A method for isolating fibroblasts derived from human induced pluripotent stem cells and its application
A technology for pluripotent stem cells and fibroblasts, which is applied in the field of fibroblasts derived from human induced pluripotent stem cells, can solve the problems of restricting industrialization and marketization, low differentiation efficiency, and complex differentiation process. The effect of long induction time, short separation time and high separation efficiency
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Embodiment 1
[0119] Example 1 Isolation of fibroblasts derived from iPSCs
[0120] 1. Experimental materials
[0121] (1) the main reagent information used in the embodiment of the present invention is shown in Table 1;
[0122] Table 1 Main reagent information used in the examples of the present invention
[0123]
[0124] (2) Preparation and storage of the reagents in the examples of the present invention
[0125] High-glucose DMEM with 10% FBS: in a 50-mL centrifuge tube, pipette 40 mL of high-glucose DMEM, add 10 mL of FBS, mix gently, and store at 4°C for a period of 2 weeks.
[0126] 2. Preparation of iPSCs with Spontaneously Differentiating Cells
[0127] The iPSC cell line used in this example (Beijing Chengnuo Medical Technology Co., Ltd.) is the iPSC derived from PBMC reprogramming. The iPSC cell line has a certain proportion of spontaneously differentiated cells when the line is established, and is located around the iPSC colony. The specific experimental method for prepa...
Embodiment 2
[0172] Example 2 Immunofluorescence detection of iPSC-derived fibroblasts
[0173] 1. Reverse transcription PCR
[0174] The purified fibroblast-like cell total RNA obtained in the above Example 1 was extracted by Trizol method, and 1 μg of total RNA was taken for reverse transcription PCR detection;
[0175] (1) Collect cells 200W cells and add 1 mL of TRIZOL to extract RNA and determine the RNA concentration. Take 1 μg of RNA and reverse it to cDNA. The reverse transcription kit is II Reverse Transcriptase (full gold, AH101-02), reverse transcription according to the manufacturer's instructions;
[0176] (2) Use PCR SuperMix (full gold, AS111-11) carries out PCR, and carries out premixing according to the reaction system as described in Table 2;
[0177] Table 2 Reaction system
[0178]
[0179] (3) then put the above-mentioned system into the PCR machine and react according to the 3-step method, the number of cycles is 45, and the reaction program is as shown in Ta...
Embodiment 3
[0202] Example 3 Flow cytometry detection of iPSC-derived fibroblasts
[0203] 1. Experimental method
[0205] Remove the purified fibroblast-like cell culture solution obtained in Example 1 with a pipette, add calcium and magnesium-free PBS, gently shake the culture bottle, discard the calcium and magnesium-free PBS, and repeat the above process once; then add 4-fold dilution Accutase, placed at 37°C, and observed cell digestion under a microscope; when most of the cells detached from the wall, tap the culture dish to collect the cell suspension into a 50mL centrifuge tube;
[0206] (2) Preparation of single cell suspension
[0207] Filter the cell suspension in the above 50mL centrifuge tube into a new 50mL centrifuge tube with a 30μm sieve, collect the cells at 300g × 5min, and discard the supernatant;
[0208] (3) Cell fixation
[0209] The collected cells were resuspended with 10 mL of calcium and magnesium-free PBS, and 50 μL were sampled ...
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