Peripheral blood hematopoietic stem cell cryopreservation solution, preparation method and application thereof

Abstract

The application provides a peripheral blood hematopoietic stem cell freezing solution, wherein each liter of the peripheral blood hematopoietic stem cell freezing solution comprises 1.5 g of poloxamer P188, 15 g of dextran, 50 g of glucose, 100 ml of dimethyl sulfoxide, 0-300 ml of human blood albumin, and the rest is a compound electrolyte solution. The application also provides a preparation method of the peripheral blood hematopoietic stem cell freezing solution and an application of the peripheral blood hematopoietic stem cell freezing solution. The peripheral blood hematopoietic stem cell freezing solution can freeze peripheral blood hematopoietic stem cells and improve the survival rate of the peripheral blood hematopoietic stem cells after freezing and resuscitation.

Description

Technical Field

[0001] This invention relates to the field of cell cryopreservation technology, and particularly to the field of cell cryopreservation solution technology, specifically to a peripheral blood hematopoietic stem cell cryopreservation solution, its preparation method, and its application. Background Technology

[0002] Cell cryopreservation is a technique that uses low temperatures to reduce the metabolic activity of cells for long-term preservation, typically using liquid nitrogen at -196°C as the storage medium. The core method involves adding cryoprotectants such as glycerol or dimethyl sulfoxide to the culture medium and using gradient cooling (e.g., initially -1 to -2°C / min) to reduce ice crystal damage. Cells in the logarithmic growth phase are often selected for cryopreservation to improve survival rates.

[0003] Peripheral blood hematopoietic stem cells (PBHSCTs) exist in the peripheral blood circulation and possess the ability to self-renew and differentiate into multiple blood cell types, including erythrocytes, leukocytes, and platelets. They play a crucial role in maintaining normal hematopoietic and immune functions. Under specific conditions such as chemotherapy or radiotherapy, they can be collected for transplantation therapy. After transplantation, they can rebuild the patient's hematopoietic and immune systems. PBHSCT has been widely developed both domestically and internationally. Compared with traditional bone marrow transplantation (BMT), it has advantages such as greater safety, convenience, and better post-transplant outcomes, and has become an effective method for treating malignant hematological diseases and solid tumors. Using PBHSCT cryopreservation solution to freeze PBHSCTs can improve the cryopreservation survival rate of PBHSCTs, ensure the number of live cells that can be reinfused from the patient's own hematopoietic stem cells, and increase the success rate of transplantation.

[0004] However, current technologies typically use a final concentration of 5% by weight dimethyl sulfoxide (DMSO), 3% by weight hydroxyethyl starch (HES), and 2% by weight human serum albumin as stem cell protectants to cryopreserve peripheral blood hematopoietic stem cells, resulting in a low cell survival rate after cryopreservation.

[0005] For example, a study by Wang Zhiguo et al. (Wang Zhiguo et al., Research on cryopreservation of peripheral blood hematopoietic stem cells, Health Must-Read (Late Issue) 000.002 (2011): 229-230) showed that when peripheral blood hematopoietic stem cells were stored in a -80℃ cryopreservation freezer with a final concentration of 5% by weight dimethyl sulfoxide (DMSO), 3% by weight hydroxyethyl starch (HES) and 2% by weight human serum albumin as stem cell protectants, the cell survival rate could be maintained at about 84.6% within six months of cryopreservation.

[0006] Some researchers also use the hematopoietic stem cell cryoprotectant from Shanghai Sansheng Biotechnology Co., Ltd., which mainly consists of dimethyl sulfoxide, dextran, albumin and nutrients. The cell survival rate of autologous stem cells collected and cryopreserved for 21 to 69 days (about 1 to 2 months) after thawing and recovery is 82 to 88% (Chen Yuekuan et al., Clinical application of autologous peripheral blood hematopoietic stem cells of patients with malignant hematological diseases cryopreserved at -80℃, Journal of the Third Military Medical University, 2009, Vol. 21, pp. 2162-2163).

