Cell cryopreservation agents, their preparation methods, applications, and cell cryopreservation methods

Abstract

This invention provides a cell cryopreservation agent, its preparation method, application, and cell cryopreservation method, relating to the field of cell technology. Through extensive experimental screening, the inventors of this invention have provided a cell cryopreservation agent with specific components and dosages. This cell cryopreservation agent can effectively preserve cell viability, while having clearly defined components. All components used are injection-grade, ensuring high clinical safety. Furthermore, it does not contain serum, effectively avoiding contamination and allergens, and meets the conditions for direct injection use.

Description

Technical Field

[0001] This invention relates to the field of cell technology, and in particular to a cell cryopreservation agent, its preparation method, application, and cell cryopreservation method. Background Technology

[0002] Currently, approximately 50 reagents are used as cryoprotectants. Based on their ability to penetrate cells, cryoprotectants are classified into permeable and non-permeable cryoprotectants. Permeable cryoprotectants include dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol formamide, whose main function is to enter cells, bind with water molecules, and reduce ice crystal formation. Non-permeable cryoprotectants include trehalose, sucrose, hydroxyethyl starch, low molecular weight dextran, and albumin, whose main function is to protect cells by mitigating cell dehydration caused by changes in osmotic pressure.

[0003] Currently, commonly used cell cryopreservation agents are typically composed of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). DMSO is a chemical substance with strong dissolving and permeating abilities. In many studies, DMSO is the most commonly used cell cryopreservation agent. Cells preserved with DMSO, after thawing, exhibit similar phenotypes, cell surface markers, and growth rates to fresh cells. However, DMSO has certain toxic effects on cells, and excessively high concentrations can cause high osmotic pressure, which is detrimental to cell thawing. Therefore, the conventional concentration of DMSO used is currently 5%-10%. FBS is an animal-derived substance with a complex composition and poses a risk of introducing contaminants and allergens, making it unsuitable for clinical application. Especially in cell therapy, the presence of animal-derived substances may lead to unknown adverse reactions.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a cell cryopreservation agent to at least solve one of the technical problems existing in the prior art.

[0006] The second objective of this invention is to provide a method for preparing the above-mentioned cell cryopreservation agent.

[0007] The third objective of this invention is to provide the application of the above-mentioned cell cryopreservation agent.

[0008] The fourth objective of this invention is to provide a cell cryopreservation method using the above-mentioned cell cryopreservation agent.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0010] The present invention provides a cell cryopreservation agent comprising, per 100 mL: 1-10 mL of dimethyl sulfoxide, 10-20 mL of dextran, 10-20 mL of human serum albumin, and the remainder being matrix buffer.

[0011] Furthermore, each 100 mL contains: 3–7 mL of dimethyl sulfoxide, 15–18 mL of dextran, 11–15 mL of human serum albumin, and the remainder is matrix buffer.

[0012] Furthermore, each 100 mL contains: 5 mL of dimethyl sulfoxide, 16.67 mL of dextran, 12.5 mL of human serum albumin, and 65.83 mL of matrix buffer.

[0013] Furthermore, the dextran is selected from dextran 40 and / or dextran 60.

[0014] Furthermore, the matrix buffer includes a compound electrolyte injection solution.

[0015] The present invention also provides a method for preparing the above-mentioned cell cryopreservation agent, wherein dimethyl sulfoxide is added to a matrix buffer solution of a specified amount, and then dextran and human serum albumin are added and mixed evenly to obtain the cell cryopreservation agent.

[0016] Furthermore, after the solution has returned to room temperature, dextran and human serum albumin are added.

[0017] The present invention also provides the application of the above-mentioned cell cryopreservation agent in cryopreservation of uterine blood stem cells.

[0018] Furthermore, this invention also provides a method for cryopreserving cells, adjusting the cell density to 7.0–8.0 × 10⁻⁶. 5 cells / ml, per 12.0–13.0 × 10 6 Cells were frozen using 15–18 mL of the cell cryopreservation agent described above.

