CN114586768A - Liver tissue preservation solution and preservation method and application thereof - Google Patents
Liver tissue preservation solution and preservation method and application thereof Download PDFInfo
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- CN114586768A CN114586768A CN202011394903.4A CN202011394903A CN114586768A CN 114586768 A CN114586768 A CN 114586768A CN 202011394903 A CN202011394903 A CN 202011394903A CN 114586768 A CN114586768 A CN 114586768A
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- 210000005228 liver tissue Anatomy 0.000 title claims abstract description 61
- 239000003761 preservation solution Substances 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 10
- 238000004321 preservation Methods 0.000 title claims abstract description 10
- 108010024636 Glutathione Proteins 0.000 claims abstract description 12
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 11
- 239000003112 inhibitor Substances 0.000 claims abstract description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 7
- 102000004039 Caspase-9 Human genes 0.000 claims abstract description 3
- 108090000566 Caspase-9 Proteins 0.000 claims abstract description 3
- 102000005927 Cysteine Proteases Human genes 0.000 claims abstract description 3
- 108010005843 Cysteine Proteases Proteins 0.000 claims abstract description 3
- 101710092490 Protein kinase 3 Proteins 0.000 claims abstract description 3
- 239000008103 glucose Substances 0.000 claims abstract description 3
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 13
- 239000012091 fetal bovine serum Substances 0.000 claims description 9
- 229960005322 streptomycin Drugs 0.000 claims description 9
- BPKSNNJTKPIZKR-UHFFFAOYSA-N 3-(1,3-benzothiazol-5-yl)-7-(2,5-dimethylpyrazol-3-yl)thieno[3,2-c]pyridin-4-amine Chemical compound Cc1cc(-c2cnc(N)c3c(csc23)-c2ccc3scnc3c2)n(C)n1 BPKSNNJTKPIZKR-UHFFFAOYSA-N 0.000 claims description 7
- VPVLPCIBKVWFDT-UHFFFAOYSA-N 4-(4-methylphenoxy)-7-(1-piperidin-4-ylpyrazol-4-yl)quinoline Chemical compound N1CCC(CC1)N1N=CC(=C1)C1=CC=C2C(=CC=NC2=C1)OC1=CC=C(C=C1)C VPVLPCIBKVWFDT-UHFFFAOYSA-N 0.000 claims description 7
- LIGGMBSSOOVGAE-UHFFFAOYSA-N 1-[5-[4-amino-7-(1-methylpyrazol-4-yl)thieno[3,2-c]pyridin-3-yl]-2,3-dihydroindol-1-yl]-2-phenylethanone Chemical compound C1=NN(C)C=C1C1=CN=C(N)C2=C1SC=C2C1=CC=C(N(CC2)C(=O)CC=3C=CC=CC=3)C2=C1 LIGGMBSSOOVGAE-UHFFFAOYSA-N 0.000 claims description 4
- UGTLDBJIOSYXRR-UHFFFAOYSA-N CNC(=O)C1=CC=C2N(C=NC2=C1)C1=CC=C(CC(=O)OC(C)(C)C)C=C1 Chemical compound CNC(=O)C1=CC=C2N(C=NC2=C1)C1=CC=C(CC(=O)OC(C)(C)C)C=C1 UGTLDBJIOSYXRR-UHFFFAOYSA-N 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- ZCDBTQNFAPKACC-UHFFFAOYSA-N n-(6-propan-2-ylsulfonylquinolin-4-yl)-1,3-benzothiazol-5-amine Chemical compound C1=C2SC=NC2=CC(NC2=CC=NC3=CC=C(C=C32)S(=O)(=O)C(C)C)=C1 ZCDBTQNFAPKACC-UHFFFAOYSA-N 0.000 claims description 4
- 229940122396 Caspase 9 inhibitor Drugs 0.000 claims description 2
- 101710156256 Myosin phosphatase Rho-interacting protein Proteins 0.000 claims description 2
- 102100033729 Receptor-interacting serine/threonine-protein kinase 3 Human genes 0.000 claims description 2
- 102000005962 receptors Human genes 0.000 claims description 2
- 108020003175 receptors Proteins 0.000 claims description 2
- 230000009977 dual effect Effects 0.000 claims 1
- 210000003494 hepatocyte Anatomy 0.000 abstract description 14
- 238000000926 separation method Methods 0.000 abstract description 5
- 230000004083 survival effect Effects 0.000 abstract description 5
- 229930182555 Penicillin Natural products 0.000 abstract description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 3
- 229940049954 penicillin Drugs 0.000 abstract description 3
- 239000012894 fetal calf serum Substances 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 238000002156 mixing Methods 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000009509 drug development Methods 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 210000005229 liver cell Anatomy 0.000 description 4