[0007] Therefore, it is desirable to provide a cryopreservation solution for peripheral blood hematopoietic stem cells that can cryopreserve peripheral blood hematopoietic stem cells and improve the survival rate of peripheral blood hematopoietic stem cells after cryopreservation and thawing. Summary of the Invention

[0008] In order to overcome the shortcomings of the prior art, one object of the present invention is to provide a peripheral blood hematopoietic stem cell cryopreservation solution that can cryopreserve peripheral blood hematopoietic stem cells and improve the survival rate of peripheral blood hematopoietic stem cells after cryopreservation and thawing, and is suitable for large-scale promotion and application.

[0009] Another objective of this invention is to provide a peripheral blood hematopoietic stem cell cryopreservation solution that is ingeniously designed, has a simple formula, is easy to prepare, and is inexpensive, making it suitable for large-scale application.

[0010] Another objective of this invention is to provide a method for preparing a cryopreservation solution for peripheral blood hematopoietic stem cells. The prepared cryopreservation solution can preserve peripheral blood hematopoietic stem cells and improve the survival rate of peripheral blood hematopoietic stem cells after cryopreservation and thawing, making it suitable for large-scale application.

[0011] Another objective of this invention is to provide a method for preparing a cryopreservation solution for peripheral blood hematopoietic stem cells, which is ingeniously designed, has simple steps, is easy to operate, and is suitable for large-scale application.

[0012] Another objective of this invention is to provide an application of a cryopreservation solution for peripheral blood hematopoietic stem cells, which is used to cryopreserve peripheral blood hematopoietic stem cells and improve the survival rate of peripheral blood hematopoietic stem cells after cryopreservation and thawing, and is suitable for large-scale promotion and application.

[0013] Another objective of this invention is to provide an application of a peripheral blood hematopoietic stem cell cryopreservation solution, which is ingeniously designed, easy to use, and suitable for large-scale promotion and application.

[0014] To achieve the above objectives, in a first aspect of the present invention, a peripheral blood hematopoietic stem cell cryopreservation solution is provided, characterized in that each liter of the peripheral blood hematopoietic stem cell cryopreservation solution comprises:

[0015] Poloxamer P188 1.5g;

[0016] 15g of dextran;

[0017] 50g of glucose;

[0018] 100ml of dimethyl sulfoxide;

[0019] Human serum albumin 0~300ml;

[0020] The remainder is a compound electrolyte solution.

[0021] Preferably, the human serum albumin is 300 ml.

[0022] Preferably, the human serum albumin is 150 ml.

[0023] Preferably, the human serum albumin is 0 ml.

[0024] In a second aspect of the present invention, a method for preparing the above-mentioned peripheral blood hematopoietic stem cell cryopreservation solution is provided, characterized by comprising the following steps:

[0025] (1) Dissolve the poloxamer P188, the dextran, and the glucose in a portion of the compound electrolyte solution to obtain a mixed solution;

[0026] (2) Add the dimethyl sulfoxide to the mixed solution and mix well, then add the human serum albumin and mix well, and finally add the remaining compound electrolyte solution and mix well.

[0027] Preferably, the compound electrolyte solution described in this part is 300 ml.

[0028] In a third aspect of the present invention, the application of the peripheral blood hematopoietic stem cell cryopreservation solution prepared by the above-described method or the method described above in the cryopreservation of peripheral blood hematopoietic stem cells is provided.

[0029] The main beneficial effects of this invention are:

[0030] 1. The peripheral blood hematopoietic stem cell cryopreservation solution of the present invention comprises, per liter: 1.5g poloxamer P188, 15g dextran, 50g glucose, 100ml dimethyl sulfoxide, 0-300ml human serum albumin, with the remainder being a compound electrolyte solution. It can cryopreserve peripheral blood hematopoietic stem cells and improve the survival rate of peripheral blood hematopoietic stem cells after cryopreservation and thawing, and is suitable for large-scale promotion and application.

[0031] 2. The peripheral blood hematopoietic stem cell cryopreservation solution of the present invention comprises, per liter: 1.5g poloxamer P188, 15g dextran, 50g glucose, 100ml dimethyl sulfoxide, 0-300ml human serum albumin, with the remainder being a compound electrolyte solution. It is ingeniously designed, has a simple formula, is easy to prepare, and is inexpensive, making it suitable for large-scale promotion and application.

