Modified Primary Culture Of Renal Tubular Epithelial Cells In SD Rat

Objective:To establish a good method for primary culture,passage and identification of renal tubule epithelial cells(RTECs) in SD rat.Methods: Kidneys were removed from four weeks old SD rats.Renal cortices were dissected visually in ice-cold dissection solution,sliced into pieces of 1~2 mm wide under sterile condition.The fragments were transferred to 0.25% trypsin solution at 37°C and digested for 20 min,the undigested renal cortices digest 20 min once again. After digestion,the supernatant was sieved through two nylon sieves(pore size 150μm and 75μm).The renal tubule segments remained in the 75-μm sieve and were resus- pended by flushing the sieve in the reverse direction with 0.9% sodium chloride, were centrifuged for 5 min at 800rpm,remove the supernatant,and then resuspended into the appropriate amount of culture medium:1:1 DMEM/F12.The renal tubule segments were purified by Percoll density gradient centrifugation.The purified renal tubule segments were seeded into plastics culture flask with DMEM/F12 medium(add 10%fetal calf serumin) at 37°C and 95%air-5%CO2 in a standard humidified incubator.The purified renal tubule segment were cultured in 24 well-plate.Cell number were tested every- day,and to draw cell growth curve.The expression of CK 18 determined by immunecyto- chemistry in cell smears of RTECs.Results:After inoculate,mass renal tubule segments were distributed under visual field, purity is over the 98%.For 24~48 hours's primary culture,most renal tubule segments were adherenced the flask bottom,a few segments can't adhered but always float in culture medium.After 24h later, a few cellular outgrowth was observed at the open ends of the tubular fragments. The culture medium was changed after 3rd day, then replaced every 2 days.From the 2rd day,most oval cellular outgrowth were observed from renal tubule segments,islands of cellular outgrowth became progre- ssively larger to form a confluent monolayer of(epithelial) polygonal cells. Cells show exponentially grow between 3~7 days.After 5 days later, the cultured cells reached almost complete confluence,display the typical cobblestone layout,show the strong transparency and refraction.The RTECs grow very quickly after passage.After 4~5 days later,the subcultured cells can passaged the second.Cells grow slowly and cannot be further passage after 3 times passage,because the cells may lack the special growth factor and extracellular matrix.The primary culture cells were identified by immunohistochemistry,which results show that:in RTECs dyeset,more than 98% of these cells CK18 expressed positive,with even size,hollow nucleus and dying cyto- plasm,there are unevenly distributed and different intensity brown particles and dots dying around nucleus and in cytoplasm;however,the PBS control groups can not show brown particles in cytoplasm.According to the results of cell morphology and positive result,the purity of primary cultured renal tubular epithelial cells reached 98%.Conclusion:Modified primary culture of renal tubule epithelial cells were established through mechanical isolation,trypsin digestion and nylon sieves filter and combined the Percoll density gradient centrifugation.According to the results of cell morph- ology and immunocytochemistry,98% cell of primary culture and passage are renal tubular epithelial cells.The RTECs were cultured by this modified method,which cells yield rich,good homogeneous growth and repeatable operation.This method provided a good experimental platform for research in vitro on interaction between calcium oxalate crystallization and renal tubular epithelial cell,for treatment drug screening.