Hepatocyte prescription freezing medium and use method thereof

Abstract

The invention discloses a liver cell prescription cryopreservation solution, which is prepared from the following raw materials: human serum albumin injection, medicinal dimethyl sulfoxide and compound electrolyte injection, and the volume concentration of the medicinal dimethyl sulfoxide is 5-7.5%. According to the invention, the human serum albumin injection is used for replacing animal-derived protein, so that cell pollution can be avoided, and adverse effects on cell therapy are reduced. And by adopting 5vol% of DMSO, the formation of ice crystals can be controlled, the damage to the internal cell structure of liver cells is avoided, and the cytotoxicity can be reduced. The invention has the advantages of simple and clear raw materials, low cost, stable components, convenient use, and high activity of liver cells after cryopreservation recovery.

Description

Technical Field

[0001] The present invention relates to the field of preservation of human body, animal and plant body or parts thereof, and in particular to a liver cell prescription freezing solution and a use method thereof. Background Art

[0002] Liquid nitrogen cryopreservation is an important method for long-term storage of cells, and cryopreservation fluid is a buffer solution that protects cells during the cryopreservation process. Stem cells have excellent self-replication and differentiation potential. There are many types of cryopreservation fluids for stem cells in the existing technology, and the technology is relatively mature, but the cost of stem cells is high and difficult to obtain. Hepatocyte transplantation can also treat certain liver failure or metabolic diseases, so there is an increasing demand for hepatocyte cryopreservation. The differentiation activity of hepatocytes is not as good as that of stem cells, and the cell activity decreases after cryopreservation. Therefore, it is very important to develop a hepatocyte cryopreservation fluid with high cell survival rate and high cell biological activity after cryopreservation.

[0003] Chinese invention patent CN 117084231 A discloses an optimized formula, preparation and cryopreservation method of a cell cryopreservation solution. The preparation raw materials include pharmaceutical grade DMSO, 20% human albumin injection, and compound electrolyte injection. The cryopreservation solution has little damage to cells and toxic side effects, is highly safe, and can be directly used for clinical transfusion and local injection. However, it is used to cryopreserve umbilical cord mesenchymal stem cells, and the cryopreservation effect on liver cells is not good. Chinese invention patent CN113383768B discloses a liver cell cryopreservation solution and its preparation method and application. The cryopreservation solution includes dimethyl sulfoxide, glucose, human recombinant albumin, vitamin C, vitamin E, hydroxyethyl starch 130 / 0.4 sodium chloride injection, cell buffer and amino acid mixture. It avoids the use of fetal bovine serum, reduces the adverse effects on cell therapy, and has low cost, stable ingredients, simple preparation method, and can be mass-produced. However, the types of raw materials are complex and the configuration is cumbersome. Summary of the invention

[0004] In order to develop a hepatocyte freezing solution with high cell survival rate and high cell biological activity after freezing, the first aspect of the present invention provides a hepatocyte prescription freezing solution, the preparation raw materials include human albumin injection, pharmaceutical grade dimethyl sulfoxide, and compound electrolyte injection, and the volume concentration of the pharmaceutical grade dimethyl sulfoxide is 5%-7.5%.

[0005] As a preferred embodiment, the purity of the human albumin injection is 10%-25%.

[0006] As a preferred embodiment, the volume ratio of the human albumin injection to the compound electrolyte injection is (0.625-1.25):(3.2-3.5).

[0007] As a preferred embodiment, the volume concentration of the human albumin injection in the cryopreservation solution is 2.5%-7.5%.

[0008] During the experiment, the inventors found that after the cells were frozen, the cell activity would decrease to a certain extent. The traditional cell freezing solution contains animal-derived proteins, which will cause a certain degree of cell contamination. The inventors used human albumin injection instead of animal-derived proteins to avoid cell contamination. In this application, it is used to freeze liver cells. Compared with common stem cells, liver cells have lower differentiation performance and have a complex cell structure. The cell structures such as liver cytoplasm, Golgi bodies, and microsomes contained in them are easily inactivated during freezing. By introducing the synergistic effect of human albumin injection and compound electrolyte injection, while balancing the osmotic pressure inside and outside the cells, nutrients are provided to the cell structure of liver cells to protect liver cells from damage.

