of 10 μΜ Y27632, 25 μΜ chloroquine, 25 μΜ SRI-011381 and the solvent of Preparation Example 1.

[0050] Comparative Example 1 A cryopreservation solution consisting of 1 μΜ ferrostatin-1 and the solvent of Preparation Example 1.

[0051] Comparative Example 2 A cryopreservation solution consisting of 100 μΜ Z-VAD-fmk and the solvent of Preparation Example 1.

[0052] Comparative Example 3 A cryopreservation solution consisting of 100 μΜ necrostatin-1 and the solvent of Preparation Example 1.

[0053] Comparative Example 4 A cryopreservation solution consisting of 25 μΜ disulfiram and the solvent of Preparation Example 1.

[0054] Comparative Example 5 A cryopreservation solution consisting of 2 μΜ beta-mercaptoethanol and the solvent of Preparation Example 1.

[0055] Application Example 1 Cryopreservation of induced pluripotent stem cells (iPSCs) iPSCs in logarithmic growth phase, with good morphology and clear clone boundaries, were digested with recombinant trypsin to form a single-cell suspension. The cell suspension was transferred to a centrifuge tube, centrifuged at 300 g for 3 minutes, resuspended in PBS and counted. The cells were divided into 2x10 6 / tube, and centrifuged to discard the supernatant. The cells were resuspended with 1 mL of the solvent of Preparation Example 1 and the cryopreservation solutions of Examples 1-4 (numbered 1-5, respectively), and Comparative Examples 1-5, and transferred to cryopreservation tubes. The cryopreservation tubes were placed in a cryopreservation box and stored in a -80°C refrigerator. After 2 weeks, the cryopreserved cells were removed and placed in a 37°C water bath for 1 minute and 45 seconds, centrifuged at 400 g for 5 minutes, and resuspended in pluripotent stem cell medium. The cells were seeded at a density of 20000 / cm 2 Figure 1 and Table 1. ​

[0056] Table 1 Cell confluency after 2 weeks of induced pluripotent stem cell cryopreservation

[0057] As can be seen from Table 1, the addition of Y27632, chloroquine, SRI-011381 to the cryopreservation solution significantly promoted the plating efficiency of iPSCs after cryopreservation recovery.

[0058] Preparation Example 2 The solvent was configured from the following volume percent components: 30% 60 mg / mL hydroxyethyl starch solution, 2.5% 10 mg / mL adenosine triphosphate disodium solution, 2.5% 50 mg / mL glucose solution, 5% 250 mg / mL sorbitol solution, 5% 200 mg / mL mannitol solution, 50% 200 mg / mL human albumin solution, and 5% dimethyl sulfoxide.

[0059] Example 5 A cryopreservation solution consisting of 100 μΜ necrostatin-1 and the solvent of Preparation Example 2.

[0060] Example 6 A cryopreservation solution consisting of 100 μΜ Z-VAD-fmk and the solvent of Preparation Example 2.

[0061] Example 7 A cryopreservation solution consisting of 250 μΜ disulfiram and the solvent of Preparation Example 2.

[0062] Example 8 A cryopreservation solution consisting of 10 μΜ necrostatin-1, 10 μΜ Z-VAD-fmk, 0.1 μΜ disulfiram, and the solvent of Preparation Example 2.

[0063] Comparative Example 6 A cryopreservation solution consisting of 100 μΜ chloroquine and the solvent of Preparation Example 2.

[0064] Comparative Example 7 A cryopreservation solution consisting of 2 μΜ β-mercaptoethanol and the solvent of Preparation Example 2.

[0065] Comparative Example 8 A cryopreservation solution consisting of 100 μΜ SRI-011381 and the solvent of Preparation Example 2.

[0066] Comparative Example 9 A cryopreservation solution consisting of 10 μΜ Y-27632 and the solvent of Preparation Example 2.

[0067] Comparative Example 10 A cryopreservation solution consisting of 1 μM ferrostatin-1 and the solvent of Preparation Example 2.