[0032] 3. The method for preparing the peripheral blood hematopoietic stem cell cryopreservation solution of the present invention includes the following steps: (1) dissolving poloxamer P188, dextran and glucose in a portion of the compound electrolyte solution to obtain a mixed solution; (2) adding dimethyl sulfoxide to the mixed solution and mixing well, then adding human serum albumin and mixing well, and finally adding the remaining compound electrolyte solution and mixing well. The prepared peripheral blood hematopoietic stem cell cryopreservation solution can cryopreserve peripheral blood hematopoietic stem cells and can improve the survival rate of peripheral blood hematopoietic stem cells after cryopreservation and thawing, which is suitable for large-scale promotion and application.

[0033] 4. The method for preparing the peripheral blood hematopoietic stem cell cryopreservation solution of the present invention includes the following steps: (1) dissolving poloxamer P188, dextran and glucose in a portion of the compound electrolyte solution to obtain a mixed solution; (2) adding dimethyl sulfoxide to the mixed solution and mixing, then adding human serum albumin and mixing, and finally adding the remaining compound electrolyte solution and mixing. The design is ingenious, the steps are simple, the operation is convenient, and it is suitable for large-scale promotion and application.

[0034] 5. The application of the peripheral blood hematopoietic stem cell cryopreservation solution of the present invention in the cryopreservation of peripheral blood hematopoietic stem cells is used to cryopreserve peripheral blood hematopoietic stem cells and improve the survival rate of peripheral blood hematopoietic stem cells after cryopreservation and thawing, which is suitable for large-scale promotion and application.

[0035] 6. The peripheral blood hematopoietic stem cell cryopreservation solution of the present invention is ingeniously designed, easy to use, and suitable for large-scale promotion and application.

[0036] These and other objects, features and advantages of the present invention will be fully realized by the following detailed description and claims, and may be achieved by the means, methods and combinations thereof specifically pointed out in the appended claims. Detailed Implementation

[0037] To improve the survival rate of peripheral blood hematopoietic stem cells after cryopreservation and thawing, the inventors conducted extensive research on the formulation of peripheral blood hematopoietic stem cell cryopreservation solutions. They discovered that adding poloxamer P188, dextran, glucose, dimethyl sulfoxide (DMSO), and human serum albumin to a compound electrolyte solution to form a peripheral blood hematopoietic stem cell cryopreservation solution can preserve peripheral blood hematopoietic stem cells and improve their survival rate after cryopreservation and thawing. Based on this, the present invention was completed.

[0038] This invention first provides a cryopreservation solution for peripheral blood hematopoietic stem cells, wherein each liter of the cryopreservation solution comprises:

[0039] Poloxamer P188 1.5g;

[0040] 15g of dextran;

[0041] 50g of glucose;

[0042] 100ml of dimethyl sulfoxide;

[0043] Human serum albumin 0~300ml;

[0044] The remainder is a compound electrolyte solution.

[0045] The amount of human serum albumin used can be determined as needed; preferably, the amount of human serum albumin is 300 ml.

[0046] The amount of human serum albumin used can be determined as needed; preferably, the amount of human serum albumin is 150 ml.

[0047] The amount of human serum albumin used can be determined as needed; preferably, the amount of human serum albumin is 0 ml.

[0048] The present invention also provides a method for preparing the above-mentioned peripheral blood hematopoietic stem cell cryopreservation solution, comprising the following steps:

[0049] (1) Dissolve the poloxamer P188, the dextran, and the glucose in a portion of the compound electrolyte solution to obtain a mixed solution;

[0050] (2) Add the dimethyl sulfoxide to the mixed solution and mix well, then add the human serum albumin and mix well, and finally add the remaining compound electrolyte solution and mix well.

[0051] The compound electrolyte solution described in this section can have any suitable volume, preferably 300 ml.

