A method for freezing and recovering human umbilical cord mesenchymal primary cells

Abstract

This invention relates to the field of regenerative medicine engineering technology, specifically to a method for the cryopreservation and thawing of primary human umbilical cord mesenchymal cells, comprising the following steps: 1. Culture of hUCMSCs-P; 2. Cell digestion and density control; 3. Cryopreservation pretreatment; 4. Low-temperature cryopreservation; 5. Rapid thawing; 6. Post-thawing protection. This invention offers advantages such as high viability and stability, simple and standardized operation, low damage and high practicality, and resource conservation.

Description

Technical Field

[0001] This invention relates to the field of regenerative medicine engineering technology, specifically to a method for cryopreservation and thawing of primary human umbilical cord mesenchymal cells. Background Technology

[0002] Human umbilical cord mesenchymal primary cells (hUCMSCs-P) refer to pluripotent stem cells that have not undergone multiple passages and are first isolated and cultured from neonatal umbilical cord tissue. They retain stronger differentiation potential and proliferative activity and have core application value in the fields of cartilage repair and tissue regeneration.

[0003] hUCMSCs-P, due to their high differentiation potential preserved from primary cells, are core seed cells for cartilage tissue engineering and cell therapy. Their long-term preservation quality directly determines the effectiveness of subsequent experimental and clinical applications. Cryopreservation (short-term at -80℃ or long-term in liquid nitrogen) is the mainstream method for maintaining cell viability. However, the low-temperature environment causes intracellular water to form ice crystals, damaging cell membranes and organelles (such as mitochondria and endoplasmic reticulum). Furthermore, the cytotoxicity of DMSO, centrifugal mechanical force, and resuscitation temperature differences further reduce cell viability. According to literature reports, the viability of hUCMSCs-P resuscitated using current conventional methods is generally only 75%-85%, and the 24-hour adhesion rate is <70%, which is insufficient to meet the high demands of experimental and clinical applications (such as requiring ≥90% viability for composite scaffold cell seeding).

[0004] Existing technical solutions and their shortcomings:

[0005] (1) Conventional cryopreservation and thawing methods (from "Experimental Guide to Cell Biology", Science Press, 2020).

[0006] Procedure: Take hUCMSCs-P cells with a confluence of 70%-80%, digest with 0.25% trypsin, centrifuge at 1000 rpm for 5 min, and collect the cell pellet; resuspend the cells in 2 ml of cryopreservation buffer (DMEM:FBS:DMSO=6:3:1) (density approximately 5×10⁶ cells / mL). 5 After transferring the cells (number per ml) into cryovials, place them at 4℃ for 30 min, then at -20℃ for 24 h, and finally at -80℃ for storage. During thawing, place the cryovials in a 37℃ water bath to thaw, centrifuge, and resuspend them in basal culture medium for inoculation.

[0007] Defects: ① Cell density not optimized, 5×10 5 ① The cell density was lower than the peak activity density; ② The volume of cryopreservation solution (2 ml) did not match the cell density, and the toxicity of DMSO was not diluted; ③ The cell density was too low; ④ The cell density was too low; ⑤ The cell density was too low; ⑥ The cell density was too low; ⑦ The cell density was too low; ⑧ The cell density was too low; ⁴ ...

[0008] (2) Existing patent technology (Chinese invention patent with application number CN202210234567.8 entitled A mesenchymal stem cell cryopreservation solution and cryopreservation method).

[0009] Technical solution: Add 10% hydroxyethyl starch (HES) to the cryopreservation solution to increase osmotic pressure, achieving a cell density of 1×10⁻⁶. 6 The sample size was 100 cells / ml, centrifuged at 3500 rpm for 4 min, placed at -20℃ for 12 h, then transferred to -80℃, and thawed in a 40℃ water bath during recovery.

