Culture solution for hypoxia culture of hepatocytes and preparation method and application thereof
A liver cell and culture medium technology, applied in the field of culture medium for hepatocyte hypoxic culture, can solve problems affecting the success or failure of surgical liver transplantation, achieve good protection, prevent hypoxic damage, and treat liver damage
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0035] An embodiment of the present invention provides a method for preparing a culture medium for hypoxic culture of hepatocytes, comprising:
[0036] S1, preparing the mesenchymal stem cell conditioned medium;
[0037] The mesenchymal stem cells of any generation in the 2-10 generation are cultured with a special medium for stem cells. The stem cell-specific medium used at this time is a conventional stem cell-specific medium, and the culture conditions at this time are conventionally known conventional culture conditions.
[0038] Wherein, mesenchymal stem cells include umbilical cord mesenchymal stem cells.
[0039] After culturing until the confluence of mesenchymal stem cells reaches 75-85%, the special medium for stem cells is replaced with a medium for hepatocytes, and then hypoxic culture is carried out. The hepatocyte culture medium used here is a conventional hepatocyte culture medium, such as DMEM.
[0040] Further, the conditions for hypoxic culture include tha...
Embodiment 1
[0058] 1. Isolation and culture of mesenchymal stem cells
[0059] Umbilical cord mesenchymal stem cells were isolated by tissue block direct attachment method: the blood in the umbilical cord was rinsed with PBS several times under a sterile operating table, and the umbilical cord was cut into 1-2 cm long fragments. The vessels (one vein and two arteries) and the outer amniotic membrane of the umbilical cord were dissected with tissue scissors and vascular forceps to prevent contamination by endothelial cells. Cut the Huatong jelly in the umbilical cord into small pieces with a volume of 0.5-1cm 3 , spread the cut small pieces of umbilical cord evenly on the bottom of a 10cm petri dish, preferably with an interval of 0.5cm. Cover the umbilical cord with 10ml of special medium for mesenchymal stem cells containing 0.1% penicillin and streptomycin double antibody and 10% FBS, and place the culture dish in 5% CO 2 , Cultured in a 37°C incubator. Observe whether there are cell...
experiment example 1
[0075] Cell CCK-8 detection
[0076] Cell viability was detected by CCK-8 kit. Changes in cell activity can reflect the damage of liver cells in hypoxic environment.
[0077] Method: HepaRG cells and L-O2 cells were seeded in a 96-well plate at a density of 5000 cells / well, added 100 μL of complete medium (DMEM+10% FBS) and cultured in a normoxic incubator (20%) for 24 hours , discard the complete medium, and wash twice with PBS.
[0078] Divide the cells into the following groups: normal oxygen culture control group ( Figure 4 and Figure 5 The first column from left to right), cells cultured in hypoxic control group ( Figure 4 and Figure 5 The second column from left to right), cell hypoxic culture + MSC-CM experimental group ( Figure 4 and Figure 5 The third column from left to right), the experimental group of hypoxic culture of cells + hypoxic culture medium of hepatocytes ( Figure 4 and Figure 5 fourth bar from left to right). Wherein, the normoxia cultu...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More