[0052] The present invention also provides the application of the peripheral blood hematopoietic stem cell cryopreservation solution prepared by the above-mentioned method for preparing the peripheral blood hematopoietic stem cell cryopreservation solution in the cryopreservation of peripheral blood hematopoietic stem cells.

[0053] To better understand the technical content of this invention, the following embodiments are provided for detailed explanation. Wherein:

[0054] Poloxamer P188 (Hubei Gedian Renfu Pharmaceutical Excipients Co., Ltd., Specification: 500g / pack)

[0055] Dextran (Shanghai Huamao Pharmaceutical Co., Ltd., Specification: 1kg / bag)

[0056] Dimethyl sulfoxide (Actylis, 2L / bottle)

[0057] Human serum albumin (Octapharma GmbH, specification: 50ml / bottle)

[0058] Compound electrolyte solution (Hebei Tiancheng Pharmaceutical Co., Ltd., specification: 500ml / bag), containing 0.31-0.38% (g / ml) of chlorine (Cl), 0.29-0.35% (g / ml) of sodium (Na), 0.017-0.021% (g / ml) of potassium (K), 0.0032-0.0039% (g / ml) of magnesium (Mg), 0.40-0.49% of gluconate (C6H11O7-), and 0.14-0.18% (g / ml) of acetate (C2H3O2-).

[0059] Cell viability detection method: AOPI fluorescence staining method, using Countstar Rigel fully automated cell fluorescence analyzer and AOPI fluorescent dye for detection.

[0060] Example 1: Preparation of Peripheral Blood Hematopoietic Stem Cell Cryopreservation Solution A

[0061] Add 7.5g poloxamer P188, 75g dextran, and 250g glucose to a reagent bottle, then add 1500ml of compound electrolyte solution, mix well until the solute is completely dissolved, add 500ml dimethyl sulfoxide (DMSO), mix well, then add 1500ml human serum albumin and mix well. Make up the volume to 5L with compound electrolyte solution and mix well. Vacuum filter sterilize (PES filter membrane pore size 0.22 microns). After filtration, obtain peripheral blood hematopoietic stem cell cryopreservation solution A.

[0062] Example 2: Preparation of Peripheral Blood Hematopoietic Stem Cell Cryopreservation Solution B

[0063] Add 7.5g poloxamer P188, 75g dextran, and 250g glucose to a reagent bottle, then add 1500ml of compound electrolyte solution, mix well until the solute is completely dissolved, add 500ml dimethyl sulfoxide (DMSO), mix well, then add 750ml human serum albumin and mix well. Make up the volume to 5L with compound electrolyte solution and mix well. Vacuum filter sterilize (PES filter membrane pore size 0.22 microns). After filtration, obtain peripheral blood hematopoietic stem cell cryopreservation solution B.

[0064] Example 3: Preparation of Peripheral Blood Hematopoietic Stem Cell Cryopreservation Solution C

[0065] Add 7.5g poloxamer P188, 75g dextran, and 250g glucose to a reagent bottle, then add 1500ml of compound electrolyte solution, mix well until the solute is completely dissolved, add 500ml dimethyl sulfoxide (DMSO), and bring the volume to 5L with compound electrolyte solution and mix well. Vacuum filter sterilize (PES filter membrane pore size 0.22 microns). After filtration, obtain peripheral blood hematopoietic stem cell cryopreservation solution C.

[0066] Example 4: Comparison of cryopreservation effects of different cryopreservation solutions

[0067] Four experimental groups were used, including three sample groups and one control group. Five peripheral blood samples (all from myeloma patients at Ruijin Hospital in Shanghai) were numbered as Sample 1 to Sample 5. 20 ml of Sample 1 to Sample 5 was taken from each sample group, and 20 ml of Sample 4 to Sample 5 was taken from the control group.

[0068] After centrifugation, the plasma from the four experimental groups was cryopreserved using peripheral blood hematopoietic stem cell cryopreservation solutions A-C (12ml each) prepared in Examples 1-3, and a hematopoietic stem cell cryoprotectant (purchased from Shanghai Sansheng Biotechnology Co., Ltd., whose composition is dimethyl sulfoxide, dextran, albumin, nutrients (M199 culture medium, glutamine, phosphate, etc.). The cryopreservation conditions were: -80℃ for 2 days, and then transferred to a liquid nitrogen tank for cryopreservation.

