LLC-PK(1) cells maintained in a new perfusion cell culture system exhibit an improved oxidative metabolism

Cultured renal proximal tubule cells dedifferentiate from an oxidative metabolism to high rates of glycolysis over time. There are many reasons why cells in culture dedifferentiate, not least being a lack of homogenous nutrient supply and poor oxygenation. To this end we have developed a new cell culture device (EpiFlow), which combines continuous perfusion of medium with continuous oxygenation of cells grown on microporous supports. LLC-PK(1) cells cultured under EpiFlow conditions were compared with the same cells grown under conventional static conditions. EpiFlow maintained cells exhibited an improved oxidative metabolism as evidenced by 1) a decreased activity of glycolytic enzymes, 2) an increase in the activity of mitochondrial phosphate-dependent-glutaminase, 3) an increase in cellular ATP content, and 4) an improved morphology (increased cell height, mitochondrial density and an increased number and height of microvilli). In addition, LLC-PK(1) cells maintained under perfusion conditions exhibited an increased sensitivity to the respiratory chain blocker antimycin A as assayed by mitochondrial membrane potential (JC-1). We conclude that LLC-PK(1) cells maintained under EpiFlow conditions develop an improved oxidative metabolism that is more comparable to the in vivo situation.