Separation culture method of primary hepatocyte of jian carp

• Main Authors: DU JINLIANG, SHEN YUJIN, JIA RUI, YIN GUOJUN, CAO LIPING, ZHAO CAIYUAN, LIU YINGJUAN
• Format: Patent
• Language: English
• Published: 21.01.2015
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS THEREOF; CULTURE MEDIA; ENZYMOLOGY; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

The invention discloses a separation culture method of the primary hepatocyte of a jian carp. The separation culture method comprises the following steps: selecting a healthy jian carp without injury, collecting blood from the caudal vein of the jian carp, then sterilizing the surface of a fish body, placing into a super clean bench, and aseptically collecting the liver of the jian carp; rinsing by using a PBS solution which contains double antibodies, adding a trypsin digestion solution, wherein the temperature of digestion treatment is 26-28 DEG C, and the time of the digestion treatment is 15-20 minutes; after digestion is finished, adding to a culture medium A to finish the digestion, filtering the trypsin digestion solution by using a filter screen of 200 meshes, and collecting filter liquor; and respectively centrifugalizing 50 grams of the filter liquor and 30 grams of the filter liquor for 5 minutes, then washing twice by using a culture medium B, removing a supernatant to obtain a precipitation, namely an extracted hepatocyte, preparing a cell suspension, and planking for culture. The experimental method disclosed by the invention is simple and fast and can save the separation time to a great extent. The separation time adopted by an experiment is about 40 minutes, and the obtained hepatocyte has the advantages of good growth condition, small erythrocyte quantity and cell survival rate of about 95%.