[0069] Cell viability was tested after 1 month, 3 months, and 6 months of cryopreservation. The results are shown in Tables 1-4 below.

[0070] Table 1. Cryopreservation effect using peripheral blood hematopoietic stem cell cryopreservation solution A

[0071]

[0072] Table 2. Cryopreservation effect using peripheral blood hematopoietic stem cell cryopreservation solution B

[0073]

[0074] Table 3. Cryopreservation effect using peripheral blood hematopoietic stem cell cryopreservation solution C

[0075]

[0076] Table 4. Cryopreservation effect using Sansheng biological cryopreservation solution

[0077]

[0078] As shown in Tables 1-4, compared with the control group, the cells cryopreserved using the cryopreservation solution of the present invention exhibit better cell viability stability. Furthermore, after one month of cryopreservation, the cell viability of cells cryopreserved using the cryopreservation solution of the present invention is similar to that of the control group, while after three and six months of cryopreservation, the cell viability of cells cryopreserved using the cryopreservation solution of the present invention is significantly higher than that of the control group.

[0079] Therefore, this invention provides a peripheral blood hematopoietic stem cell cryopreservation solution by adding poloxamer P188, dextran, glucose, dimethyl sulfoxide (DMSO), and human serum albumin to a compound electrolyte solution. This solution can cryopreserve peripheral blood hematopoietic stem cells and significantly improve the survival rate of peripheral blood hematopoietic stem cells after cryopreservation and thawing, with a six-month survival rate of over 90%.

[0080] In summary, the peripheral blood hematopoietic stem cell cryopreservation solution of the present invention can cryopreserve peripheral blood hematopoietic stem cells and improve the survival rate of peripheral blood hematopoietic stem cells after cryopreservation and thawing. It is ingeniously designed, has a simple formula, is easy to prepare, and is inexpensive, making it suitable for large-scale promotion and application.

[0081] Therefore, it is evident that the objective of this invention has been fully and effectively achieved. The function and structural principles of this invention have been demonstrated and explained in the embodiments. Any modifications can be made to the implementation methods without departing from these principles. Therefore, this invention includes all modified embodiments based on the spirit and scope of the claims.

Claims

1. A peripheral blood hematopoietic stem cell cryopreservation solution, characterized by, Each liter of the peripheral blood hematopoietic stem cell cryopreservation solution comprises: Poloxamer P188 1.5g; 15g of dextran; 50g of glucose; 100ml of dimethyl sulfoxide; Human serum albumin 0~300ml; The remainder is a compound electrolyte solution.

2. The peripheral blood hematopoietic stem cell cryopreservation solution of claim 1, wherein, The human serum albumin was 300ml.

3. The peripheral blood hematopoietic stem cell cryopreservation solution of claim 1, wherein The human serum albumin was 150 ml.

4. The peripheral blood hematopoietic stem cell cryopreservation solution of claim 1, wherein, The human serum albumin was 0 ml.

5. A method for preparing a peripheral blood hematopoietic stem cell cryopreservation solution as claimed in claim 1, characterized by, Includes the following steps: (1) Dissolve the poloxamer P188, the dextran, and the glucose in a portion of the compound electrolyte solution to obtain a mixed solution; (2) Add the dimethyl sulfoxide to the mixed solution and mix well, then add the human serum albumin and mix well, and finally add the remaining compound electrolyte solution and mix well.

6. The method for preparing a peripheral blood hematopoietic stem cell cryopreservation solution according to claim 5, wherein The compound electrolyte solution described in this section is 300 ml.

7. The application of the peripheral blood hematopoietic stem cell cryopreservation solution prepared according to any one of claims 1 to 4 or the method of preparing the peripheral blood hematopoietic stem cell cryopreservation solution according to any one of claims 5 to 6 in the cryopreservation of peripheral blood hematopoietic stem cells.

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