- 206010019851 Hepatotoxicity Diseases 0.000 description 3
- 230000007686 hepatotoxicity Effects 0.000 description 3
- 231100000304 hepatotoxicity Toxicity 0.000 description 3
- 239000002547 new drug Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 206010048610 Cardiotoxicity Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 231100000259 cardiotoxicity Toxicity 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000037450 immune related reaction Effects 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 229940127554 medical product Drugs 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a liver tissue preservation solution and a preservation method and application thereof. The liver tissue preservation solution is a high-glucose DMEM culture medium containing cysteine protease 9 (Caspase-9) inhibitor, reduced glutathione, receptor action protein kinase 3 (RIP 3) inhibitor, fetal calf serum and penicillin double antibody. The obtained liver tissue preservation solution is used for preserving liver tissues, the success rate of obtaining primary hepatocytes by separating and extracting the liver tissues preserved by the liver tissue preservation solution is greatly improved, the survival rate and the purity of the obtained primary hepatocytes are high, and the separation of the primary hepatocytes of the human liver tissues is realized by obtaining materials in different places.
Description
Technical Field
The invention relates to the technical field of biological reagents, in particular to a liver tissue preservation solution and a preservation method and application thereof.
Background
Studies show that the failure rate of new Drug clinical trials in the II and III stages reaches 25% and 14% respectively [ Harrison RK. Phase II and Phase III failures: 2013-. Another study showed that the leading cause of withdrawal from the market in 1950-2014 was adverse reactions, with hepatotoxicity being the leading cause, followed by immune-related reactions, again cardiotoxicity [ Onkpoya IJ, Heneghan CJ, Aronson JK. Post-marking with driver of 462 medical products subcase of additive drug reactions: a systematic review of the world competence, BMC Med.2016; 14:10 ]. Therefore, finding the risk of possible failure of drug development more rapidly at the early stage of new drug development can improve the success rate of drug development and save the cost of drug development.
The compound can be further screened by using the primary hepatocytes to perform the hepatotoxicity evaluation of the candidate compound before the non-clinical safety evaluation of the new drug, so that the risk of failure in the non-clinical safety evaluation and clinical research can be reduced.
At present, primary hepatocytes such as mice and rats are separated by a liver-regulating perfusion method. However, due to species differences, hepatotoxicity evaluations using primary hepatocytes derived from human liver tissue are more predictive of risk for subsequent studies.
At present, most of liver tissues of a human body, which can be clinically obtained, are broken liver tissues, the liver tissues are rich in lysosomes and the like, various enzymes released by apoptotic or necrotic cells in the broken liver tissues after being isolated are easy to cause damages to other cells, the success rate of primary liver cell separation after storage and transportation is extremely low, and the separated liver cells cannot adhere to the wall and die quickly. Cell isolation should be performed immediately after ex vivo to obtain primary hepatocytes that are viable and of purity suitable for testing. This clearly limits the acquisition and application of primary hepatocytes.
Disclosure of Invention
The invention aims to provide a liver tissue preservation solution which can relieve the damage of cells of liver tissues after being isolated, further can be stored and transported, and improves the success rate of primary hepatocyte isolation.