[0019] Furthermore, the cells include uterine blood stem cells.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] Through extensive experimental screening, the inventors of this invention have provided a cell cryopreservation agent with specific components and dosage. This cell cryopreservation agent can effectively preserve cell viability, while its composition is clearly defined. All components used are injection-grade, ensuring high clinical safety. Furthermore, it does not contain serum, effectively avoiding contamination and allergens, and meets the conditions for direct injection use. Detailed Implementation

[0022] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0023] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The inventors of this invention conducted extensive experiments by researching the cryopreservation formulations of FDA-approved marketed cell-based drugs and finally identified a cell cryopreservation agent comprising, per 100 mL: 1-10 mL of dimethyl sulfoxide, 10-20 mL of dextran, 10-20 mL of human serum albumin, and the remainder being matrix buffer.

[0026] Dimethyl sulfoxide (DMSO) is a polar organic solvent that can dissolve many water-soluble and fat-soluble organic substances and has always been known as a "universal solvent". As an essential cryopreservation agent, each 100 mL of cell cryopreservation agent in this invention contains 1 to 10 mL of dimethyl sulfoxide, for example, but not limited to 1 mL, 2 mL, 5 mL, 8 mL or 10 mL.

[0027] Dextran, including dextran 40 and dextran 60, serves as a non-permeable extracellular cryoprotectant. During freezing, it reduces intracellular ice crystal formation, and during thawing, it alleviates cell swelling caused by changes in osmotic pressure. The combined use of dextran 40 and DMSO exhibits synergistic or additive cryoprotective effects. Furthermore, dextran 40 infusion is clinically used to expand blood volume, and no side effects have been observed in humans. In the cryopreservation and thawing of peripheral blood stem cells, comparisons of data before and after cryopreservation show that the combined cryoprotectant of dextran 40 and DMSO effectively protects stem cells under deep hypothermia in the short term. In this invention, each 100 mL of cell cryopreservation medium contains 10–20 mL of dextran, for example, but not limited to, 10 mL, 12 mL, 15 mL, 18 mL, or 20 mL.

[0028] Human serum albumin is produced by extracting plasma from healthy individuals immunized with hepatitis B vaccine using a low-temperature ethanol protein separation method and then inactivating the virus. It protects cells from environmental stresses and prevents cell adhesion to the cryopreservation tube walls during storage, thus providing cell protection. In this invention, each 100 mL of cell cryopreservation medium contains 10–20 mL of human serum albumin, for example, but not limited to, 10 mL, 12 mL, 15 mL, 18 mL, or 20 mL.

[0029] The matrix buffer can stabilize the pH of the cell cryopreservation agent, and Peptide A is preferred as the matrix buffer of this invention. Peptide A, as an isotonic compound electrolyte, has the advantages of being calcium ion-free, having a pH of 7.4, good acid resistance, a composition similar to extracellular fluid, and being lactate-free. It is a highly physiological crystalloid extracellular fluid replenishment solution and is an ideal replenishment solution for fluid and electrolyte loss caused by surgical procedures such as burns, trauma, and diseases.

[0030] By selecting the specific components and dosages mentioned above, this cell cryopreservation agent can effectively preserve cell viability. At the same time, the composition is clearly defined, all components used are injection grade, and it has high clinical safety. Furthermore, it does not contain serum, which can effectively avoid contamination and allergens, meeting the conditions for direct injection use.

[0031] It should be noted that all raw materials used in this invention are of medical pharmacopoeia injection grade. Examples include compound electrolyte injection (Bopuli A) and dextran 40 glucose injection.

[0032] In some preferred embodiments, each 100 mL contains: 3-7 mL of dimethyl sulfoxide, 15-18 mL of dextran, 11-15 mL of human serum albumin, and the remainder being matrix buffer.

[0033] In some preferred embodiments, each 100 mL contains: 5 mL of dimethyl sulfoxide, 16.67 mL of dextran, 12.5 mL of human serum albumin, and 65.83 mL of matrix buffer.