[0009] In addition, the introduction of DMSO into the cell freezing solution can reduce the formation of ice crystals and avoid cell freezing damage, but DMSO has certain toxicity, and the amount of DMSO added needs to be optimized to balance the effects of cell damage and cytotoxicity. The use of 5vol% DMSO can not only control the formation of ice crystals and avoid damaging the cell structure inside the hepatocytes, but also reduce cytotoxicity. The inventors further discovered that the use of higher concentrations of DMSO will also reduce the freezing survival rate of hepatocytes. The possible reason is speculated to be: hepatocytes are complete cell structures with biological activity, and high concentrations of DMSO have certain toxicity to hepatocytes, which leads to a decrease in cell viability after freezing and is prone to death during the freezing process.

[0010] As a preferred embodiment, the diameter of the hepatocytes used for cryopreservation is 14-20 μm.

[0011] As a preferred embodiment, the frozen cell density of the hepatocytes is 3×10 6 -3×10 7 Pieces / mL.

[0012] A second aspect of the present invention provides a method for using a hepatocyte prescription cryopreservation solution, comprising the following steps:

[0013] S1: Take the cultured allogeneic cells, digest and dissociate them into single cells, add staining solution to the cell suspension, and count them;

[0014] S2: taking the stained cell suspension into a centrifuge tube according to the count, centrifuging, discarding the supernatant, adding the hepatocyte prescription freezing solution, pipetting evenly, and dispensing into internal rotation freezing tubes;

[0015] The S3 cryovials were transferred to a programmed cooling box and stored in a -80°C refrigerator for 12 h before being transferred to liquid nitrogen for storage.

[0016] As a preferred embodiment, the method for resuscitating hepatocytes after freezing the hepatocytes comprises the following steps:

[0017] M1 transfers the cryotubes into a dry ice box;

[0018] The temperature of the M2 water bath is set to 35-38°C. Take out the cryotubes from the dry ice box and place them in the water bath to melt. After the cells are completely thawed, transfer the cryotubes to the biosafety cabinet.

[0019] M3 opened the cryovial in the biosafety cabinet and transferred the cells into a centrifuge tube containing UI-P medium;

[0020] After M4 culture, centrifugation, staining, and counting;

[0021] M5 takes the stained cells according to the count and inoculates the cells in a cell culture flask, transfers them to an incubator for culture, and observes the cell adhesion growth state.

[0022] As a preferred embodiment, the volume ratio of cells to UI-P culture medium in step M2 is 1:(8-10), preferably, the volume ratio of cells to UI-P culture medium in step M2 is 1:9.

[0023] As a preferred embodiment, the inoculated cell density is 1.0×10 4 Pieces / cm 2 .

[0024] As a preferred embodiment, the cell culture conditions are a temperature of 37° C. and 5 vol% CO 2 .

[0025] As a preferred embodiment, the dyeing solution is 0.2 wt % trypan blue dyeing solution.

[0026] As a preferred embodiment, the centrifugation in steps S2 and M4 is 200g for 5 min.

[0027] As a preferred embodiment, after the cryopreserved hepatocytes are revived, the immediate survival rate of the hepatocytes is greater than 90%.

[0028] As a preferred embodiment, after the frozen hepatocytes are cultured for 3 days, the confluence of the hepatocytes is ≥ 70%.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The hepatocyte cryopreservation solution of the present invention uses human albumin injection to replace animal-derived protein to avoid cell contamination and reduce adverse effects on cell therapy.

[0031] (2) The hepatocyte cryopreservation solution of the present invention uses 5 vol% DMSO to control ice crystal formation and avoid damaging the cell structure inside the hepatocytes, and also to reduce cytotoxicity.