[0068] Application Example 2 Cryopreservation of natural killer cells (NK) The NK cells were expanded for 13 days using the NK Cell Expansion Kit (Nanjing Keygen Biotech Co., Ltd.), counted, and resuspended at a density of 1 x 10 8 / mL in cryopreservation tubes, centrifuged to discard the supernatant, and resuspended with 1 mL of the solvent of Preparation Example 2 and the cryopreservation solutions of Examples 5-8 (labeled 6-10, respectively) and Comparative Examples 6-10, respectively, and transferred to cryopreservation tubes. The cryopreservation tubes were placed in a cryopreservation box and stored in a -80 °C refrigerator. After 2 weeks, the cryopreserved cells were removed and placed in a 37 °C water bath for 1 minute and 45 seconds, centrifuged at 300 g for 10 minutes, and resuspended with NK cell culture medium at a density of 2 x 10 6 / mL. After 24 hours, the number of viable cells was determined by trypan blue staining, and the 24-hour recovery rate was calculated according to the formula recovery rate (%) = number of viable cells / number of cells inoculated x 100%, and the results are shown in Table 2. Figure 2 and Table 2.

[0069] Table 2 24-hour recovery rate of natural killer cells after 2 weeks of cryopreservation

[0070] As can be seen from Table 2, the cryopreservation solutions containing necrostatin-1, Z-VAD-fmk, and disulfiram significantly promoted the recovery rate of NK cells.

[0071] Preparation Example 3 The solvent was prepared from the following components in the indicated volume percentages: 30% dextran solution at 60 mg / mL, 7.5% adenosine solution at 3 mg / mL, 10% glucose solution at 50 mg / mL, 10% mannitol solution at 200 mg / mL, 30% human albumin solution at 200 mg / mL, 7.5% propylene glycol, and 5% glycerol.

[0072] Example 9 A cryopreservation solution consisting of 1 μM ferrostatin-1 and the solvent of Preparation Example 3.

[0073] Example 10 A cryopreservation solution consisting of 20 μM Y27632 and the solvent of Preparation Example 3.

[0074] Example 11 A cryopreservation solution consisting of 2 μM β-mercaptoethanol and the solvent of Preparation Example 3.

[0075] Example 12 A cryopreservation solution consisting of 0.05 μM ferrostatin-1, 5 μM Y27632, 0.05 μM β-mercaptoethanol and the solvent of Preparation 3.

[0076] Comparative Example 11 A cryopreservation solution consisting of 100 μM chloroquine and the solvent of Preparation 3.

[0077] Comparative Example 12 A cryopreservation solution consisting of 50 μM necrostatin-1 and the solvent of Preparation 3.

[0078] Comparative Example 13 A cryopreservation solution consisting of 50 μM Z-VAD-fmk and the solvent of Preparation 3.

[0079] Comparative Example 14 A cryopreservation solution consisting of 100 μM SRI-011381 and the solvent of Preparation 3.

[0080] Comparative Example 15 A cryopreservation solution consisting of 10 μM disulfiram and the solvent of Preparation 3.

[0081] Application Example 3 Cryopreservation of mesenchymal stromal cells (MSCs) MSCs in logarithmic growth phase and good morphology were digested with recombinant trypsin to form a single cell suspension, the cell suspension was transferred to a centrifuge tube, centrifuged at 400 g for 3 minutes, resuspended in PBS for counting, and 1 x 10 7 / tube, centrifuged to discard the supernatant, and resuspended with 1 mL of the solvent of Preparation 3 and the cryopreservation solutions of Examples 9-12 (numbered 11-15 in order) and Comparative Examples 11-15, respectively, and transferred to cryopreservation tubes. The cryopreservation tubes were placed in a cryopreservation box and stored in a -80°C refrigerator. After 2 weeks, the cryopreserved cells were removed and placed in a 37°C water bath for 1 minute and 45 seconds, centrifuged at 400 g for 5 minutes, resuspended in mesenchymal stem cell medium, and inoculated at a density of 13000 / cm 2 The cell confluence was analyzed by real-time dynamic imaging of living cells using a Cellcyte X, and the results are shown in Figure 3 and Table 3.

[0082] Table 3 Cell confluence of mesenchymal stromal cells after 2 weeks of cryopreservation

[0083] As can be seen from Table 3, the cryopreservation solutions added with ferrostatin-1, Y27632, and β-mercaptoethanol all significantly promoted the adhesion rate of MSCs.