[0034] By further adjusting and optimizing the dosage of the above components, the resulting cell cryopreservation agent has a better cryopreservation effect on cells and has less impact on cell state after thawing.

[0035] Secondly, the present invention provides a method for preparing the above-mentioned cell cryopreservation agent, comprising adding dimethyl sulfoxide to a matrix buffer solution of a specified amount, then adding dextran and human serum albumin, and mixing evenly to obtain the cell cryopreservation agent.

[0036] This method is simple, easy to prepare, and requires no specific equipment or personnel, making it suitable for widespread application. During the preparation process, dimethyl sulfoxide (DMSO) generates heat; adding DMSO first allows this heat to dissipate before adding other components, which helps protect the activity of human serum albumin.

[0037] In some preferred embodiments, dextran and human serum albumin are added after the solution has returned to room temperature.

[0038] In some specific implementations, the main preparation process includes:

[0039] Calculation of the required volume V for each component: V = volume of cell cryopreservation agent * r (volume percentage of each component in the cell cryopreservation agent), V is rounded to one decimal place. The density of matrix buffer and dextran is considered to be 1 g / ml, and the volume is obtained by weighing during the preparation process. The corresponding volumes of DMSO and human serum albumin are obtained by measuring with a pipette.

[0040] 1. Add matrix buffer

[0041] (1) Wipe and disinfect the matrix buffer solution and transfer it to the clean workbench. Start the electronic balance and prepare a storage bottle after tareing.

[0042] (2) Pour the matrix buffer into a 225ml centrifuge tube, and then gradually add it to the storage bottle, and so on until it is close to the required volume (i.e. weight); then use a pipette to draw up to the final volume.

[0043] 2. Add DMSO

[0044] After wiping and disinfecting the DMSO, transfer it to the clean workbench. Use hemostatic forceps to remove the cap, wipe and disinfect the rubber stopper, and remove it. Remove the storage bottle from the electronic balance, and use a pipette to gradually add DMSO slowly into the storage bottle to prevent the DMSO from directly contacting the bottle wall. After adding the DMSO, blow it 10 times to mix it. Place the storage bottle on the electronic balance and record the weight of the liquid at this time.

[0045] 3. Add dextran

[0046] (1) Wipe and disinfect with dextran and transfer to a clean workbench. Take a disposable blood transfusion set (with needle) and cut the stopper with scissors. Calculate the final weight of dextran added, m1 = V dextran * 1g / ml.

[0047] (2) Remove the pull ring at the bag opening, insert the puncture device into the rubber stopper, squeeze the bag to take out some solution into the 50ml centrifuge tube, squeeze the bottle again to let the solution flow into the storage bottle until it is close to the required concentration, and then use a pipette to add to the final volume (i.e. weight), and mix by blowing and blowing 10 times.

[0048] 4. Add human serum albumin

[0049] Human serum albumin is wiped and disinfected and transferred to a clean workbench. The cap is removed with hemostatic forceps, the rubber stopper is wiped and disinfected and then removed. The storage bottle is removed from the electronic balance. Human serum albumin is gradually added to the storage bottle using a pipette. After the addition is complete, the bottle is blown 10 times to mix.

[0050] 5. Solution filtration

[0051] (1) Preparation of filtration device: Prepare an iron stand, take a disposable sterile syringe, place the syringe on the clamp, take a 40μm cell filter and place it at the end of the syringe, and place a storage bottle at the bottom to collect the filtrate.

[0052] (2) Filtration: Take out the mixed solution step by step from the storage bottle, filter it through the filtration device, and collect all the filtrate as cell cryopreservation agent.

[0053] Thirdly, based on the beneficial effects of the cell cryopreservation agent provided by the present invention, the present invention also provides the application of the cell cryopreservation agent in cryopreserving uterine blood stem cells.

[0054] Furthermore, a fourth aspect of the present invention provides a method for cryopreserving cells, comprising adjusting the cell density to 7.0–8.0 × 10⁻⁶. 5 cells / ml, per 12.0–13.0 × 10 6 Cells were cryopreserved using 15–18 mL of cell cryopreservation medium.

