Long-term culture of normal human articular chondrocytes
2020-12-08 10:17:45
Long-term culture of normal human articular chondrocytes
Experimental Materials:
1. Source of chondrocytes: 20-24 weeks of spontaneous abortion of the femur and tibia;
2. Washing solution: 1×PBS containing no Ca2+ and Mg2+, adding 100 IU/ml penicillin, 100 μg/ml streptomycin, pH 7.2;
3. Digestion solution I: 2mg/ml trypsin and 2mg/ml collagenase mixed digestive juice, prepared with PBS solution;
4. Digestive juice II: 0.5mg/ml collagenase, prepared with culture solution I;
5. Culture medium I: DMEM medium supplemented with 10% fetal bovine serum;
6. Culture medium II: DMEM medium supplemented with 10% fetal bovine serum, penicillin 1000 IU/ml, streptomycin 1 mg/ml, amphotericin 2.5 μg/ml, glutamine 2 mmol/L, 1% vitamin additive ( Vitamin supplements) and vitamin C 50μg/ml;
7. 10% poly HEMA: Weigh 6g of 2-hydroxyethylmethacrylate (poly HEMA), add it to 50ml of 95% ethanol, and shake it slowly in a constant temperature shaking water bath at 37 °C overnight. The resulting solution was centrifuged (2500 r/min, 10 min) the next day to remove undissolved particles, which was a dry liquid. Mix 1 ml of dry liquid with 9 ml of 95% ethanol and dilute to 10% (v/v) poly HEMA solution;
8. Screen: nylon mesh with a pore size of 100 μm;
experimental method:
1. Under aseptic conditions, the cartilage of the femoral head and the tibial plateau of the spontaneously aborted fetus from 20 to 24 weeks is taken. Wash the blood with PBS. Remove fibrous tissue as much as possible;
2. Place the cartilage in digestive juice I and incubate at 37 ° C for 1 h;
3. Discard the incubation solution. The cartilage block was fully chopped, and the digestive juice II was added and incubated at 37 ° C overnight;
4. Filter with a nylon mesh and add the culture solution I to the filtrate. After centrifugation for 5 min at 250 g, the supernatant was discarded;
5. Blow out the cell pellet with medium I, resuspend the cells, and centrifuge. Repeat 4 times. The resulting cell mass is made into a suspension with a culture solution;
6. Count the cells. An average of approximately 3.0 x 108 chondrocytes are obtained per gram of cartilage;
7. Place 0.9 ml of 10% poly HEMA solution in a plastic dish of 60 mm diameter and place it horizontally on a ventilated clean bench to dry (overnight);
8. The poly HEMA-coated petri dish was irradiated with a sterile UV lamp for 30 min before use;
9. Inoculate a suspension containing 5 x 106 chondrocytes into a Petri dish coated with poly HEMA, and incubate in a CO2 incubator at 37 ° C, 5% CO 2 , and saturated humidity;
10. Change the liquid every 3 - 4d. Cultivation can last for more than half a year;
Experimental Materials:
1. Source of chondrocytes: 20-24 weeks of spontaneous abortion of the femur and tibia;
2. Washing solution: 1×PBS containing no Ca2+ and Mg2+, adding 100 IU/ml penicillin, 100 μg/ml streptomycin, pH 7.2;
3. Digestion solution I: 2mg/ml trypsin and 2mg/ml collagenase mixed digestive juice, prepared with PBS solution;
4. Digestive juice II: 0.5mg/ml collagenase, prepared with culture solution I;
5. Culture medium I: DMEM medium supplemented with 10% fetal bovine serum;
6. Culture medium II: DMEM medium supplemented with 10% fetal bovine serum, penicillin 1000 IU/ml, streptomycin 1 mg/ml, amphotericin 2.5 μg/ml, glutamine 2 mmol/L, 1% vitamin additive ( Vitamin supplements) and vitamin C 50μg/ml;
7. 10% poly HEMA: Weigh 6g of 2-hydroxyethylmethacrylate (poly HEMA), add it to 50ml of 95% ethanol, and shake it slowly in a constant temperature shaking water bath at 37 °C overnight. The resulting solution was centrifuged (2500 r/min, 10 min) the next day to remove undissolved particles, which was a dry liquid. Mix 1 ml of dry liquid with 9 ml of 95% ethanol and dilute to 10% (v/v) poly HEMA solution;
8. Screen: nylon mesh with a pore size of 100 μm;
experimental method:
1. Under aseptic conditions, the cartilage of the femoral head and the tibial plateau of the spontaneously aborted fetus from 20 to 24 weeks is taken. Wash the blood with PBS. Remove fibrous tissue as much as possible;
2. Place the cartilage in digestive juice I and incubate at 37 ° C for 1 h;
3. Discard the incubation solution. The cartilage block was fully chopped, and the digestive juice II was added and incubated at 37 ° C overnight;
4. Filter with a nylon mesh and add the culture solution I to the filtrate. After centrifugation for 5 min at 250 g, the supernatant was discarded;
5. Blow out the cell pellet with medium I, resuspend the cells, and centrifuge. Repeat 4 times. The resulting cell mass is made into a suspension with a culture solution;
6. Count the cells. An average of approximately 3.0 x 108 chondrocytes are obtained per gram of cartilage;
7. Place 0.9 ml of 10% poly HEMA solution in a plastic dish of 60 mm diameter and place it horizontally on a ventilated clean bench to dry (overnight);
8. The poly HEMA-coated petri dish was irradiated with a sterile UV lamp for 30 min before use;
9. Inoculate a suspension containing 5 x 106 chondrocytes into a Petri dish coated with poly HEMA, and incubate in a CO2 incubator at 37 ° C, 5% CO 2 , and saturated humidity;
10. Change the liquid every 3 - 4d. Cultivation can last for more than half a year;
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