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CN-122012377-A - Method for extracting and freezing mouse liver cells

CN122012377ACN 122012377 ACN122012377 ACN 122012377ACN-122012377-A

Abstract

The application relates to the technical field of biology, in particular to a method for extracting and freezing mouse liver cells. The method comprises the steps of perfusing and digesting a mouse cadaver through a lower vena cava, cutting off a portal vein, clamping an upper vena cava, perfusing with perfusion liquid for 6-8min, circularly perfusing with digestion liquid for 10-15min, separating and purifying cells, taking out the liver, tearing liver membranes in the precooling stop digestion liquid at 3-5 ℃, filtering with a screen, collecting filtrate, centrifuging the filtrate for the first time, supplementing a complete culture medium, centrifuging for the second time, collecting cell sediment, washing 2-3 times with the complete culture medium, centrifuging again, reserving cell sediment at the bottom to obtain purified liver parenchymal cells, re-suspending the purified liver parenchymal cells with precooled frozen stock solution to a final concentration of 1-2×10 7 cells/mL, and sub-packaging and freezing. The method has the advantages of stable operation, high cell yield, good activity and high recovery rate after freezing.

Inventors

  • CUI YINGYING
  • ZHANG XIAOYUN
  • ZHAO RUIYING

Assignees

  • 博康生物工程(山东)有限公司

Dates

Publication Date
20260512
Application Date
20260224

Claims (10)

  1. 1. The method for extracting and freezing the liver cells of the mice is characterized by comprising the following steps: S1, liver perfusion and digestion, namely, catheterizing a mouse cadaver through a lower vena cava, cutting off a portal vein, clamping an upper vena cava, firstly perfusing with perfusion fluid for 6-8min, and then circularly perfusing with digestive fluid for 10-15min; S2, separating and purifying cells, namely taking out the liver after circulatory perfusion, tearing liver membranes in a precooling stop digestive juice at 3-5 ℃, filtering by a screen, collecting filtrate, centrifuging the filtrate for the first time, supplementing a complete culture medium, centrifuging for the second time, collecting cell precipitates, washing 2-3 times by using the complete culture medium, centrifuging again, and reserving cell precipitates at the bottom to obtain purified hepatic parenchymal cells; s3, re-suspending the purified hepatic parenchymal cells to a final concentration of 1-2 multiplied by 10 7 cells/mL by using pre-cooled freezing solution, and sub-packaging and freezing.
  2. 2. The method for extracting and cryopreserving liver cells of mice according to claim 1, wherein the perfusion fluid is 1 xKrebs-Ringer buffer, the temperature of the perfusion fluid is 36-38 ℃, and the flow rate of the perfusion fluid is 5-6ml/min.
  3. 3. The method for extracting and cryopreserving liver cells of mice according to claim 1, wherein the digestion solution consists of 100CDU/ml type IV collagenase and 1 x Krebs-Ringer buffer, the temperature of the digestion solution is 36-38 ℃, and the flow rate is 7-8ml/min.
  4. 4. The method of claim 1, wherein the stop digestive juice is Williams' E solution containing 1.5-2.5% fetal bovine serum and 4-6mM CaCl 2 .
  5. 5. The method for extracting and cryopreserving liver cells of mice according to claim 1, wherein the primary centrifugation is performed at a centrifugal force of 45-55 Xg for 3-5min at a centrifugal temperature of 3-5 ℃.
  6. 6. The method for extracting and cryopreserving liver cells of mice according to claim 1, wherein the complete medium is DMEM medium containing 8-12% fetal bovine serum.
  7. 7. The method for extracting and cryopreserving liver cells of mice according to claim 1, wherein the secondary centrifugation is carried out under the conditions that 35-40% of Percoll gradient liquid is prepared in a centrifuge tube in advance, cell suspension is added to the upper layer of the gradient liquid, the volume ratio of the gradient liquid to the cell suspension is 2-3:1, the centrifugation temperature is 3-5 ℃, the centrifugal force is 750-850 Xg, and the centrifugation time is 10-15min.
  8. 8. The method for extracting and cryopreserving liver cells of mice according to claim 1, wherein the re-centrifugation is performed at a centrifugal force of 45-55 Xg for 3-5min.
  9. 9. The method for extracting and cryopreserving liver cells of mice according to claim 1, wherein the cryopreservation solution comprises 40-50 parts by mass of Williams' E, 30-40 parts by mass of fetal bovine serum, 4-6 parts by mass of DMSO, 4-6 parts by mass of trehalose and Trolox with a final concentration of 45-55 mu M.
  10. 10. The method for extracting and freezing the liver cells of the mice according to claim 1, wherein the freezing method is that the liver cells are preserved for 30-40min at 4 ℃, are preserved for 30-40min at 1-2 ℃ to-20 ℃ and are preserved for 8-9h at 1-2 ℃ to-80 ℃ and are then preserved in liquid nitrogen.