[0055] By matching the cell cryopreservation agent provided by this invention with specific cell density and cell number, the cryopreservation effect can be optimized.

[0056] The cryopreservation effect is better when the frozen cells are uterine blood stem cells.

[0057] In some specific implementations, cell cryopreservation:

[0058] Take a 1000ml storage bottle, weigh and tare the cells; use a 5ml pipette to draw 10ml of cell cryopreservation medium in two separate applications, pipetting 20 times to resuspend the cells evenly. Combine the cell suspensions in the storage bottle. Wash each centrifuge tube once with approximately 20ml of cell cryopreservation medium, combining the washings in the storage bottle; gradually prepare to 800g. Place a 20-100μm cell filter on a syringe for filtration. Take a sample for cell counting. Adjust the cell count to 7.72×10⁻⁶. 5 cells / ml, each bag contains 16.2ml of cells, with a cell count of 12.5 × 10⁻⁶. 6 Cells are filled into containers. Finally, they are pre-frozen using a programmed temperature drop device and then transferred to a liquid nitrogen tank for storage.

[0059] The present invention will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0060] Example 1: Screening of Formulas for Uterine Blood Stem Cell Injection

[0061] Experimental procedure:

[0062] 1. Cell treatment

[0063] Select uterine blood stem cells with cell number 03503-2-5-P5, and obtain approximately 7.0 × 10⁶ cells. 8 The culture supernatant of hematopoietic stem cells was centrifuged at 300g for 6 minutes, with an upward and downward speed of 9 and 7 respectively (centrifugation speed settings: upward speed 9, downward speed 7). The cells were discarded, and the supernatant was resuspended in PBS. The cells were then centrifuged again at 300g for 6 minutes, with an upward and downward speed of 9 and 7 respectively. The supernatant was discarded, and the cells were resuspended in PBS to a final volume of 280ml. After mixing, a sample was taken for counting. The result was 4.2 × 10⁻⁶ cells. 8 The uterine blood stem cells were evenly distributed into 14 50ml centrifuge tubes, and the remaining cells were discarded. The experimental group information was labeled, with each group containing approximately 3.0 × 10⁶ uterine blood stem cells. 7 Cells were washed with an appropriate amount of PBS, diluted to 48 ml / tube, and centrifuged at 300 g for 6 min, ↑9, ↓7. The supernatant was discarded, and the cell pellets in each tube were kept for later use.

[0064] 2. Preparation of cryoprotectant

[0065] The dosage of excipients for each group in the formulation for preparing 100ml of cryopreservation preparation is shown in the table below:

[0066]

[0067] The configuration method is as follows:

[0068] Calculation of the required volume V for each component: V = volume of cell cryopreservation agent V * r, V is rounded to one decimal place. Among them, the compound electrolyte injection and dextran 40 glucose injection are aqueous solutions with a density of 1 g / ml. The volume obtained by weighing during the preparation process is the volume. The corresponding volumes of dimethyl sulfoxide and human serum albumin are obtained by measuring with a pipette.

[0069] S 1. Add compound electrolyte injection (Bopuli A)

[0070] (1) Wipe and disinfect the compound electrolyte solution and transfer it to the clean workbench. Start the electronic balance and prepare a storage bottle for later use.

[0071] (2) Remove the secondary packaging bag, cut it open from the outlet with scissors, pour the compound electrolyte into a 225ml centrifuge tube, and then gradually add it to the storage bottle, adding it in this way until it is close to the required volume (i.e. weight); then use a pipette to draw up the final volume.

[0072] S2. Add Dimethyl Sulfoxide

[0073] After wiping and disinfecting the Dimethyl Sulfoxide, transfer it to the clean workbench. Use hemostatic forceps to remove the cap, wipe and disinfect the rubber stopper, and then remove it. Remove the storage bottle from the electronic balance. Use a pipette to gradually add Dimethyl Sulfoxide to the storage bottle, preventing Dimethyl Sulfoxide from directly contacting the bottle wall. After adding, blow and mix 10 times. Place the storage bottle on the electronic balance and record the weight of the liquid at this time.

