Development of a Primary Culture System of Rat Kidney Proximal Tubule Cells for Transport Studies

ABSTRACT

Primary culture of proximal tubule cells have provided useful model for the study of proximal tubule cell function, especially transport studies. Kidney is excised from the anesthetized rat, cortex is separated and proximal tubule cells are isolated by using DNAse-collagenase and separated by using different mesh size sieves and collagen coated surfaces of flasks containing 15% Dulbecco’s Modified Eagle’s medium (DME) to exclusively avoid other cells or cell debris. The cells are then seeded onto rat-tail collagen- coated flasks containing 15 % DME. After one day, the above medium is changed and the cells are suspended with new 15% DME medium. After two days, the above medium is replaced with routine serum free culture medium containing growth factors, antibiotics and cholera toxin in different proportions. The medium is replaced once in 2 days. The cells are confluent in 6-8 days. The cells are then sub cultured in rat-tail collagen coated 24 well plates. These cells are confluent and ready for doing transport experiments after 2 or 3 days. Our histochemical methods reveal that the cells are positive for gamma glutamyl transpeptidase and glucose-6-phosphatase and hence they are of proximal tubule origin. Fluoroskan methods show cellular absorption of fluorescein methotrexate is concentration dependent and hence these cells exhibit original transport properties of the intact proximal tubule. Hence, the above methods are well suited to grow good quality proximal tubule cells and to assess its property for transport studies.

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