A cell cold storage protection solution and its preparation method and application

Abstract

The present invention discloses a cell cold storage protection solution and a preparation method and application thereof, and belongs to the field of cell preservation technology. The present invention provides a cell cold storage protection solution, the formula composition includes a compound electrolyte solution, red blood cell preservation solution, heparin sodium, human serum albumin, human platelet lysate and glucose. When preparing the cell cold storage protection solution, mix and prepare it under sterile conditions at room temperature. When the cell cold storage protection solution of the present invention is used to refrigerate a stem cell preparation, the activity of the stem cells is still above 85% after refrigeration for 96 hours, and the growth, proliferation and differentiation potential of the stem cells can be maintained.

Description

Technical Field

[0001] The invention belongs to the technical field of cell preservation, and in particular relates to a cell cold storage protection solution and a preparation method and application thereof. Background Art

[0002] In recent years, cell biological therapy technology has gradually replaced traditional drug therapy and played an important role in various disease areas. Cell therapy refers to the use of cells with certain specific functions, which are processed by bioengineering methods such as in vitro amplification and special culture to make these cells have therapeutic effects such as enhancing immunity, killing pathogens and tumor cells, promoting tissue and organ regeneration and body recovery, thereby achieving the purpose of treating diseases.

[0003] MSCs or fibroblasts often need to be transported over long distances during clinical use. During transportation, the activity of cells will be affected by the transportation environment and will decrease. For example, if the cells used have been frozen and revived, the cell viability and activity are relatively low, and the storage time in sodium chloride injection is relatively short. In the process of clinical application, it often takes 10 hours or even 2 to 3 days of transportation time, which is not conducive to the transportation and application of cells.

[0004] Sodium chloride injection is the most commonly used cell resuspension solution and also a commonly used cell transport preservation solution. It is a clinical product. Its advantage is that after the cell resuspension is prepared in the laboratory, it does not need to be processed again and can be used directly in the clinic. Sodium chloride injection can be used in short-term transportation, and it guarantees the activity of cells for about 10 hours. However, in the long-term transportation, the activity of cells will gradually decline over time, and it cannot be transported over long distances. Summary of the invention

[0005] The purpose of the present invention is to provide a cell cold storage protection solution and a preparation method and application thereof, so that the cell biological activity can be kept stable and controllable when the cell preparation is transported and stored for a long time under cold chain conditions.

[0006] The invention provides a cell cold storage protection solution, which comprises the following components: a compound electrolyte solution, a red blood cell preservation solution, heparin sodium, human serum albumin, human platelet lysate and glucose.

[0007] Preferably, the composition comprises the following components in percentage by volume: 30-60% compound electrolyte solution, 10-35% red blood cell preservation solution, 3-30% heparin sodium aqueous solution, 10-30% human albumin solution, 2-15% human platelet lysate and 5-20% glucose aqueous solution.

[0008] Preferably, the concentration of heparin sodium in the heparin sodium aqueous solution is 100 to 500 units / mL;

[0009] Preferably, the mass percentage of human albumin in the human albumin solution is 20%.

[0010] Preferably, the mass concentration of glucose in the glucose aqueous solution is 20 g / 100 mL.

[0011] The present invention also provides a method for preparing the cell cold storage protection solution, which comprises mixing a compound electrolyte solution, a red blood cell preservation solution, sodium heparin, human albumin, a human platelet lysate and glucose in a sterile environment to obtain the cell cold storage protection solution.

[0012] The present invention also provides the use of the cell cold storage protection solution in cold chain preservation and / or cold chain transportation of cell samples.

[0013] Preferably, the cell sample comprises a stem cell sample.

[0014] Preferably, the cell sample comprises neural stem cells, mesenchymal stem cells or adipose stromal cells.

[0015] Preferably, the temperature of the cold chain preservation and / or cold chain transportation is 2-8°C.

[0016] Beneficial effects: The present invention provides a cell cold preservation solution, the formula composition includes a compound electrolyte solution, red blood cell preservation solution, heparin sodium, human serum albumin, human platelet lysate and glucose. When preparing the cell cold preservation solution, mix and prepare it under sterile conditions at room temperature. When the cell cold preservation solution of the present invention is used to cold store the stem cell preparation, the activity of the stem cells is still above 85% after 96 hours of cold storage, and the growth, proliferation and differentiation potential of the stem cells can be maintained. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The cell status diagram of hUC-MSCs in the cell cold storage protection solution experimental group after 0h, 24h, 48h, 72h, and 96h of attachment;

[0018] Figure 2 The cell status diagram of hUC-MSCs in the sodium chloride injection control group after 0h, 24h, 48h, 72h, and 96h of attachment;

[0019] Figure 3 These are the differentiation results of hUC-MSCs in the cell cold storage protection solution experimental group after being stored for 0h, 24h, 48h, 72h, and 96h. DETAILED DESCRIPTION

[0020] The invention provides a cell cold storage protection solution, which comprises the following components: a compound electrolyte solution, a red blood cell preservation solution, heparin sodium, human serum albumin, human platelet lysate and glucose.