[0074] S3. Add dextran 40 glucose injection

[0075] (1) Wipe and disinfect the dextran 40 glucose injection and transfer it to a clean workbench. Take a disposable blood transfusion set (with needle) and cut the stopper with scissors. Calculate the final weight of the added dextran 40 glucose injection m1 = V dextran * 1g / ml.

[0076] (2) Remove the pull ring at the bag opening, insert the puncture device into the rubber stopper, squeeze the bag to take out some solution into the 50ml centrifuge tube, squeeze the bottle again to let the solution flow into the storage bottle until it is close to the required concentration, and then use a pipette to add to the final volume (i.e. weight), and mix by blowing and blowing 10 times.

[0077] S4. Add human serum albumin

[0078] Human serum albumin is wiped and disinfected and transferred to a clean workbench. The cap is removed with hemostatic forceps, the rubber stopper is wiped and disinfected and then removed. The storage bottle is removed from the electronic balance. Human serum albumin is gradually added to the storage bottle using a pipette. After the addition is complete, the bottle is blown 10 times to mix.

[0079] S5. Solution filtration

[0080] (1) Preparation of filtration device: Prepare an iron stand, take a disposable sterile syringe, place the syringe on the clamp, take a 40μm cell filter and place it at the end of the syringe, and place a storage bottle at the bottom to collect the filtrate.

[0081] (2) Filtration: Take out the mixed solution step by step from the storage bottle, filter it through the filtration device, and collect all the filtrate as cell cryopreservation agent.

[0082] 3. Cell preparation filling and programmed cooling

[0083] Add the corresponding excipients to each tube according to the information, mix well and fill the tubes. Store the filled cells at 2-8℃. After filling, take one bag of cells from each experimental group to test cell number, viability, osmotic pressure, pH value and other parameters. All other cells are subjected to controlled cooling. After cooling, they are transferred to liquid nitrogen for storage.

[0084] 4. Cell thawing

[0085] After 7 days of cryopreservation in liquid nitrogen, each experimental group took one bag, thawed it in a 37°C water bath, and then measured the cell count, viability, osmotic pressure, and pH value.

[0086] 5. Relevant testing

[0087] 5.1 Assay for cell viability and cell number using the trypan blue exclusion assay

[0088] The trypan blue rejection assay was used to determine cell viability and cell concentration before cryopreservation and after thawing 7 days after cryopreservation.

[0089] 5.2 pH value and osmotic pressure detection

[0090] The pH value of the uterine blood stem cell injection solution was measured using a pH meter before cryopreservation and after thawing 7 days after cryopreservation. The osmotic pressure of the uterine blood stem cell injection solution was measured using an osmometer before cryopreservation and after thawing 7 days after cryopreservation.

[0091] 6. Results

[0092] 6.1 Analysis of Cell Viability, Quantity, and Agglomeration Rate Before and After Cryopreservation

[0093] After mixing the cells from each group, take an appropriate volume of cell suspension into a 1.5 ml centrifuge tube. Using a pipette, pipette 20 μl of trypan blue and 20 μl of cell suspension into another 1.5 ml centrifuge tube, mix well, and then add 20 μl of the mixture to the Countstar cell counting chamber. Insert the counting chamber into the counter, set the counting parameters in the Countstar counter software, and then perform the instrumentation to detect cell concentration and viability. The detection results are shown in the table below:

[0094] Cell viability, number, and clumping rate before and after cryopreservation

[0095]

[0096] Results of cell count, viability, and clumping rate after 7 days of cryopreservation

[0097] Sample number <![CDATA[Cell count (×10 7 cells)]]> Cell viability (%) Clumping rate (%) 1 1.22 91.15 12.04 2 1.42 96.37 14.53 3 1.51 95.61 13.65 4 1.03 90.23 13.36 5 1.37 95.8 12.05 6 1.43 92.26 11.98 7 1.16 92 17.61 8 1.30 92.84 9.78 9 1.28 91.26 14.74 10 1.18 91.36 12.59 11 1.31 98.02 10.89 12 1.31 96.34 13.88 13 1.30 96.97 15.32 14 1.02 90.37 15.08

[0098] Analysis of the cell count results before and after cryopreservation using trypan blue staining showed that the viability of each group of cells was above 90% before cryopreservation, and the viability was also above 90% after 7 days of cryopreservation.