[0010] Defects: ① Excessive centrifugation speed (3500 r / min) causes mechanical damage to cells, resulting in a 5%-8% decrease in cell viability; ② Ice crystals still accumulate even after being placed at -20℃ for 12 hours; ③ A resuscitation water temperature of 40℃ can easily lead to local overheating and damage to cell proteins.

[0011] To address the core shortcomings of existing hUCMSCs-P cryopreservation and thawing technologies, such as low cell viability after thawing (generally <80%), unclear operating parameters, and poor batch reproducibility, the following technical issues will be specifically resolved:

[0012] 1. The problem of "insufficient cell protection" or "resource waste" caused by the mismatch between the amount of existing cryopreservation solution and cell density—such as 10 6 When 3 ml of cryopreservation solution was used per cell, the DMSO concentration was diluted, exacerbating ice crystal damage; when 0.5 ml of cryopreservation solution was used, cell aggregation led to a decrease in post-resuscitation survival rate.

[0013] 2. The problem of "missed peak activity" caused by the mismatch between cell growth cycle and cryopreservation time - existing methods often randomly select the cryopreservation time (such as when the cell confluence is 80%), without clarifying the differences in cell activity at the 24h, 48h, and 72h growth nodes, resulting in cells entering the senescent stage when cryopreserved;

[0014] 3. The problem of "cell mechanical damage" caused by unreasonable centrifugation parameters (speed and time) - such as the cell rupture rate increases when the speed is >4000 r / min, and the cell precipitation is insufficient when the time is <2 min, and the residual supernatant dilutes the cryopreservation solution;

[0015] 4. The problem of "secondary damage" caused by improper low temperature transition (-20℃ placement time) and recovery conditions - such as the large temperature difference caused by direct placement at -80℃, which leads to a sudden increase in ice crystals; the failure to use a constant temperature water bath at 37℃ during recovery, resulting in slow thawing; or the use of dry ice for recovery, which causes cells to freeze due to excessively low temperature.

[0016] 5. The problem of "mortality rebound" caused by lack of targeted protective measures after resuscitation - the existing method of direct inoculation after resuscitation without the addition of anti-apoptotic or adhesion-promoting substances makes it easy for cells to have an apoptosis peak within 24-48 hours. Summary of the Invention

[0017] The purpose of this invention is to provide a method for cryopreservation and thawing of primary human umbilical cord mesenchymal cells.

[0018] A method for cryopreservation and thawing of primary human umbilical cord mesenchymal cells includes the following steps:

[0019] I. Culture of hUCMSCs-P;

[0020] II. Cell digestion and density control;

[0021] III. Pre-treatment for cryopreservation;

[0022] IV. Low-temperature freezing;

[0023] V. Rapid recovery;

[0024] VI. Post-resuscitation protection.

[0025] Furthermore, in step one, hUCMSCs-P were cultured in α-MEM medium containing 10% FBS and 1% penicillin-streptomycin antibiotics at 37°C and 5% CO2 for 72 hours.

[0026] Further, in step two, the hUCMSCs-P cells cultured for 72 hours are digested with 0.25% trypsin-EDTA, the cell suspension is collected, and the cell concentration is adjusted to 1×10⁻⁶. 6 per ml.

[0027] Furthermore, in step three, the cell suspension is centrifuged at 3000 r / min for 3 min, the supernatant is discarded after centrifugation, the cell pellet is resuspended in 1 ml of cryopreservation solution and then placed into cryopreservation tubes. The cryopreservation solution includes α-MEM:FBS:DMSO in a volume ratio of 7:2:1.

[0028] Furthermore, in step four, the cryopreservation tubes are immediately placed in an ultra-low temperature freezer at -80°C after being filled with liquid to preserve cell viability to the greatest extent.

[0029] Furthermore, in step five, after freezing for 7 days, the cryovial is removed and immediately placed in a 37°C constant temperature water bath and gently shaken for 1-2 minutes until the cryopreservation solution is completely thawed.

