Liver precursor like cell line, construction method and application of liver precursor like cell line to field of bioartificial livers
A construction method and cell line technology, applied in the field of biology and bioartificial liver, can solve the problems of increasing the cost of induction and differentiation culture, and cannot guarantee the differentiation of liver, so as to achieve a good effect of liver differentiation
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Embodiment 1
[0063] This embodiment provides the first construction method.
[0064] The culture plates involved in this embodiment are all six-well culture plates with the same specifications, and are pre-plated with Matrigel, and the plated density of Matrigel is 0.87 microliters / square centimeter.
[0065] The cell culture incubator involved in this embodiment has a constant temperature inside of 37° C., saturated humidity, and a carbon dioxide concentration of 5%.
[0066] In the first construction method, except for the process carried out in the cell culture incubator, other operations are carried out in a normal temperature sterile operating bench.
[0067] The first construction method is specifically:
[0068] S11: Human primary hepatocyte culture was mixed with 1×10 4 Inoculate the first culture plate at the seeding density per square centimeter, and after adding 2 milliliters of TEM medium to each culture well of the first culture plate, place the first culture plate in the ce...
Embodiment 2
[0082] This embodiment provides a second construction method, the difference between the second construction method and the first construction method in Example 1 is:
[0083] The plating density of the Matrigel was 1.74 microliters / square centimeter.
[0084] In the step S11, the inoculation density of the human primary hepatocyte culture is 2×10 4 pieces / square centimeter.
[0085] In the step S12, the number of passages of the subculture is 5, and the passage ratio is 1:4.
[0086] In the step S21, the seeding density of the cell culture to be transfected is 1×10 5 pieces / square centimeter.
[0087] In the step S31, the ratio of the number of lentiviruses in the lentivirus suspension to the number of adherent cells is 50:1, and after the infection, the time to continue culturing in the cell culture incubator is 48 hours .
[0088] In the step S41, TEM medium and hygromycin are added to the fifth culture plate, the time of the expansion culture is 2 days, the passage ra...
Embodiment 3
[0090] This embodiment provides a third construction method, the difference between the third construction method and the first construction method in Example 1 is:
[0091] The plating density of the matrigel is 1 microliter / square centimeter.
[0092] In the step S11, the seeding density of the human primary hepatocyte culture is 0.5×10 4 per square centimeter, and the time for the proliferation culture is 3 days.
[0093] In the step S12, the number of passages of the subculture is 10, and the passage ratio is 1:6.
[0094] In the step S21, the seeding density of the cell culture to be transfected is 1.5×10 4 pieces / square centimeter.
[0095] In the step S31, the lentiviral suspension is a lentiviral suspension expressing the telomerase reverse transcriptase gene, and the inoculation density of the digestion culture is 1×10 4 cells / cm2, the duration of the infection is 10 hours, the ratio of the number of the lentivirus to the adherent cells is 40:1, and the concentrat...
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