[0099] 6.2 Analysis of osmotic pressure and pH values ​​before and after cryopreservation

[0100] 6.2.1 Analysis of osmotic pressure results before and after cryopreservation

[0101] Osmotic pressure testing method: Use a standard solution to calibrate the osmometer. Take 60 μl of each group of test samples with a sampler and inject it into the test tube (ensure there are no visible air bubbles). Push the test tube into the support to the stop position so that the temperature probe is completely immersed in the test sample in the test tube. After the test is completed, press "Save" to save the current test results; press "Print" to enter the printing interface where you can set the sample name and sample number.

[0102] Osmotic pressure before and after cryopreservation

[0103] Sample number Before cryopreservation (mOsmol) After 7 days of cryopreservation (mOsmol) 1 1139 1141 2 1168 1177 3 1168 1168 4 1660 1646 5 1687 1689 6 1687 1690 7 2251 2275 8 2245 2277 9 2305 2304 10 758 771 11 1158 1167 12 1161 1166 13 1162 1171 14 1168 1173

[0104] The osmotic pressure test results showed that the osmotic pressure of each experimental group increased with the increase of DMSO content in the cell cryopreservation agent formulation.

[0105] 6.2.2 Analysis of pH values ​​before and after freezing

[0106] pH measurement method: Take 2 ml of the cell suspension to be tested from each group into a 15 ml centrifuge tube. Calibrate the pH meter using PBS. Place the electrode in the sample solution to be tested, ensuring the solution fully submerges the electrode, and press the read button to start the measurement. The decimal point on the screen will flash. Automatic measurement endpoint A is the instrument's default setting. Once the electrode output stabilizes, the display automatically locks and shows the pH value of the sample solution. The results are shown in the table below.

[0107] pH values ​​before and after freezing

[0108] Sample number Before cryopreservation (mOsmol) After 7 days of cryopreservation (mOsmol) 1 6.98 6.88 2 6.87 6.83 3 6.91 6.82 4 7.01 6.92 5 6.89 6.85 6 6.93 6.82 7 6.9 6.94 8 6.92 6.87 9 6.95 6.86 10 6.83 6.79 1 1 6.92 6.82 12 6.77 6.73 13 6.86 6.72 14 6.91 6.84

[0109] The pH values ​​of the cell preparations in each experimental group before and after freezing were between 6.72 and 7.01. The normal blood pH value is between 7.3 and 7.4. If the blood pH value is less than 7.0 or greater than 7.6, it is not conducive to the normal operation of physiological functions. Therefore, the pH value needs to be adjusted to meet the requirements of injectable products in clinical applications.

[0110] 7. Conclusion

[0111] Based on the results of trypan blue staining cell viability tests, pH values, and osmotic pressure tests before and after cryopreservation, the formulations of Group 3 and Group 5, which have relatively low osmotic pressure, relatively high cell viability, relatively small decrease in cell viability after 7 days of cryopreservation, and relatively low cost, are recommended as preferred cell cryopreservation formulations for uterine blood stem cell cryopreservation. Considering the potential toxicity and adverse reactions of high-concentration DMSO in clinical use, the formulation of Group 3 is preferred for uterine blood stem cell cryopreservation. Its formula is 5% DMSO + 12.5% ​​human serum albumin infusion solution + 16.67% dextran 40 glucose injection + 65.83% pulex A.