[0021] The components of the cell cold storage protection solution of the present invention include a compound electrolyte solution, and the volume of the compound electrolyte solution is preferably 30-60% of the volume of the cell cold storage protection solution, and more preferably 40%. The compound electrolyte solution of the present invention preferably contains a variety of substances such as sodium chloride, potassium chloride, calcium chloride, and magnesium chloride, which can replenish nutrients for cell energy metabolism in time, which is conducive to better maintaining cell life activities. The compound electrolyte solution of the present invention is preferably a commercially available product, and the trade name is Bomeili A.

[0022] The components of the cell cold preservation solution of the present invention include red blood cell preservation solution, and the volume of the red blood cell preservation solution is preferably in the range of 10-35% of the volume of the cell cold preservation solution, more preferably 15-30%, and most preferably 20%. The red blood cell preservation solution of the present invention is a commercial drug, and the instructions show that the preservation time of red blood cells in vitro is 35 days.

[0023] The components of the cell cold storage protection solution of the present invention include heparin sodium, and the heparin sodium exists in the form of solution when used. The concentration of heparin sodium in the heparin sodium aqueous solution is 100-500 units / mL, more preferably 150-400 units / mL, and most preferably 200 units / mL. The heparin sodium aqueous solution of the present invention is preferably 3-30% of the volume of the cell cold storage protection solution, more preferably 3-15%, and most preferably 5%. The heparin sodium of the present invention can effectively avoid the coagulation phenomenon caused by the addition of platelet lysate.

[0024] The components of the cell cold preservation solution of the present invention include human albumin, and the human albumin is preferably present in the form of a solution when used, and the concentration of the human albumin solution is preferably 20%. The volume percentage of the human albumin solution in the cell cold preservation solution of the present invention is preferably 10-30%, more preferably 10-20%, and most preferably 20%. The human albumin of the present invention is a marketed drug for clinical intravenous use, and has a protective effect on cell activity due to its high protein content.

[0025] The components of the cell cold storage protection solution of the present invention include human platelet lysate, and the human platelet lysate exists in liquid form. The volume percentage of the human platelet lysate in the cell cold storage protection solution of the present invention is preferably 2-15%, more preferably 2-10%, and most preferably 5%. The human platelet lysate of the present invention is a commercial human platelet extract, which contains various cytokines such as platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). The human platelet lysate used in the embodiments of the present invention is preferably a platelet lysate that is a commercial reagent, and the brand is helios.

[0026] The components of the cell cold preservation solution of the present invention include glucose, and the glucose is added in the form of a glucose aqueous solution, and the concentration of glucose in the glucose aqueous solution is 20g / 100mL. The volume percentage of the glucose aqueous solution in the cell cold preservation solution of the present invention is preferably 5-20%, more preferably 5-10%, and most preferably 10%. The glucose concentration of the present invention is 20%, which provides energy for cells and helps maintain cell activity.

[0027] The present invention also provides a method for preparing the cell cold storage protection solution, which comprises mixing a compound electrolyte solution, a red blood cell preservation solution, sodium heparin, human albumin, a human platelet lysate and glucose in a sterile environment to obtain the cell cold storage protection solution.

[0028] The cell cold storage protection solution of the present invention is preferably mixed under sterile conditions at room temperature (18-25° C.) to complete the preparation.

[0029] The present invention also provides the use of the cell cold storage protection solution in cold chain preservation and / or cold chain transportation of cell samples.

[0030] The cell cold storage protection solution of the present invention can be used for long-term cold storage of cell samples, and the cell samples preferably include stem cell samples, and more preferably include neural stem cells, mesenchymal stem cells or adipose stromal cells. The temperature of the cold chain preservation and / or cold chain transportation of the present invention is preferably 2 to 8°C. When the stem cell preparation is refrigerated using the cell cold storage protection solution of the present invention, the activity of the stem cells is still above 85% after 96 hours of cold storage, and the growth, proliferation and differentiation potential of the stem cells can be maintained.

[0031] In order to further illustrate the present invention, a cell cold storage protection solution and its preparation method and application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the protection scope of the present invention.

