Can anyone suggest tips for isolating and culturing kidney tubules from ADULT rat.
Protocols include collagenase and mild segregation.
It works quite good for young rats/embryos, but not so well for adult one: cells(tubules) have poor survivability and adhesion.
I am in the process of tuning methodic - can use some advice.
Thank you.
Isolation and primary culture of human proximal tubule cells.
Vesey DA1, Qi W, Chen X, Pollock CA, Johnson DW.
Abstract
Primary cultures of renal proximal tubule cells (PTC) have been widely used to investigate tubule cell function. They provide a model system where confounding influences of renal haemodynamics, cell heterogeneity, and neural activity are eliminated. Additionally they are likely to more closely resemble PTC in vivo than established kidney cell lines, which are often virally immortalised and are of uncertain origin. This chapter describes a method used in our laboratories to isolate and culture pure populations of human PTC. The cortex is dissected away from the medulla and minced finely. Following collagenase digestion, the cells are passed through a sieve and separated on a Percoll density gradient. An almost pure population of tubule fragments form a band at the base of the gradient. Cultured in a hormonally defined serum-free growth media, they form a tightly packed monolayer that retains the differentiated characteristics of PTC for up to three passages.
http://www.ncbi.nlm.nih.gov/pubmed/19148604
Isolation, propagation and characterization of primary tubule cell culture from human kidney.
Qi W1, Johnson DW, Vesey DA, Pollock CA, Chen X.
Abstract
Proximal tubule cells (PTC) are the major cell type in the cortical tubulointerstitium. Because PTC play a central role in tubulointerstitial pathophysiology, it is essential to prepare pure PTC from kidney tissue to explore the mechanisms of tubulointerstitial pathology. The authors have successfully refined and characterized primary cultures of human PTC using Percoll density gradient centrifugation as a key PTC enrichment step. The cells obtained by this method retain morphological and functional properties of PTC and are minimally contaminated by other renal cells. In particular, the primary isolates have characteristics of epithelial cells with uniform polarized morphology, tight junction and well-formed apical microvilli. Cytokeratin is uniformly and strongly expressed in the isolates. Brush border enzyme activities and PTC transport properties are retained in the isolates. This method therefore provides an excellent in vitro model for the physiologic study of the human proximal tubule.
http://www.ncbi.nlm.nih.gov/pubmed/17371339
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