[0112] Experimental Example 2: Study on the Activity Trend of Formulation after Production and Freezing

[0113] 1. Experimental Design

[0114] When using the third-group formulation for cryopreservation of uterine blood stem cells, and during batch production according to the relevant procedures for uterine blood mesenchymal stem cell injection (SC01009), samples were taken at various time points during drug preparation, filling, and after thawing for cell counting and apoptosis detection. Specific sampling points and requirements are shown in the table below.

[0115]

[0116] 2. Experimental Procedure

[0117] 2.1 Cell Preparation

[0118] Production was carried out in accordance with the above experimental design requirements, and samples were taken for testing.

[0119] 2.2 Cell Count

[0120] Trypan blue rejection assay for cell viability and cell concentration.

[0121] 2.3 Apoptosis detection

[0122] 2.3.1 Sample processing for apoptosis detection

[0123] Dilute 10×Annexin V Binding Buffer with ultrapure water to obtain 1×Annexin V Binding Buffer.

[0124] The collected sample cell suspension (cell quantity: 1×10⁶) 6 Centrifuge at 300g for 5 min at 4℃, discarding the supernatant after centrifugation. Wash cells once with 1 mL of pre-chilled 1×Annexin V Binding Buffer. Centrifuge at 300g for 5 min at 4℃, discarding the supernatant after centrifugation. Add 1 mL of pre-chilled 1×Annexin V Binding Buffer to each tube and gently pipette to resuspend the cells. Label four EP tubes for each sample: one blank control tube, one Annexin V single-staining tube, one PI single-staining tube, and one sample double-staining tube, adding 150 μl of cell suspension to each tube. Do not add dye to the blank control tube. Add 2.5 μl of Annexin V to the Annexin V single-staining tube and the sample double-staining tube. Mix gently and incubate at 4℃ in the dark for 10 min. Add another 1.5 μl of PI to the PI single-staining tube and the sample double-staining tube, and mix gently. Add 200 μl of 1×Annexin V Binding Buffer to each EP tube, mix well, and analyze within 1 hour.

[0125] 2.3.2 Apoptosis detection

[0126] Before loading samples, check the liquid levels in all flow flasks to ensure the instrument can operate normally. Turn on the flow cytometer and wait for the indicator lights to stop flashing before turning on the computer and software. Run ultrapure water for 15 minutes at Fast speed before loading samples. After the ultrapure water run is complete, click Delete Events to remove the background. Gently mix the sample and place it at the loading needle. Set the sampling conditions (select low or medium speed; high speed is generally not used), select the sample location, name the sample, and click "run". Click on the x-axis and set it to FSC-H and SSC-H respectively. Use the icon to find the optimal field of view for the cell population. Based on the cell population location, click the threshold; it is recommended to set the FSC-H threshold to 1,000,000. Use the tool to draw a gate to define the target cell population, set the collection limit to 10,000 cells within the gate, and continue sampling according to the set conditions. Load subsequent samples sequentially. After loading, click "Dot Plot" to draw a two-color scatter plot, setting the axis parameters as FITC on the x-axis and PE on the y-axis. Click "Gate" on the graph, select the corresponding gate, choose the "Include" relationship, and click "Apply". The normal cell group can be used as a threshold to remove spectral overlap and set the position of the cross gate. Click "Set Color Compensation", select the fluorescence parameters to be compensated, enter the compensation values ​​to adjust the fluorescence compensation of the FITC and PI channels, and click "Save & Close". After adjusting the compensation according to the Annexin V and PI single-stain tubes, apply it to the sample tube cells to observe the final apoptosis of the cells being tested.

[0127] 2.3.3 Data Analysis

[0128] On a bivariate flow cytometry scatter plot, the upper left quadrant represents mechanically damaged cells (Annexin V- / PI+). The lower left quadrant represents normal viable cells (Annexin V- / PI-). Normal cells have intact cell membranes and no everted PS (presumably a cytokine molecule), resulting in double-negative results. The upper right quadrant represents late-stage apoptotic cells (Annexin V+ / PI+). Late-stage apoptotic cells not only have everted PS but also have incomplete cell membranes, resulting in double-positive results. The lower right quadrant represents early-stage apoptotic cells (Annexin V+ / PI-). In the early stages of apoptosis, PS can flip from the inside of the cell membrane to the surface, exposing them to the extracellular environment. They can then bind to Annexin V labeled with FITC, resulting in a positive result; while an intact cell membrane prevents PI from entering the cell, resulting in a negative result.

