CN113383768A - Hepatocyte freezing solution and preparation method and application thereof - Google Patents

Abstract

The invention relates to a frozen liver cell stock solution and a preparation method and application thereof, wherein the frozen stock solution comprises the following components: dimethyl sulfoxide, glucose, human recombinant albumin, vitamin C, vitamin E, hydroxyethyl starch 130/0.4 sodium chloride injection, cell buffer solution and amino acid mixed solution. The frozen stock solution avoids the use of fetal calf serum, reduces adverse effects on cell therapy, and has the advantages of low cost, stable components, simple preparation method, and large-scale production.

Description

Hepatocyte freezing solution and preparation method and application thereof

Technical Field

The invention relates to the technical field of cell freezing solution, in particular to a liver cell freezing solution and a preparation method and application thereof.

Background

The cell therapy is that cells with certain specific functions are utilized, and after the cells are treated by in vitro amplification, special culture and the like by adopting a bioengineering method, the cells have the treatment effects of enhancing immunity, killing pathogens and tumor cells, promoting regeneration of tissues and organs, recovering organisms and the like, so that the purpose of treating diseases is achieved. The technical development of the method enables a new method for treating diseases such as tumors, leukemia and the like.

Wherein the L-02 cell line was identified in 1980 (Yexizhen et al, 1980). The cell is a normal liver cell line, has typical liver cell morphological characteristics, has similar cell functions to normal liver cells, and is a seed cell in liver disease cell treatment. L-02 cells can play an important auxiliary role in the treatment of liver diseases through cell therapy technology.

At present, the frozen stock solution of the liver cells contains fetal calf serum. However, since cells are in contact with the human body in the cell therapy and fetal calf serum has uncertain factors therein, the cell therapy effect may be affected. Even if the frozen stock solution is removed by centrifugation, the influence of the fetal calf serum cannot be completely removed, and the effect of subsequent cell therapy can be influenced to a certain extent.

Disclosure of Invention

In view of the above, the present invention provides a frozen hepatocyte storage solution, which contains no fetal bovine serum, and can further reduce unsafe factors in clinical hepatocyte treatment, and maintain the cell survival rate and biological activity of hepatocytes.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

a hepatocyte freezing solution comprises the following components: the volume fraction of the injection is 5-10% of dimethyl sulfoxide, the volume fraction of the injection is 10-15% of hydroxyethyl starch 130/0.4 sodium chloride, 0.5-2.0 g/L of glucose, 35-50 g/L of human recombinant albumin, 30-120 mu mol/L of vitamin C, 10-50 mu mol/L of vitamin E and the balance of a mixed solution, wherein the volume ratio of a cell buffer solution to an amino acid mixed solution in the mixed solution is 1: 1-2.

Preferably, the cell buffer has the following components: 6800-8000 mg/L NaCl, 300-400 g/L KCl, 140-200 mg/L CaCl₂95-100 mg/L anhydrous MgSO₂、100～130mg/L Na₂HPO₄、40～55mg/L KH₂PO₄And the balance of sterile water, wherein the pH value of the cell buffer solution is 7.1-7.6, and the osmotic pressure of the cell buffer solution is 250-290 mOsm/L.

Preferably, the amino acid mixture contains: 5-7.5 mg/L glycine, 7-9 mg/L L-alanine, 40-50 mg/L L-valine, 45-55 mg/L L-leucine, 45-55 mg/L L-isoleucine, 25-35 mg/LL-phenylalanine, 8-13 mg/L L-proline, 8-13 mg/L L-tryptophan, 8-13 mg/L L-serine, 30-40 mg/L L-leucine, 12-20 mg/L L-methionine, 280-300 mg/L L-glutamine, 25-35 mg/L L-cystine, 12-15 mg/L L-asparagine, 120-130 mg/L L-arginine, 70-75 mg/L L-lysine, 40-45 mg/L L-histidine, 12-15 mg/L L-aspartic acid, 13-15 mg/L L-glutamic acid, 0.5-1.8 g/L sodium bisulfite and 11-15 g/L sorbitol, wherein the solvent of the amino acid mixed solution is sterile water.

The invention also provides a method for preparing the hepatocyte freezing solution, which comprises the following steps:

s1, dissolving the raw materials of each component in the cell buffer solution in sterile water, uniformly stirring, adjusting the pH value to 7.1-7.6, and sterilizing to obtain the cell buffer solution;

s2, dissolving various amino acids and sodium bisulfite in a sterile water solution containing sorbitol, and uniformly stirring to obtain an amino acid mixed solution;

s3, mixing the cell buffer solution obtained in the step S1 with the amino acid mixed solution obtained in the step S2 in proportion, stirring uniformly, adding glucose, human recombinant albumin, vitamin C, vitamin E and hydroxyethyl starch 130/0.4 sodium chloride injection, stirring uniformly, enabling the osmotic pressure to be 250-290 mOsm/L, sterilizing, and mixing with dimethyl sulfoxide to obtain the hepatocyte freezing solution.