[0032] (3) The hepatocyte cryopreservation solution of the present invention adopts human albumin injection with a purity of 10-25%, and the cell viability after resuscitation is not less than 90%, which meets the requirement that the cell viability after cryopreservation is not less than 80%.

[0033] (4) The hepatocyte cryopreservation solution of the present invention has a high adherent growth confluence rate and high biological activity of the hepatocytes after hepatocytes are revived from cryopreservation.

[0034] (5) The hepatocyte cryopreservation solution of the present invention has simple and clear raw materials, low cost, stable ingredients, convenient use, and high activity of hepatocytes after cryopreservation and resuscitation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a picture of the cells in Example 1-12 growing on the wall after being cultured for 3 days after being thawed from cryopreservation.

[0036] In the figure: AM corresponds to Examples 1-12 and Comparative Example 1 respectively; a: cryopreserved for 7 days; b: cryopreserved for 14 days.

[0037] Figure 2 This is a bar graph of live cells immediately after and after centrifugation when the cells were revived after being frozen for 7 days;

[0038] Figure 3 This is a bar graph of live cells immediately after and after centrifugation when the cells were revived after 14 days of cryopreservation;

[0039] Figure 4 The figure shows the live cell histogram immediately after and after centrifugation when the cells were revived after being frozen for 7 days and 14 days. DETAILED DESCRIPTION

[0040] Example

[0041] The specific formula of a hepatocyte cryopreservation solution is shown in Table 1 below.

[0042] Table 1

[0043]

[0044] 20% HAS (human albumin injection) was purchased from JetBelin, and compound electrolyte injection was purchased from Shanghai Better Medical Co., Ltd.

[0045] A method for using a prescription hepatocyte cryopreservation solution comprises the following steps:

[0046] S1: Take the cultured allogeneic cells, digest and dissociate them into single cells, add staining solution to the cell suspension, and count them;

[0047] S2 counts were based on a cell density of 3 × 10 6 Take the stained cell suspension in a centrifuge tube, centrifuge, discard the supernatant, add the hepatocyte prescription freezing solution, pipette evenly, and dispense into internal rotation freezing tubes;

[0048] The S3 cryovials were transferred to a programmed cooling box and stored in a -80°C refrigerator for 12 h before being transferred to liquid nitrogen for storage.

[0049] Store in liquid nitrogen for 7 days and thaw after 14 days.

[0050] The method for resuscitating the hepatocytes after freezing the hepatocytes comprises the following steps:

[0051] M1 transfers the cryotubes into a dry ice box;

[0052] The temperature of the M2 water bath was set to 37°C. The cryotubes in the dry ice box were taken out and placed in the water bath to melt. After the cells were completely thawed, the cryotubes were transferred to the biosafety cabinet.

[0053] M3 opened the cryovial in the biosafety cabinet and transferred the cells into a centrifuge tube containing UI-P medium;

[0054] After M4 culture, centrifugation, staining, and counting;

[0055] M5 takes the stained cells according to the count and inoculates the cells in a cell culture flask, transfers them to an incubator for culture, and observes the cell adhesion growth state.

[0056] The volume ratio of cells to UI-P culture medium in step M2 is 1:9.

[0057] The material information of the UI-P culture medium is shown in the table below.

[0058]

[0059] The preparation method of the UI-P medium is as follows:

[0060] Place the beaker in a biosafety cabinet, place the magnetic stirrer in the beaker, measure the reagents according to the solution ingredient list and put them into the beaker, then place it on a magnetic stirrer, adjust the speed to 400 rpm, stir and mix thoroughly for 1 hour, use a PES membrane disposable sterile bottle top filter, set the pressure of the aspirator to ≤0.05 MPa for filtration, and package for use.

[0061] The staining solution is 0.2 wt % trypan blue staining solution.

[0062] The centrifugation of step S2, M4 is 200g, 5min.

[0063] The inoculation cell density was 1.0×10 4 Pieces / cm 2 .

[0064] The cell culture conditions are 37°C and 5 vol% CO2.