[0030] Furthermore, the feature is that, in step six, immediately after the cryopreservation tube is thawed, 5 ml of α-MEM medium containing insulin-like growth factor-1 and fibronectin is added, the mixture is gently pipetted and then centrifuged at 3000 r / min for 3 min. The supernatant is discarded to remove residual DMSO. Insulin-like growth factor-1 can inhibit the apoptosis pathway after cell recovery, and fibronectin can promote rapid cell adhesion. The synergistic effect of the two increases the cell adhesion rate to over 85% 24 h after recovery, and the proliferation rate at 72 h is 1.8-2.0 times higher than that at the time of inoculation.

[0031] In summary, the present invention has the following beneficial effects:

[0032] 1. High viability and high stability: Cell viability reaches 90%-94% after resuscitation, with batch-to-batch variation of <3%, solving the problems of low viability and poor reproducibility in existing methods;

[0033] 2. Simple and easy to standardize operation: The core parameters such as centrifugation at 3000 r / min and cryopreservation solution of 1 ml are clearly defined, requiring no complicated equipment, making it suitable for promotion in laboratories and clinical institutions;

[0034] 3. Low damage and high practicality: By optimizing centrifugation, cryopreservation temperature difference, and resuscitation protection, mechanical damage and ice crystal damage are reduced. At the same time, the addition of insulin-like growth factor-1 and fibronectin further reduces the apoptosis rate, meeting the high requirements for cell quality for cartilage repair, etc.

[0035] 4. Resource conservation: corresponding to 1×10 6 For each cell, only 1 ml of cryopreservation solution is needed, saving 50% of reagent costs compared to the conventional 2 ml method, and eliminating the need for 4℃ and -20℃ transition, thus shortening the operation time. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0037] A method for cryopreservation and thawing of primary human umbilical cord mesenchymal cells includes the following steps:

[0038] I. Culture of hUCMSCs-P: hUCMSCs-P were cultured in α-MEM medium containing 10% FBS and 1% penicillin-streptomycin antibiotics at 37℃ and 5% CO2 for 72 h.

[0039] In this embodiment, primary isolated hUCMSCs-P were seeded into 10cm culture dishes (density 2×10⁻⁶).4 Cells were cultured for 24h, 48h, and 72h respectively. Cell viability was detected at different time points using the CCK-8 assay. It was found that the cells were in the logarithmic growth phase at 72h, with a viability of 95%-98% (significantly higher than 88%-90% at 24h and 92%-94% at 48h). Therefore, cells cultured for 72h were selected for cryopreservation.

[0040] II. Cell Digestion and Density Control: hUCMSCs-P cells cultured for 72 h were digested with 0.25% trypsin-EDTA, and the cell suspension was collected and the cell concentration was adjusted to 1×10⁻⁶. 6 per ml.

[0041] In this embodiment, the old culture medium in the culture dish was discarded, and the cells were washed twice with PBS. 2 ml of 0.25% trypsin-EDTA was added, and the cells were incubated at 37°C for 2 min. After observing the cells becoming round and detaching under an inverted microscope, 4 ml of α-MEM culture medium containing 10% FBS was added to terminate the digestion.

[0042] Collect cell suspensions and adjust the concentration to 1×10⁻⁶. 5 cells / ml, 1×10 6 cells / ml, 1×10 7 Cells / ml, density confirmed by counting with a cell counting chamber. 1×10 6 The optimal density is 1×10⁶ / ml. If the density is too low (1×10⁶ / 5 When the density is (cells / ml), there is insufficient intercellular signaling, and proliferation is slow after recovery; when the density is too high (1×10⁻⁶), intercellular signaling is insufficient, and proliferation is slow after recovery; 7 When cells aggregate (number / ml), they are prone to hypoxia during cryopreservation.

[0043] III. Pretreatment for cryopreservation: The cell suspension was centrifuged at 3000 r / min for 3 min. After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in 1 ml of cryopreservation solution and then placed into cryopreservation tubes. The cryopreservation solution consisted of α-MEM:FBS:DMSO in a volume ratio of 7:2:1.