In the application of the frozen hepatocyte culture solution provided by the invention in freezing hepatocyte, the number of hepatocyte cells stored in every 1mL of frozen culture solution is 1-1.5 multiplied by 10 under aseptic condition⁷And storing the cells in a cell freezing tube reactor, and freezing according to a gradient cooling method.

The invention has the beneficial effects that: the invention forms the frozen stock solution aiming at the liver cells with stable components, long nutrient substances and strong buffer capacity of a solution penetration system by mutually matching various components. When the frozen stock solution is used for freezing liver cells, various nutrients can be provided for the liver cells, the liver cells are protected, and the activity of the cells can be maintained. Moreover, the hepatocyte freezing and storing liquid completely avoids using fetal calf serum, so that hepatocytes are frozen and thawed, and contact with serum of other organisms in the subsequent culture process, factors which possibly cause adverse biological reactions are removed, and the effect of the hepatocyte freezing and storing liquid can be better exerted in the cell treatment process; meanwhile, adverse reactions of organisms can not be caused in the process of improving the disease course of a patient, so that the side effects of the patients on the organisms are reduced, and the patients can play better effects in subsequent clinical use. The hepatocyte freezing solution has the advantages of low cost, safety, no toxic or side effect, simple and convenient preparation process, capability of being prepared in large quantities in a short time and large-scale production.

Detailed Description

The technical solutions of the present invention are described in detail below by specific examples, which are only used for explaining the present invention and are not used for limiting the scope of the present invention.

Example 1

The components of the hepatocyte freezing solution of the present example are: 5 percent (volume fraction) of dimethyl sulfoxide, 0.5/L of glucose, 35g/L of human recombinant albumin, 30 mu mol/L of vitamin C, 10 mu mol/L of vitamin E, 10 percent (volume fraction) of hydroxyethyl starch 130/0.4 sodium chloride injection and the balance of mixed solution, wherein the volume ratio of the cell buffer solution to the amino acid mixed solution in the mixed solution is 1:1.

The cell buffer solution comprises the following components in percentage by weight: 6800mg/L NaCl, 300g/L KCl, 140mg/L CaCl₂95mg/L anhydrous MgSO₂、100mg/L Na₂HPO₄、40mg/L KH₂PO₄The pH of the cell buffer was adjusted to 7.1 with the balance being sterile water, and the osmolality was 250 mOsm/L.

The amino acid mixed solution comprises the following components in percentage by weight: 5mg/L glycine, 7mg/L L-alanine, 40mg/L L-valine, 45mg/L L-leucine, 45mg/L L-isoleucine, 25mg/L L-phenylalanine, 8mg/L L-proline, 8 mg/LL-tryptophan, 8mg/L L-serine, 30 mg/LL-tyrosine, 12mg/L L-methionine, 280mg/L L-glutamine, 25mg/L L-cystine, 12mg/L L-asparagine, 120mg/L L-arginine, 70mg/L L-lysine, 40mg/L L-histidine, 12mg/L L-aspartic acid, 13mg/L L-glutamic acid, 0.5g/L sodium bisulfite, 11g/L sorbitol.

Example 2

The frozen hepatocyte culture solution comprises 8% (volume fraction) of dimethyl sulfoxide, 1.0g/L of glucose, 40g/L of human recombinant albumin, 110 mu mol/L of vitamin C, 35 mu mol/L of vitamin E, 12% (volume fraction) of hydroxyethyl starch 130/0.4 sodium chloride injection and the balance of mixed solution, wherein the volume ratio of a cell buffer solution to an amino acid mixed solution in the mixed solution is 1: 1.5.

