Human synoviocyte primary culture experiment
Human synoviocyte primary culture experiment
Primary culture of human synoviocytes can be applied to: (1) cell preservation; (2) for physiological and morphological studies of the corresponding cells; (3) for research on related diseases.
Operation method
enzymatic digestion
Principle
Human synovium was removed from the organism, treated with trypsin, chelating agent (commonly used EDTA), dispersed into single cells, and placed in α-MEM growth medium for culture to allow the cells to survive, grow, and multiply.
Materials and Instruments
Synovial tissue Move I. Preparation of experimental materials Caveat 1. Use α-MEM or RPMI1640 or DMEM with 10% fetal bovine serum for whole culture. Cannot use calf serum, fetal calf serum with domestic can be;2. Ca2+-free PBS must be used. Common Problems Preparation of experimental materials 1. synovial tissue: surgically excised tissue on the operating table, ask the nurse to put the specimen into low-temperature saline, and at the completion of the surgery, put the specimen into PBS ice-water solution and put it into a sterilized container to bring back to the laboratory. 2. Reagents: 4 mg/ml type I collagenase (α-MEM preparation), culture medium (α-MEM with 10% fetal bovine serum). 3. Instruments: surgical instruments. For more product details, please visit Aladdin Scientific website.
Type I collagenase, fetal bovine serum, saline, PBS.
Ultra-clean table Dissecting shears Filters Centrifuge tubes Counting plates Culture flasks
1. Synovial tissue: The surgically excised tissue will be on the operating table and the nurse will be asked to put the specimen into low-temperature saline, and at the completion of the surgery, the specimen will be put into PBS ice-water solution and put into a sterilized container to bring back to the laboratory.
2. Reagents: 4 mg/ml type I collagenase (α-MEM preparation), culture medium (α-MEM with 10% fetal bovine serum).
3. Instruments: surgical instruments.
II. Methods
1. The excised tissues were asked to be placed by the nurse on the operating table in cold saline (at rest) and at the completion of the procedure the specimens were placed in PBS ice water solution and placed in sterilized containers to be taken back to the laboratory.
2. Place the specimen in a petri dish on an ultra-clean table and wash with α-MEM.
3. Obtain a tissue excision, carefully identify the white smooth synovial tissue at the articular reflex and hyperplasia, the hyperplastic vascular opacities attached to the articular cartilage surfaces are also synovial tissue (can be digested as a separate tube), carefully excise the fatty tissue, wash it twice with α-MEM (to remove erythrocytes), and place it in a small cyanide vial to be sharply chopped up with dissecting scissors.
4. Incubate at 37°C for 120 minutes (shaking and mixing several times during this period) with double the volume of obtained tissue digested with α-MEM containing 4 mg/ml type I collagenase.
5. The first tube of cells was collected after 30 min: the cell suspension was filtered through a 70 um cell strainer, centrifuged at 1500-2000 rpm for 6 min, and the supernatant for collagenase type I was added to the unfiltered tissue and continued to be used for digestion.
6. Cells at the bottom of the centrifuge tubes were washed twice with α-MEM centrifugation, each time with α-MEM resuspension followed by centrifugation at 1500-2000 rpm for 6 min, the last time the presence of cells was observed with a tally plate. Cells were finally suspended in α-MEM containing 10% fetal bovine serum and inoculated into culture flasks for culture.
7. The second tube of cells was collected after 60 clocks: the cell suspension was filtered through a 70 um cell strainer and centrifuged at 1500-2000 rpm for 6 minutes; the cells were washed twice with α-MEM, and each time they were resuspended in α-MEM and then centrifuged at 1500-2000 rpm for 6 minutes, and the last time the cells were observed with a tally plate and counted. Cells were finally suspended in alpha-MEM containing 10% fetal bovine serum and inoculated into culture flasks for culture.
8. A third tube of cell collection was done if necessary; and primary synoviocyte culture was done immediately.
9. Change the fluid every 2-3 days.