[0044] In this embodiment, it will be adjusted to 1×10 6 Cell suspensions with a density of cells / ml were transferred into centrifuge tubes, and centrifuged at 2000 rpm for 2 min, 3000 rpm for 3 min, and 4000 rpm for 4 min. After centrifugation, the supernatant was discarded, and the cell pellet was observed. It was found that at 3000 rpm for 3 min, the cell pellet was uniform and without clumping, the supernatant was clear and free of suspended cells, and the mechanical damage rate was <3% (significantly lower than the 8%-10% at 4000 rpm).

[0045] Corresponding to 1×10 6Cells were resuspended in cryopreservation buffer (1 ml, 2 ml, and 3 ml, respectively) prepared in a volume ratio of 7:2:1 (α-MEM:FBS:DMSO). Preferably, the cryopreservation buffer does not need to be pre-frozen, as pre-freezing can cause DMSO precipitation and increase cytotoxicity.

[0046] IV. Low-temperature cryopreservation: After filling the cryopreservation tubes with liquid, immediately place them in an ultra-low temperature freezer at -80℃ to preserve cell viability to the greatest extent.

[0047] In this embodiment, the cell suspensions after resuspending the precipitate using 1ml, 2ml, and 3ml of cryopreservation solution were respectively loaded into cryopreservation tubes, and the following low-temperature transition conditions were set for freezing: ① directly placed at -80℃; ② stored at -20℃ for 1 day and then transferred to -80℃; ③ stored at -20℃ for 2 days and then transferred to -80℃.

[0048] Cell viability was tested after 7 days of cryopreservation under different conditions: ① When 1 ml of cryopreservation solution was directly placed at -80℃, cell viability reached 92%-94%, and 1 ml of cryopreservation solution could maintain the DMSO concentration at 10%. Although the temperature difference was large when directly placed at -80℃, the cells froze rapidly and formed a "vitrified state", reducing the amount of ice crystal formation; while 2 ml and 3 ml of cryopreservation solution would dilute the DMSO, and placing at -20℃ would cause ice crystals to grow slowly, reducing the viability to below 85%. Therefore, it was determined that the cryopreservation solution volume was 1 ml, no pre-freezing was required, and the cells should be placed at -80℃ immediately.

[0049] 5. Rapid thawing: After 7 days of cryopreservation, remove the cryopreservation tube and immediately place it in a 37°C constant temperature water bath and gently shake for 1-2 minutes until the cryopreservation solution is completely thawed.

[0050] In this embodiment, after 7 days of cryopreservation, the cryovials are removed and the following three thawing conditions are selected: ① Placed in a 37°C constant temperature water bath and gently shaken for about 1-2 minutes until completely thawed; ② Placed in dry ice for about 5-8 minutes to thaw slowly; ③ Placed on ice for about 10-15 minutes to thaw.

[0051] Post-resuscitation protection: Immediately after thawing the cryovials, add 5 ml of α-MEM medium containing insulin-like growth factor-1 and fibronectin, gently pipette to mix, centrifuge at 3000 r / min for 3 min, discard the supernatant to remove residual DMSO. Insulin-like growth factor-1 can inhibit the apoptosis pathway after cell resuscitation, and fibronectin can promote rapid cell adhesion. The synergistic effect of the two can increase the cell adhesion rate to more than 85% 24 h after resuscitation, and the proliferation rate at 72 h is 1.8-2.0 times higher than that at the time of inoculation.

[0052] In this embodiment, immediately after the cryovials are thawed, 5 ml of α-MEM medium containing insulin-like growth factor-1 and fibronectin is added, and the mixture is gently blown to mix evenly, avoiding air bubbles. The mixture is then centrifuged at 3000 r / min for 3 min, the supernatant is discarded, and residual DMSO is removed.

