METHOD FOR PRODUCING AND CULTURING FIBROBLAST-LIKE CELLS FROM MILK TOOTH PULP Russian patent published in 2020 - IPC C12N5/00 

FIELD: biotechnology.

SUBSTANCE: invention represents a method for obtaining and culturing fibroblast-like cells from milk tooth pulp, involving sampling, preparation, enzymatic treatment of material, separation of cell suspension during centrifugation, where in case of appearing of milk tooth loss in child its parents are given a sterile penicillin bottle with Hank’s solution with antibiotics, in which the dropped tooth is placed; after bottle delivery to laboratory, tooth is removed with sterile instrument into Petri dish and washed three times with sterile Hank’s solution with antibiotics; the tooth is transferred into a new test tube with sterile Hank’s solution with antibiotics and placed in a medical refrigerator for 24 hours; in solution left in transportation flask one adds three drops of cow's fruit serum and placed in thermostat, incubating at 37° for 24 hours, after which sampling sterility is monitored; if there are no signs of material contamination, 0.2 ml of 0.1 % of collagenase from crab pancreas LLC "BioloT" is injected into the insulin syringe; syringe needle is inserted into the milk tooth canal and the canal content is aspirated into the syringe; transferring the contents into a centrifugation test tube and incubating for two minutes at room temperature; enzyme action is inactivated by adding 2 ml of sterile 0.02 % Versene solution into the test tube and 1 ml of 10 % complete growth medium α-MEM with glutamine; centrifuging the tube in a cold centrifuge at 4 °C, rotation speed 1200 rpm for 5 minutes; supernatant is utilized; cell pellet is re-suspended in a complete culture medium α-MEM with glutamine, including 10 % FBS, 8 mcg/ml gentamycin; calculating the number of recovered viable cells in the Goryaev chamber using 0.4 % trypan blue; cells are inoculated in dose of 1×105 viable cells/cm² in culture flask with complete medium α-MEM with glutamine, including 10 % FBS, 8 mcg/ml gentamycin; culturing is carried out in CO₂-incubator at temperature 37 °C, 5 % CO₂ and constant moisture content; every second day, another similar nutrient medium is added into 0.2 ml flask; first change of medium is carried out on 7th day; conditioned culture medium from a vial with cells is selected by a pipette, centrifuged in a cold centrifuge at temperature 4 °C, rotational speed 1500 rpm for 10 minutes, then returned to culture flask and fresh medium is added in ratio of 1:1; when the confluent monolayer is 80 % complete, the cells are reseeded at ratio of 1:2; 1 PF.

EFFECT: invention enables to obtain and cultivate fibroblast-like cells from the pulp of milk teeth.

1 cl, 1 tbl, 1 dwg