[0065] Comparative Example 1

[0066] A freezing solution, the raw materials for preparation include DMSO 10vol%, FBS (fetal bovine serum) 20vol%, DMEM / F-12 medium 70vol%, and the rest of the implementation is the same as the example. DMEM / F-12 medium is purchased from Gibco. FBS is purchased from Corning.

[0067] Performance Testing

[0068] The immediate survival of cells revived after 7 days of cryopreservation is shown in Table 2 below;

[0069] The survival of cells after thawing and centrifugation after 7 days of cryopreservation is shown in Table 3 below;

[0070] The immediate survival of cells revived after 14 days of cryopreservation is shown in Table 4 below;

[0071] The survival of cells after thawing and centrifugation after 14 days of cryopreservation is shown in Table 5 below;

[0072] The bar graphs of live cells immediately after and after centrifugation when the cells were revived after 7 days of freezing are shown in Figure 2 ; In the figure: Ⅰ is the average total number of live cells in real time for 7 days; Ⅱ is the average total number of live cells after centrifugation for 7 days;

[0073] The histogram of live cells immediately after and after centrifugation when the cells were revived after 14 days of freezing is shown in Figure 3 ; In the figure: Ⅲ is the average total number of live cells in real time at 14 days; Ⅳ is the average total number of live cells after centrifugation at 14 days;

[0074] The bar graphs of live cells immediately after and after centrifugation when cells were revived after 7 and 14 days of freezing are shown in Figure 4 ;

[0075] The cell groups from left to right are Examples 1-12.

[0076] Using the cryopreservation solution of Example 1-12, the cells in Comparative Example 1 were cryopreserved and revived. The cell adherence growth chart after 3 days of culture is shown in FIG. Figure 1 .

[0077] Figure 1 Middle: Example 1: After 7 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 70%; cultured for 75 hours; discarded after taking pictures. After 14 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 80%; cultured for 72 hours; discarded after taking pictures.

[0078] Example 2: After 7 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 70%; cultured for 75 hours; discarded after taking a photo. After 14 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 80%; cultured for 72 hours; discarded after taking a photo.

[0079] Example 3: After 7 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 75%; cultured for 75 hours; discarded after taking a photo. After 14 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 80%; cultured for 72 hours; discarded after taking a photo.

[0080] Example 4 After 7 days of cryopreservation, the revived cells grew attached to the wall and were spindle-shaped; confluence: 75%; cultured for 75 hours; discarded after taking photos. After 14 days of cryopreservation, the revived cells grew attached to the wall and were spindle-shaped; confluence: 75%; cultured for 72 hours; discarded after taking photos.

[0081] Example 5: After 7 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 85%; cultured for 75 hours; discarded after taking a photo. After 14 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 85%; cultured for 72 hours; discarded after taking a photo.

[0082] Example 6 After 7 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 80%; cultured for 75 hours; discarded after taking pictures. After 14 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 80%; cultured for 72 hours; discarded after taking pictures.

[0083] Example 7: Cells revived after 7 days of cryopreservation adhered to the wall and grew in a spindle shape; confluence: 75%; cultured for 75 hours; discarded after taking photos. Cells revived after 14 days of cryopreservation adhered to the wall and grew in a spindle shape; confluence: 80%; cultured for 72 hours; discarded after taking photos.

[0084] Example 8 After 7 days of cryopreservation, the revived cells grew attached to the wall and were spindle-shaped; confluence: 75%; cultured for 75 hours; discarded after taking photos. After 14 days of cryopreservation, the revived cells grew attached to the wall and were spindle-shaped; confluence: 80%; cultured for 72 hours; discarded after taking photos.

[0085] Example 9: Cells revived after 7 days of cryopreservation adhered to the wall and grew in a spindle shape; confluence: 10%; cultured for 75 hours; discarded after taking photos. Cells revived after 14 days of cryopreservation adhered to the wall and grew in a spindle shape; confluence: 20%; cultured for 72 hours; discarded after taking photos.