The cell buffer solution comprises the following components in percentage by weight: 7000mg/L NaCl, 350g/L KCl, 170mg/L CaCl₂97mg/L of anhydrous MgSO₂、115mg/L Na₂HPO₄、50mg/L KH₂PO₄And the balance of sterile water, the pH of the cell buffer solution is adjusted to 7.4, and the osmotic pressure content is 275 mOsm/L;

the amino acid mixed solution comprises the following components in percentage by weight: 7.0mg/L glycine, 8.5mg/L L-alanine, 48mg/L L-valine, 52mg/L L-leucine, 50mg/L L-isoleucine, 33mg/L L-phenylalanine, 12mg/L L-proline, 11mg/L L-tryptophan, 12mg/L L-serine, 35mg/L L-leucine, 15mg/L L-methionine, 290mg/L L-glutamine, 30mg/L L-cystine, 13mg/L L-asparagine, 125mg/L L-arginine, 73mg/L L-lysine, 42mg/L L-histidine, 13mg/L L-aspartic acid, 14mg/L L-glutamic acid, 0.8g/L sodium bisulfite, 13g/L sorbitol.

Example 3

The components of the frozen liver cells of this example were: 10 percent (volume fraction) of dimethyl sulfoxide, 2.0g/L of glucose, 50g/L of human recombinant albumin, 120 mu mol/L of vitamin C, 50 mu mol/L of vitamin E, 15 percent (volume fraction) of hydroxyethyl starch 130/0.4 sodium chloride injection and the balance of mixed solution, wherein the volume ratio of the cell buffer solution to the amino acid mixed solution in the mixed solution is 1:2.

The cell buffer solution contains 7500mg/L NaCl, 380g/LKCl and 180mg/L CaCl₂98mg/L anhydrous MgSO₂、125mg/L Na₂HPO₄、53mg/L KH₂PO₄And the balance being sterile water, the pH of the cell buffer being 7.6 and the osmotic pressure being 290mOsm/L；

The amino acid mixed solution comprises the following components in percentage by weight: 7.0mg/L glycine, 8.5mg/L L-alanine, 48mg/L L-valine, 52mg/L L-leucine, 52mg/L L-isoleucine, 33mg/L L-phenylalanine, 12mg/L L-proline, 12mg/L L-tryptophan, 12mg/L L-serine, 38mg/L L-leucine, 18mg/L L-methionine, 2295mg/L L-glutamine, 33mg/L L-cystine, 15mg/L L-asparagine, 128mg/L L-arginine, 75mg/L L-lysine, 44mg/L L-histidine, 15mg/L L-aspartic acid, 15mg/L L-glutamic acid, 1.5g/L sodium bisulfite, 15g/L sorbitol.

Comparative example 1

The frozen stock solution of hepatocytes of this example did not contain the hydroxyethyl starch 130/0.4 sodium chloride injection, and the rest were the same as in example 1.

Comparative example 2

The frozen stock solution of hepatocytes of this example did not contain the hydroxyethyl starch 130/0.4 sodium chloride injection, and the rest were the same as in example 2.

Comparative example 3

The frozen hepatocytes of this example did not contain the hydroxyethyl starch 130/0.4 sodium chloride injection, and the rest were the same as in example 3.

Comparative example 4

In the cryopreservation of hepatocytes of this example, the volume ratio of the cell buffer solution to the amino acid mixture in the mixed solution was 1:0.5, and the other steps were the same as in example 1.

Comparative example 5

In the cryopreservation of hepatocytes of this example, the volume ratio of the cell buffer solution to the amino acid mixture in the mixed solution was 1:2.5, and the rest was the same as in example 2.

Comparative example 6

In the cryopreservation of hepatocytes of this example, the volume ratio of the cell buffer solution to the amino acid mixture in the mixed solution was 1:3, and the rest was the same as in example 3.

Comparative example 7

The amount of hydroxyethyl starch 130/0.4 sodium chloride injection in the frozen stock solution of hepatocytes of this example was 30% (volume fraction), and the rest was the same as in example 1.

Comparative example 8

The frozen stock solution in this comparative example contained 5% by volume of dimethyl sulfoxide and 95% by volume of fetal bovine serum.

Comparative example 9

The frozen stock solution in this comparative example contained 8% by volume of dimethyl sulfoxide and 92% by volume of fetal bovine serum.

Comparative example 10

The frozen stock solution in this comparative example contained 10% by volume of dimethyl sulfoxide and 90% by volume of fetal bovine serum.

