Normoxia Influences Cell Growth and Total Mitochondrial Volume in Cell Culture

Abstract

Cancer cell lines are routinely grown in hyperoxic conditions (21% O2) that they would never experience physiologically. The partial pressure of O2 in the kidney cortex is 40-50 mmHg (4-6% O2, Welch et al, 2001). The experiments described here have tested the hypothesis that growing a Human Embryonic Kidney cell line (HEK 293) at more appropriate physiological PO2 will result in differences in the cell growth and also in the mitochondrial content of the cells. HEK 293 cells were cultured in DMEM supplemented with 10% fetal calf serum and penicillin / streptomycin. Cells were seeded at a density of 7 x 106 cells / flask and grown for 96 hours in incubators set at 5% CO2 and 5%, 10% and 21% O2. Cells were then counted using a hemocytometer and final cell density was recorded. Cells were loaded with mitotracker (Invitrogen) and the nucleus stained with Hoechst 33342. Images were acquired using a DeltaVision microscope (Applied Precision). Multiple z- sections were acquired and images were deconvolved. 3D reconstruction and quantification of mitochondrial volume was assessed using the scientific software module Imaris XT (version 7.0, Bitplane, Zurich Switzerland).Results indicate that HEK 293 cells grown at physiological concentrations of O2 (5%) grow slower than those grown in hyperoxic conditions (10% and 21%) and have a larger volume of mitochondria. These results indicate that there are differences between cells grown at physiological PO2 and those grown at inappropriate levels of O2. Therefore, a consideration of the physiological environment that cell lines were originally acquired from should be made before culturing cells in vitro. Thanks to BioSTAR program for support. Welch et al, (2001) Kidney International; 59: 230-37 J. Chika. Morah, Barbara. L. Barr, Huong-Thao. Tran, Julia. E. Paulet and Christopher. N. Wyatt.