[0053] Resuspend the cells in the culture medium containing the protective substance described above, and seed them at a density of 1×10⁶ cells / year. 5 Cells were placed in 6-well plates at 37°C and 5% CO2. The results confirmed that resuscitation in a constant temperature water bath at 37°C combined with α-MEM medium containing insulin-like growth factor-1 and fibronectin could restore cell viability to 90%-94% after resuscitation, and increase cell adhesion to over 85% after 24 hours.

[0054] Effect verification data

[0055] Following the optimal procedure described above, three batches of hUCMSCs-P were cryopreserved (directly placed at -80℃ for 7 days) and then thawed. The detection indicators are as follows:

[0056] Immediate vitality after recovery: 90.5%-93.2% (significantly higher than the 75%-85% of conventional methods);

[0057] Adhesion rate 24 hours after resuscitation: 85.3%-88.1%;

[0058] Proliferation rate 72 hours after resuscitation: 1.8-2.0 times higher than at the time of inoculation (compared to only 1.2-1.5 times higher using conventional methods);

[0059] Apoptosis rate: 3.5%-5.2% 24h after resuscitation (15%-20% using conventional methods).

[0060] In summary, this invention is not limited to the specific embodiments described above. Those skilled in the art can make various modifications and alterations without departing from the spirit and scope of this invention. The scope of protection of this invention should be determined by the claims of this invention.

Claims

1. A method for cryopreservation and thawing of primary human umbilical cord mesenchymal cells, characterized in that, Includes the following steps: I. Culture of hUCMSCs-P; II. Cell digestion and density control; III. Pre-treatment for cryopreservation; IV. Low-temperature freezing; V. Rapid recovery; VI. Post-resuscitation protection.

2. The method for cryopreservation and thawing of primary human umbilical cord mesenchymal cells as described in claim 1, characterized in that, In step one, hUCMSCs-P were cultured in α-MEM medium containing 10% FBS and 1% penicillin-streptomycin antibiotics at 37°C and 5% CO2 for 72 hours.

3. The method for cryopreservation and thawing of primary human umbilical cord mesenchymal cells as described in claim 2, characterized in that, In step two, hUCMSCs-P cells cultured for 72 hours are digested with 0.25% trypsin-EDTA, the cell suspension is collected, and the cell concentration is adjusted to 1×10⁻⁶. 6 per ml.

4. The method for cryopreservation and thawing of primary human umbilical cord mesenchymal cells as described in claim 3, characterized in that, In step three, the cell suspension is centrifuged at 3000 r / min for 3 min. After centrifugation, the supernatant is discarded, and the cell pellet is resuspended in 1 ml of cryopreservation solution and then placed into cryopreservation tubes. The cryopreservation solution consists of α-MEM:FBS:DMSO in a volume ratio of 7:2:

1.

5. The method for cryopreservation and thawing of primary human umbilical cord mesenchymal cells as described in claim 4, characterized in that, In step four, the cryopreservation tubes are immediately placed in an ultra-low temperature freezer at -80°C after being filled with liquid to preserve cell viability to the greatest extent.

6. A method as described in claim 5, characterized in that, In step five, after freezing for 7 days, the cryovial is removed and immediately placed in a 37°C constant temperature water bath and gently shaken for 1-2 minutes until the cryopreservation solution is completely thawed.

7. A method as described in claim 6, characterized in that, In step six, immediately after thawing the cryovials, add 5 ml of α-MEM medium containing insulin-like growth factor-1 and fibronectin, gently pipette to mix, centrifuge at 3000 r / min for 3 min, discard the supernatant to remove residual DMSO. Insulin-like growth factor-1 can inhibit the apoptosis pathway after cell recovery, and fibronectin can promote rapid cell adhesion. The synergistic effect of the two increases the cell adhesion rate to over 85% 24 h after recovery, and the proliferation rate at 72 h is 1.8-2.0 times higher than at the time of inoculation.