[0086] Example 10 After 7 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 75%; cultured for 75 hours; discarded after taking a photo. After 14 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 75%; cultured for 72 hours; discarded after taking a photo.

[0087] Example 11 After 7 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 80%; cultured for 75 hours; discarded after taking pictures. After 14 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 80%; cultured for 72 hours; discarded after taking pictures.

[0088] Example 12 After 7 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 80%; cultured for 75 hours; discarded after taking a photo. After 14 days of cryopreservation, the revived cells adhered to the wall and grew in a spindle shape; confluence: 80%; cultured for 72 hours; discarded after taking a photo.

[0089] The cell adhesion of Comparative Example 1 after 7 days of cryopreservation was not significantly different from that of Example 1, and the cell recovery after 14 days of cryopreservation was significantly worse than that of Example 1.

[0090] Confluence = area of ​​culture vessel inhabited by attached cells / total area of ​​culture vessel × 100%.

[0091] In summary, the cell proliferation rate of the cryopreservation solution of Example 5 was the best, and the confluence was the highest at the same observation time, while the cell proliferation rate of the cryopreservation solution of Example 9 was the worst.

[0092] Table 2

[0093]

[0094] Table 3

[0095]

[0096]

[0097] Table 4

[0098]

[0099]

[0100] Table 5

[0101]

Claims

1. A hepatocyte cryopreservation solution, characterized in that: The preparation raw materials include human albumin injection, pharmaceutical-grade dimethyl sulfoxide and compound electrolyte injection. The volume concentration of the pharmaceutical-grade dimethyl sulfoxide is 5%-7.5%.

2. The hepatocyte cryopreservation solution according to claim 1, characterized in that: The purity of the human albumin injection is 10%-25%.

3. The hepatocyte cryopreservation solution according to claim 1, characterized in that: The volume ratio of the human albumin injection to the compound electrolyte injection is (0.625-1.25):(3.2-3.5).

4. The hepatocyte cryopreservation solution according to claim 1, characterized in that: The volume concentration of the human albumin injection in the freezing solution is 2.5%-7.5%.

5. The hepatocyte cryopreservation solution according to claim 1, characterized in that: The diameter of the hepatocytes used for cryopreservation was 14-20 μm.

6. The hepatocyte cryopreservation solution according to claim 1, characterized in that: The cryopreserved cell density of the hepatocytes was 3×10 6 -3×10 7 Pieces / mL.

7. A method for using the hepatocyte cryopreservation solution according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1: Take the cultured allogeneic cells, digest and dissociate them into single cells, add staining solution to the cell suspension, and count them; S2: taking the stained cell suspension into a centrifuge tube according to the count, centrifuging, discarding the supernatant, adding the hepatocyte prescription freezing solution, pipetting evenly, and dispensing into internal rotation freezing tubes; The S3 cryovials were transferred to a programmed cooling box and stored in a -80°C refrigerator for 12 h before being transferred to liquid nitrogen for storage.

8. The method for using the hepatocyte cryopreservation solution according to claim 7, characterized in that: The method for resuscitating the hepatocytes after freezing the hepatocytes comprises the following steps: M1 transfers the cryotubes into a dry ice box; The temperature of the M2 water bath is set to 35-38°C. Take out the cryotubes from the dry ice box and place them in the water bath to melt. After the cells are completely thawed, transfer the cryotubes to the biosafety cabinet. M3 opened the cryovial in the biosafety cabinet and transferred the cells into a centrifuge tube containing UI-P medium; After M4 culture, centrifugation, staining, and counting; M5 takes the stained cells according to the count and inoculates the cells in a cell culture flask, transfers them to an incubator for culture, and observes the cell adhesion growth state.

9. The method for using the hepatocyte cryopreservation solution according to claim 7, characterized in that: After the cryopreserved hepatocytes were thawed, the immediate survival rate of the hepatocytes was >90%.

10. The method for using the hepatocyte cryopreservation solution according to claim 8, characterized in that: After the frozen hepatocytes were cultured for 3 days, the confluence of the hepatocytes was ≥ 70%.

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