[0084] Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained on the basis of the above embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A cell cryopreservation solution, comprising one or more of induced pluripotent stem cell cryopreservation solution, natural killer cell cryopreservation solution, and mesenchymal matrix cell cryopreservation solution, characterized in that, The induced pluripotent stem cell cryopreservation solution comprises a first death inhibitor and a first solvent; the first death inhibitor is one or more of Y27632, chloroquine, and SRI-011381. The natural killer cell cryopreservation solution includes a second death inhibitor and a second solvent; the second death inhibitor is one or more of necrostatin-1, Z-VAD-fmk, and disulfiram. The mesenchymal stem cell cryopreservation solution includes a third death inhibitor and a third solvent; the third death inhibitor is one or more of ferrostatin-1, Y27632, and β-mercaptoethanol. The first solvent, the second solvent, and the third solvent each comprise the following components by volume percentage: 20-80% colloidal solution, 5-20% energy substance, 5-60% hypertonic solution, and 5-15% permeability protectant.

2. The cell cryopreservation solution according to claim 1, characterized in that, The concentration of Y27632 in the cryopreservation solution for the induced pluripotent stem cells is 5-20 μM; The concentration of chloroquine in the cryopreservation solution for the induced pluripotent stem cells was 25-200 μM. The concentration of SRI-011381 in the induced pluripotent stem cell cryopreservation solution is 10-200 μM.

3. The cell cryopreservation solution according to claim 1, characterized in that, The concentration of necrostatin-1 in the natural killer cell cryopreservation solution is 10-100 μM; The concentration of Z-VAD-fmk in the natural killer cell cryopreservation solution is 10-100 μM; The concentration of disulfiram in the natural killer cell cryopreservation solution is 0.1-25 μM.

4. The cell cryopreservation solution according to claim 1, characterized in that, The concentration of ferrostatin-1 in the mesenchymal matrix cell cryopreservation solution was 0.05-1 μM; The concentration of Y27632 in the mesenchymal matrix cell cryopreservation solution was 5-20 μM; The concentration of β-mercaptoethanol in the mesenchymal matrix cell cryopreservation solution is 0.05-2 mM.

5. The cell cryopreservation solution according to any one of claims 1 to 4, characterized in that, The colloidal solution comprises hydroxyethyl starch and / or dextran; The energy substance includes one or more of adenosine triphosphate disodium, adenosine, and glucose; The hypertonic solution includes one or more of sorbitol, mannitol, and human albumin; The permeation protectant includes one or more of dimethyl sulfoxide, propylene glycol, and glycerol.

6. The cell cryopreservation solution according to claim 5, characterized in that, The colloidal solution comprises 30-90 mg / mL hydroxyethyl starch solution and / or 30-90 mg / mL dextran solution; The energy substance includes one or more of the following: 5-20 mg / mL adenosine triphosphate disodium, 1-10 mg / mL adenosine, and 50-500 mg / mL glucose; The hypertonic solution comprises one or more of the following: 200-300 mg / mL sorbitol, 150-250 mg / mL mannitol, and 50-200 mg / mL human albumin.

7. The use of the cell cryopreservation solution according to any one of claims 1 to 6 in one or more of the following: (1) Cell cryopreservation; (2) Improving cell fusion after cryopreservation and thawing; (3) Reducing programmed cell death after cryopreservation and thawing; (4) Improving the recovery rate of cells after cryopreservation and thawing.

8. The application according to claim 7, characterized in that, The cell cryopreservation temperature is ≤-80℃.

9. The application according to claim 7, characterized in that, The cell confluence includes cell confluence within 24 hours; the programmed cell death includes programmed cell death within 24 hours; and the recovery rate includes the recovery rate within 24 hours.

10. A cell cryopreservation method, comprising one or more of the following: induced pluripotent stem cell cryopreservation method, natural killer cell cryopreservation method, and mesenchymal matrix cell cryopreservation method, characterized in that, The method for cryopreserving induced pluripotent stem cells includes the following steps: mixing the induced pluripotent stem cell cryopreservation solution according to any one of claims 1 to 6 with induced pluripotent stem cells at a ratio of 1×10 6 ~5×10 7 Mix / mL and store frozen at ≤-80℃; The natural killer cell cryopreservation method includes the following steps: mixing the natural killer cell cryopreservation solution according to any one of claims 1 to 6 with natural killer cells at a ratio of 1×10⁻⁶. 7 ~5×10 8 Mix / mL and store frozen at ≤-80℃; The mesenchymal stromal cell cryopreservation method comprises the following steps: mixing the mesenchymal stromal cell cryopreservation solution according to any one of claims 1 to 6 with mesenchymal stromal cells at a ratio of 1×10 6 ~5×10 7 Mix / m and freeze at ≤-80℃.

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