In order to achieve the aim, the invention provides a liver tissue preservation solution which comprises a high-glucose DMEM medium containing cysteine protease 9 (Caspase-9) inhibitor, reduced glutathione, receptor-activated protein kinase 3 (RIP 3) inhibitor, fetal bovine serum and streptomycin double antibody.
The Caspase-9 inhibitor is Z-LEHD-FMK or Z-LEHD-FMKTFA, and the concentration of the Z-LEHD-FMK or Z-LEHD-FMKTFA is 1uM-5 uM.
The concentration of the reduced glutathione is 60-120 mg/L.
The RIP3 inhibitor is any one of GSK840, GSK843, GSK2593074A, GSK872 and HS1371, the concentration of GSK840 is 0.5-1uM, the concentration of GSK843 is 1-3uM, the concentration of GSK2593074A is 1-3nM, the concentration of GSK872 is 1-3uM, and the concentration of HS1371 is 5-15 uM.
The fetal bovine serum concentration is 10-20%.
The concentration of the streptomycin double antibody is 1-2%.
The liver tissue preservation solution is used for transportation and preservation of liver tissues.
The liver tissue preservation method is that the isolated liver tissue is placed in the liver tissue preservation solution of claims 1-7 for cooling preservation.
The volume ratio of the liver tissue preservation solution to the liver tissue is not less than 5: 4.
Compared with the prior art, the invention has the following beneficial effects:
at present, no special human liver tissue preservation solution exists. The tissue preservation solution of the general-purpose tissue preservation solution is not applicable to liver tissues. The characteristic of the invention for liver tissue is characterized in that apoptosis and necrosis related factor inhibitor and antioxidant reduced glutathione are added in DMEM, thus eliminating or reducing the damage in the process of liver tissue preservation and transportation, realizing the separation of primary cells of human liver tissue by taking materials from different places and improving the success rate of primary liver cell separation.
The liver tissue preservation solution provided by the invention is used for preserving the crushed liver tissue for 24 hours, primary liver cells are successfully separated and obtained, the survival rate is over 90%, and the purity is over 96%.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.
The test methods used in the following examples are all conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: preparation of liver tissue preserving fluid
Z-LEHD-FMK is dissolved in DMSO to prepare a 1mM solution, reduced glutathione is dissolved in deionized water to prepare a 10mg/ml solution, and GSK843 is dissolved in DMSO to prepare a 3mM solution.
Preparing a 100ml volumetric flask, adding 60ml of DMEM, then adding 10ml of fetal bovine serum, and uniformly mixing; then 1ml of the streptomycin double antibody is added and mixed evenly. Adding 1ml of 10mg/ml reduced glutathione, and mixing uniformly. 100ul of 1mM Z-LEHD-FMK solution was added and mixed well. Finally, 100ul of 3mM GSK843 solution was added and mixed well.
And (4) fixing the volume to 100ml by using a DMEM medium, and evenly mixing the solution by inversion to obtain the liver tissue preservation solution.
TABLE 1 formula of liver tissue preservation solution
| Reagent | Dosage of | Final concentration |
| GSK843（3mM） | 100ul | 3uM |
| Z-LEHD-FMK(1mmM) | 100ul | 1uM |
| Reduced glutathione (10mg/ml) | 1ml | 100mg/L |
| FBS | 10ml | 10% |
| Penicillin streptomycin double antibody | 1ml | 1% |
| DMEM | 87.8ml | 88.8% |
Example 2: preparation of liver tissue preserving fluid
Z-LEHD-FMKTFA is dissolved in DMSO to prepare a 2.5mM solution, reduced glutathione is dissolved in deionized water to prepare a 10mg/ml solution, and HS1371 is dissolved in DMSO to prepare a 10mM solution.
Preparing a 100ml volumetric flask, adding 50ml of DMEM, then adding 20ml of fetal bovine serum, and uniformly mixing; then 2ml of the streptomycin double antibody is added and mixed evenly. Adding 10mg/ml reduced glutathione 1.2ml, mixing well. Add 2.5mM Z-LEHD-FMKTFA solution 100ul and mix well. Finally, 100ul of 10mM HS1371 solution was added and mixed well.