[0032] Example 1

[0033] 1. Preparation method of human umbilical cord mesenchymal stem cells

[0034] With the informed consent of the parturient, one umbilical cord tissue of a full-term baby delivered by normal delivery or cesarean section was collected under aseptic operation, and primary cells were extracted and separated by the tissue block adhesion method. The cells were expanded and cultured in vitro to the fifth generation using α-MEM medium containing 10% FBS, digested and centrifuged, and human umbilical cord mesenchymal stem cells (hUC-MSCs) were harvested in a centrifuge tube.

[0035] 2. Preparation method of human adipose-derived mesenchymal stem cells

[0036] With the informed consent of the donor, 50 ml of abdominal fat was aspirated, primary cells were extracted by collagenase digestion, and 10% FBS α-MEM medium was used for in vitro expansion culture to the fifth generation. After digestion and centrifugation, human adipose-derived mesenchymal stem cells (hAD-MSCs) were harvested in a centrifuge tube.

[0037] 3. Saving method

[0038] The human umbilical cord mesenchymal stem cells and human adipose mesenchymal stem cells were divided into two parts respectively, one part was added with cell cold preservation solution, and the other part was added with sodium chloride injection and human serum albumin.

[0039] 3.1. Storage of cells in cryopreservation solution

[0040] Prepare cell cold preservation solution according to the following method: add 45 ml of compound electrolyte solution, 20 ml of red blood cell preservation solution, 5 ml of 200 units / ml heparin sodium, 15 ml of 20% human albumin, 5 ml of human platelet lysate, and 10 ml of 20% glucose solution in sequence, mix well under sterile conditions, and take an appropriate amount of the cell cold preservation solution to suspend the above cell pellet.

[0041] 3.2. Storage of Sodium Chloride Injection

[0042] Take an appropriate amount of the above cell precipitate, add sodium chloride injection, mix well under sterile conditions, then add human albumin solution with a final concentration of 5%, and mix well.

[0043] 4. Cell viability

[0044] hUC-MSCs and hAD-MSCs were prepared according to the above method. The two cells were placed in the cell cold storage protection solution and sodium chloride injection control group, respectively, and the cell concentration was adjusted to 2×10 5 Each formula was prepared into 100 ml, and all were stored at 2-8°C in the dark. Samples were taken at 0h, 24h, 48h, and 72h, and the cell viability was detected by AO / PI staining and a cell counter. The results are shown in Table 1.

[0045] Table 1 Changes in cell viability at different storage times

[0046]

[0047] 5. Immunophenotype

[0048] (1) Main equipment: flow cytometer (manufacturer: BD), centrifuge (manufacturer: Thermo).

[0049] (2) Main reagents: CD73, CD90, CD105, CD14, CD19, CD34, CD45, HLA-DR monoclonal antibodies (manufacturer: BD; catalog numbers: 550257, 555596, 560839, 557153, 555412, 555821, 555482, 555560).

[0050] (3) Experimental methods:

[0051] hUC-MSCs were taken at a cell concentration of 5×10 5 12 tubes were dispensed per tube, with 1 living cell per tube. Different fluorescently labeled CD73, CD90, CD105, CD14, CD19, CD34, CD45, HLA-DR monoclonal antibodies and isotype controls were added respectively, mixed well, and incubated at 4°C in the dark for 30 min.

[0052] After incubation, 1 ml of sodium chloride injection was added to each tube, mixed, centrifuged at 1800 rpm for 7 min, supernatant discarded, washed repeatedly once, and then 0.5 ml of sodium chloride injection was added to each tube, mixed, and detected using a BD Calibur flow cytometer, and data analysis was performed using Flowjo.7.6 software.

[0053] (4) Immunophenotypic criteria:

[0054] The positive rates of CD73, CD90 and CD105 were all ≥95%

[0055] The positive rates of CD14, CD19, CD34, CD45 and HLA-DR were all ≤2%

[0056] (5) Test results

[0057] The results are shown in Table 2. The immune phenotypes of the cell cold storage protection solution experimental group at 0h, 24h, 48h, 72h, and 96h all met the standards; the immune phenotypes of the sodium chloride injection experimental group at 0h, 24h, and 48h all met the standards, but the immune phenotypes at 72h and 96h were significantly different and did not meet the standards.