The cryopreservation solutions in examples 1 to 3 and comparative examples 1 to 10 were respectively taken, and the same L-02 cells were subjected to cryopreservation recovery test, wherein the test process is as follows: respectively placing the freezing medium and the control preserving medium with the same volume in different freezing tubes, clearly marking, adding the same amount of hepatocytes into each 1ml of the freezing medium by freezing 1 × 107L-02 cells, and freezing by gradient cooling; then, respectively recovering after 3d, 7d and 30d, observing under a mirror after the cells are attached to the wall, observing the survival rate of the recovered hepatocytes, recording and comparing, wherein the test results are shown in the following table:

as can be seen from the above table, the frozen stock solution for liver cells provided by the present invention has no obvious difference from the traditional frozen stock solution in the survival rate, and various components in the frozen stock solution are all available for human bodies or are clinically used, and have no influence on the subsequent cell therapy. Meanwhile, as the frozen stock solution does not contain fetal calf serum, the factors which possibly cause adverse reactions are reduced for subsequent clinical experiments, and the vitamin C, E is added, the liver cells are protected, and other side effects cannot be caused, the proportion of the hydroxyethyl starch 130/0.4 sodium chloride injection and the mixed solution of the cell buffer solution and the amino acid has obvious protection effect on the survival rate of the cells, and the reason is as follows: the hydroxyethyl starch injection and the cell buffer solution in a certain proportion maintain the colloid osmotic pressure and the crystal osmotic pressure of cells together, but the cells are dehydrated or swelled due to overhigh or overlow, and the hydroxyethyl starch injection belongs to the field of clinical medication and does not bring harm to human bodies when used for clinical artificial liver treatment in the follow-up process. However, higher hydroxyethyl starch levels result in decreased survival; and when the content of the hydroxyethyl starch injection is too much (such as volume fraction is more than or equal to 50%), the mixing capability with other components is poor, and the system is easy to delaminate.

It should be noted that the above-mentioned embodiments are merely illustrative and not restrictive, and any modifications, equivalents, improvements and the like which come within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (5)

1. A hepatocyte cryopreservation solution, comprising: the volume fraction of the injection is 5-10% of dimethyl sulfoxide, the volume fraction of the injection is 10-15% of hydroxyethyl starch 130/0.4 sodium chloride, 0.5-2.0 g/L of glucose, 35-50 g/L of human recombinant albumin, 30-120 mu mol/L of vitamin C, 10-50 mu mol/L of vitamin E and the balance of a mixed solution, wherein the volume ratio of a cell buffer solution to an amino acid mixed solution in the mixed solution is 1: 1-2.

2. The hepatocyte cryopreservation solution of claim 1, wherein the composition of the cell buffer solution is: 6800-8000 mg/L NaCl, 300-400 g/L KCl, 140-200 mg/L CaCl₂95-100 mg/L anhydrous MgSO₂、100～130mg/L Na₂HPO₄、40～55mg/L KH₂PO₄And the balance of sterile water, wherein the pH value of the cell buffer solution is 7.1-7.6, and the osmotic pressure of the cell buffer solution is 250-290 mOsm/L.

3. The hepatocyte cryopreservation solution according to claim 1 or 2, wherein the amino acid mixture solution contains: 5-7.5 mg/L glycine, 7-9 mg/L L-alanine, 40-50 mg/L L-valine, 45-55 mg/L L-leucine, 45-55 mg/L L-isoleucine, 25-35 mg/L L-phenylalanine, 8-13 mg/L L-proline, 8-13 mg/L L-tryptophan, 8-13 mg/L L-serine, 30-40 mg/L L-leucine, 12-20 mg/L L-methionine, 280-300 mg/L L-glutamine, 25-35 mg/L L-cystine, 12-15 mg/L L-asparagine, 120-130 mg/L L-arginine, 70-75 mg/L L-lysine, 40-45 mg/L L-histidine, 12-15 mg/L L-aspartic acid, 13-15 mg/L L-glutamic acid, 0.5-1.8 g/L sodium bisulfite and 11-15 g/L sorbitol, wherein the solvent of the amino acid mixed solution is sterile water.

4. A method for preparing a frozen stock solution of liver cells, comprising the steps of:

s1, dissolving the raw materials of each component in the cell buffer solution in sterile water, uniformly stirring, adjusting the pH value to 7.1-7.6, and sterilizing to obtain the cell buffer solution;

s2, dissolving various amino acids and sodium bisulfite in a sterile water solution containing sorbitol, and uniformly stirring to obtain an amino acid mixed solution;

s3, mixing the cell buffer solution obtained in the step S1 with the amino acid mixed solution obtained in the step S2 in proportion, stirring uniformly, adding glucose, human recombinant albumin, vitamin C, vitamin E and hydroxyethyl starch 130/0.4 sodium chloride injection, stirring uniformly, enabling the osmotic pressure to be 250-290 mOsm/L, sterilizing, and mixing with dimethyl sulfoxide to obtain the hepatocyte freezing solution.

5. The use of the frozen stock solution for hepatocytes according to claim 1, wherein the number of hepatocytes to be stored per 1mL of the frozen stock solution is 1 to 1.5X 10⁷And (4) respectively.