And (4) fixing the volume to 100ml by using a DMEM medium, and evenly mixing the solution by inversion to obtain the liver tissue preservation solution.
TABLE 2 formula of liver tissue preservation solution
| Reagent | Dosage of | Final concentration |
| HS1371（10mM） | 100ul | 10uM |
| Z-LEHD-FMKTFA(2.5mM) | 100ul | 2.5uM |
| Reduced glutathione (10mg/ml) | 1.2ml | 120mg/L |
| FBS | 20ml | 20% |
| Penicillin streptomycin double antibody | 2ml | 2% |
| DMEM | 76.6ml | 76.6% |
Example 3: separation and extraction of primary hepatocytes from human liver tissue fragments preserved by liver tissue preservation solution
Clinically obtained crushed liver tissues were preserved for 24 hours using the liver tissue preservation solutions prepared in tables 1 and 2, respectively, while DMEM containing 10% FBS and 1% streptomycin double antibody was used as a control.
The crushed liver tissue preserved in the liver tissue preservation solution for 24 hours was isolated and cultured by the method provided in application publication No. CN 108070551 a.
The three preservation solutions are preserved for 24 hours for separating, extracting and culturing primary hepatocytes, and the survival rate and the purity of the obtained primary hepatocytes are shown in table 3.
TABLE 3 survival rate and purity of primary hepatocytes isolated, extracted and cultured after human liver tissue is preserved in the formula liver tissue preservation solution, the formula liver tissue preservation solution and the comparison preservation solution
| Preserving fluid | Survival rate | Purity of |
| Formula liver tissue preservation solution | 90.2% | 97.4% |
| Formula liver tissue preservation solution | 92.5% | 96.1% |
| Reference preservative fluid | Failure to adhere to isolated hepatocytes after plating | Unsuccessfully isolated and cultured cells |
The above description is only a preferred embodiment of the present invention, but the scope of the present invention is not limited to this embodiment. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A liver tissue preservation solution is characterized by comprising a high-glucose DMEM medium containing cysteine protease 9 (Caspase-9) inhibitor, reduced glutathione, receptor-activated protein kinase 3 (RIP 3) inhibitor, fetal bovine serum and a streptomycin double antibody.
2. The liver tissue preservation solution according to claim 1, wherein the Caspase-9 inhibitor is Z-LEHD-FMK or Z-LEHD-FMKTFA, and the concentration of Z-LEHD-FMK or Z-LEHD-FMKTFA is 1uM to 5 uM.
3. The liver tissue preservation solution according to claim 1, wherein the concentration of the reduced glutathione is 60-120 mg/L.
4. The liver tissue preservation solution according to claim 1, wherein the RIP3 inhibitor is any one of GSK840, GSK843, GSK2593074A, GSK872 and HS1371, the concentration of GSK840 is 0.5-1uM, the concentration of GSK843 is 1-3uM, the concentration of GSK2593074A is 1-3nM, the concentration of GSK872 is 1-3uM, and the concentration of HS1371 is 5-15 uM.
5. The liver tissue preservation solution according to claim 1, wherein the concentration of fetal bovine serum is 10-20%.
6. The liver tissue preservation solution according to claim 1, wherein the concentration of the dual anti-streptomycin antibody is 1-2%.
7. Use of the preservation solution according to any one of claims 1 to 6 for the preservation of liver tissue.
8. A method for storing a liver tissue, comprising the step of storing a separated liver tissue in the liver tissue storage solution according to any one of claims 1 to 7 under reduced temperature.
9. The preservation method according to claim 8, wherein the volume ratio of the liver tissue preservation solution to the liver tissue is not less than 5: 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202011394903.4A CN114586768A (en) | 2020-12-03 | 2020-12-03 | Liver tissue preservation solution and preservation method and application thereof |
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| CN202011394903.4A CN114586768A (en) | 2020-12-03 | 2020-12-03 | Liver tissue preservation solution and preservation method and application thereof |
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Cited By (2)
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