[0129] The cell count results can reveal the changes in cell viability throughout the entire production process of the uterine blood stem cell preparation and whether the final preparation meets the quality standards for uterine blood mesenchymal stem cell injection (cell viability ≥ 80%).

[0130] The apoptosis results show the changing trend of cell apoptosis throughout the entire production process of uterine blood stem cell preparations.

[0131] 3. Experimental Results

[0132] 3.1 Analysis of Cell Count Results

[0133] During the filling process of uterine blood mesenchymal stem cell preparation, the cell viability first decreased and then increased, and remained generally stable with a slight decrease (around 4%-6%). It can be considered that time has no significant effect on cell viability throughout the process.

[0134] Cell counting data results during formulation filling

[0135]

[0136] 3.2 Analysis of apoptosis results

[0137] During the production of mesenchymal stem cell preparations from uterine blood, the percentage of normal viable cells decreased slightly, while the percentage of apoptotic cells increased slightly. Overall, cryopreservation and thawing had an impact on cell viability, but it was not significant.

[0138]

[0139] 4. Conclusion

[0140] Cell viability measurements obtained by automated cell counting and flow cytometry apoptosis detection differ due to their different experimental principles. Automated cell counting uses trypan blue staining to count cells, excluding those that have lost their intact cell membranes (including dead cells and some late-apoptotic cells). Flow cytometry, on the other hand, excludes cells that have undergone early apoptosis, late apoptosis, and dead cells. Therefore, automated cell counting tends to yield higher values, while flow cytometry apoptosis results in a lower proportion of normal viable cells, with the greatest difference observed after cell thawing. Flow cytometry apoptosis detection more accurately reflects the cell state at the time of thawing. From this perspective, thawing after cryopreservation may affect cell state to some extent, but the effect is not significant.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for cryopreserving uterine blood stem cells, characterized in that, Adjust the cell density to 7.0~8.0×10⁻⁶. 5 Cells / ml, per 12.0~13.0×10 6 Cells were cryopreserved using 15-18 mL of cell cryopreservation medium; The cell cryopreservation agent described herein comprises, per 100 mL: 1-10 mL of dimethyl sulfoxide, 10-20 mL of dextran, 10-20 mL of human serum albumin, and the remainder being matrix buffer; in: The matrix buffer is Pulse A; The preparation method of the cryopreservation agent is as follows: add dimethyl sulfoxide to the matrix buffer solution of the formula amount, and then add dextran and human serum albumin. After mixing evenly, the cell cryopreservation agent is obtained.

2. The cryopreservation method according to claim 1, characterized in that, Each 100 mL contains: 3-7 mL of dimethyl sulfoxide, 15-18 mL of dextran, 11-15 mL of human serum albumin, and the remainder of matrix buffer.

3. The cryopreservation method according to claim 2, characterized in that, Each 100 mL contains: 5 mL of dimethyl sulfoxide, 16.67 mL of dextran, 12.5 mL of human serum albumin, and 65.83 mL of matrix buffer.

4. The cryopreservation method according to any one of claims 1-3, characterized in that, The dextran is selected from dextran 40 and / or dextran 60.

5. The cryopreservation method according to claim 1, characterized in that, In the preparation method of the cryopreservative, dextran and human serum albumin are added after the solution has been brought back to room temperature.

6. The cryopreservation method according to claim 1, characterized in that, The cell density was adjusted to 7.72 × 10⁻⁶. 5 Cells / ml, per 12.5 × 10 6 The cells were cryopreserved using 16.2 mL of cell cryopreservation medium.

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