Description

Method for extracting and freezing mouse liver cells Technical Field The application relates to the technical field of biology, in particular to a method for extracting and freezing mouse liver cells. Background The liver is the most important detoxification organ in the organism, the metabolism, synthesis, secretion and storage capacity of liver cells are directly related to the health degree of the organism, primary liver cells are ideal tools for researching liver diseases, wherein mouse liver parenchymal cells are important tools in biomedical research, and the liver cells are widely applied to the fields of drug metabolism and toxicity tests, liver physiology pathology research, virology research, cell transplantation treatment and the like and have higher application value in scientific research. However, in vitro application of primary hepatocytes is always limited by two main technical bottlenecks, namely that cells are easy to damage in the separation process, the efficiency of liver cells obtained by a common collagenase digestion method and a mechanical separation method is influenced by various factors, such as perfusion pressure and flow rate, digestion time, collagenase concentration and activity, so that the activity and function of cells are low, the difference of cell mass among different batches is large, and the primary cells are difficult to culture and preserve for a long time, and the conventional freezing method can cause serious ice crystal damage and osmotic stress, so that the cell activity and function after resuscitation cannot be ensured. Therefore, the method for obtaining the appropriate liver parenchymal cell model is very important for researching pathogenesis of liver diseases, pharmacology of medicines, molecular genetics and the like. The preparation method of the mouse liver parenchyma cells, which is standardized, has strong repeatability, can obtain high activity and high purity, is suitable for long-term freezing and has important practical application value. Disclosure of Invention Aiming at the problems, the application provides a method for extracting and freezing mouse liver cells, which has the advantages of stable operation, high cell obtaining rate, good activity and high recovery rate after freezing. In order to achieve the above purpose, the application provides a method for extracting and cryopreserving mouse liver cells, which comprises the following steps: S1, liver perfusion and digestion, namely, catheterizing a mouse cadaver through a lower vena cava, cutting off a portal vein, clamping an upper vena cava, firstly perfusing with perfusion fluid for 6-8min, and then circularly perfusing with digestive fluid for 10-15min; S2, separating and purifying cells, namely taking out the liver after circulatory perfusion, tearing liver membranes in a precooling stop digestive juice at 3-5 ℃, filtering by a screen, collecting filtrate, centrifuging the filtrate for the first time, supplementing a complete culture medium, centrifuging for the second time, collecting cell precipitates, washing 2-3 times by using the complete culture medium, centrifuging again, and reserving cell precipitates at the bottom to obtain purified hepatic parenchymal cells; s3, re-suspending the purified hepatic parenchymal cells to a final concentration of 1-2 multiplied by 10 7 cells/mL by using pre-cooled freezing solution, and sub-packaging and freezing. Further, the perfusion liquid is 1 XKrebs-Ringer buffer, the temperature of the perfusion liquid is 36-38 ℃, and the flow rate of the perfusion liquid is 5-6ml/min. Further, the digestion solution consists of 100CDU/ml type IV collagenase and 1 XKrebs-Ringer buffer, the temperature of the digestion solution is 36-38 ℃, and the flow rate is 7-8ml/min. Further, the stop digestion solution is a Williams' E solution containing 1.5-2.5% fetal bovine serum and 4-6mM CaCl 2. Further, the mesh has a pore size of 70 μm or 100. Mu.m. Further, the primary centrifugation is carried out at a centrifugal force of 45-55 Xg for 3-5min and at a centrifugal temperature of 3-5 ℃. Further, the complete medium is DMEM medium containing 8-12% fetal calf serum. Further, the secondary centrifugation is carried out under the condition that 35-40% of Percoll gradient liquid is prepared in a centrifuge tube in advance, the cell suspension is added to the upper layer of the gradient liquid, the volume ratio of the gradient liquid to the cell suspension is 2-3:1, the centrifugation temperature is 3-5 ℃, the centrifugal force is 750-850 Xg, and the centrifugation time is 10-15min. Further, the re-centrifugation is carried out at a centrifugal force of 45-55 Xg for 3-5min. Further, the frozen stock solution comprises the following materials, by mass, 40-50 parts of Williams' E, 30-40 parts of fetal bovine serum, 4-6 parts of DMSO, 4-6 parts of trehalose and Trolox with a final concentration of 45-55 mu M. Further, the freezing preservation method comprises the steps