[0058] Table 2 Changes in cell immune phenotypes at different times of refrigerated storage

[0059]

[0060] 6. Cell morphology

[0061] hUC-MSCs were diluted with cell cold storage solution and sodium chloride injection solution to a cell concentration of 5×10 5The viable cells / tube were cultured and the cell growth and proliferation were counted after 0h, 24h, 48h, 72h, and 96h of culture. Figure 1 and Figure 2 As shown, only a small number of cells adhered to the wall during the culture process in the sodium chloride injection control group, while the cells in the cell cold storage protection solution experimental group gradually proliferated and reached a confluence of more than 90% after 96 hours of culture.

[0062] 7. Induce differentiation into osteoblasts

[0063] (1) Main equipment: biological safety cabinet (manufacturer: Beijing Donglianhal Instrument Manufacturing Co., Ltd.; model: BSC-1100-LⅡA2), CO2 incubator (manufacturer: Thermo; model: 371), inverted microscope (manufacturer: Olympus Corporation; model: CKX41)

[0064] (2) Main reagents: osteogenic differentiation induction medium (manufacturer: Stemcell; catalog number: 05465), 0.2% Alizarin Red staining solution (manufacturer: Beijing Solebow Technology Co., Ltd.; catalog number: G1450), high glucose medium (manufacturer: Gibco; catalog number: 12800017), fetal bovine serum (manufacturer: BI; catalog number: 04-001-1ACS)

[0065] (3) Experimental method: hUC-MSCs were collected from the sodium chloride injection control group and the cell cold storage protection solution experimental group after being stored under cold conditions for 0 h, 24 h, 48 h, 72 h, and 96 h, respectively. They were first expanded and cultured until the cell confluence was about 80%. After successful expansion and culture, 2 × 10 5 Live cells / well were inoculated into 6-well cell culture plates, and high-glucose medium containing 10% fetal bovine serum was added to 2 ml / well. The plates were cultured in a 37°C, 5% CO2 incubator until the cell confluence reached about 80%, and then the osteogenic differentiation induction medium was replaced at 2 ml / well. The first medium change was recorded as the first day of differentiation induction. Thereafter, the medium was changed once every 3 days for 21 days of differentiation induction.

[0066] After the induction of differentiation is completed, discard the supernatant, add sodium chloride injection to wash once, 3ml / time; add 4% tissue cell fixative, 2ml / well, fix for 30min, add sodium chloride injection to wash twice, 3ml / time; add 0.2% Alizarin Red staining solution, 2ml / well, stain for 30min, add sodium chloride injection to wash twice, 3ml / time; enlarge 40 times under a microscope and take pictures with a digital camera.

[0067] The experimental results are as follows Figure 3As shown, in the sodium chloride injection control group, hUC-MSCs stored under refrigerated conditions for 0h, 24h, 48h, 72h, and 96h were taken for expansion and culture until 24h and 48h. There were very few cells attached to the wall, and no cell proliferation was observed, so the induction differentiation experiment was not performed; in the cell cold storage protection solution experimental group, hUC-MSCs stored under refrigerated conditions for 0h, 24h, 48h, 72h, and 96h were taken for expansion and culture, and the samples taken out at each time point were able to grow and proliferate. The cell confluence was about 80% after culturing for 48h. The samples at each time point were subjected to the induction differentiation experiment into osteoblasts, and the results showed that they were all able to differentiate into osteocytes. There was no significant difference in the osteoblast differentiation ability of the samples at each time point in the cell cold storage protection solution experimental group, indicating that the cell activity and biological function can be maintained unchanged until 96h.

[0068] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A stem cell cryopreservation protection solution, characterized in that: It is composed of the following components in percentage by volume: 30-60% compound electrolyte solution, 10-35% red blood cell preservation solution, 3-30% heparin sodium aqueous solution, 10-30% human albumin solution, 2-15% human platelet lysate and 5-20% glucose aqueous solution; The concentration of heparin sodium in the heparin sodium aqueous solution is 100-500 units / mL; The mass percentage of human albumin in the human albumin solution is 20%; The mass concentration of glucose in the glucose aqueous solution is 20 g / 100 mL.

2. The method for preparing the stem cell cryopreservation protection solution according to claim 1, characterized in that: The compound electrolyte solution, red blood cell preservation solution, heparin sodium, human albumin, human platelet lysate and glucose are mixed in a sterile environment to obtain the cell cold storage protection solution.

3. Use of the stem cell cold storage protection solution according to claim 1 in cold chain storage and / or cold chain transportation of cell samples, characterized in that: The cell sample includes a stem cell sample.

4. The use according to claim 3, characterized in that: The cell samples include neural stem cells, mesenchymal stem cells or adipose stromal cells.

5. The application according to claim 3, characterized in that: The temperature of the cold chain preservation and / or cold chain transportation is 2~8°C.

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