Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration

Abstract

We describe a simple protocol for determining the oxygen consumption of cells in static culture. The protocol is based on a noninvasive oxygen-sensing microplate and a simple mathematical model derived from Fick's Law. The applicability of the model is confirmed by showing the correlation of computed oxygen consumption rate (OCR) values to actual cell densities ascertained by direct cell counting and/or MTT for HL60 and U937 cells cultured in suspension. Correlation between computed OCR and these other indications of cell number was quite good, as long as the cultures were not diffusion-limited for oxygen. The impact of the geometric factors of media depth and well size were confirmed to be consistent with the model. Based on this demonstrated correlation, we also developed a simple, completely noninvasive algorithm for ascertaining the per-cell oxygen utilization rate (OUR), which is the ratio of OCR to cell number, and a fundamental cell characteristic. This is accomplished by correlating the known seed densities to extrapolated determinations of OCR at time zero. Such determinations were performed for numerous cell types, in varying well sizes. Resulting OUR values are consistent with literature values acquired by far more painstaking methods, and ranged from <0.01 fmol.min(-1).cell(-1) for bacteria to 0.1-10 fmol.min(-1).cell(-1) for immortalized mammalian and insect cell lines to >10 fmol.min(-1).cell(-1) for primary hepatocytes. This protocol for determining OCR and OUR is extremely simple and broadly applicable and can afford rapid, informative, and noninvasive insight into the state of the culture.

Model‐based analysis and design of a microchannel reactor for tissue engineering

“…We use the published data for reactor geometry and oxygen consumption of hepatocytes (Allen and Bhatia, 2003;Roy et al, 2001) for our calculations. Further, the oxygen uptake is reported to vary up to 2 orders of magnitude for wide variety of cell types (Guarino et al, 2004) and hence we vary the parameter Da to simulate different cell types. Similarly, the uptake of the cellsecreted autocrine factor is varied based on estimation of the range of autocrine ligand secretion rates as reported earlier (Oehrtman et al, 1998).…”

“…Microchannel reactors fabricated from PDMS are also recommended in literature (Leclerc et al, 2003) as The parameters for the cell proliferation model (K 1 , K 2 , K 3 ) were estimated roughly from the data on the doubling times of cells in media of different glucose/serum concentrations as reported by Guarino et al (2004) and were chosen so as to have the maximum growth rate at dimensionless concentration value C ¼ 0.5. they support diffusion of oxygen due to their large gas permeability. We used our model to investigate the significance of gas diffusion as related to the design and operation of the bioreactor.…”

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

“…Importantly, skeletal muscle mitochondria play a specific role in exercise capacity 2 , aging 3 , degenerative disease 4 and Type II diabetes 5 . As an aging society, and Type II Diabetes being the 7 th leading cause of death in the United States 6 , the need for methods that assess mitochondrial function has become increasingly more prevalent in biomedical research 7,8 . Specifically, the measurement of oxygen consumption has exceptional utility in the assessment of mitochondrial function since it represents the coordinated function between mitochondrial and nuclear genomes to express functional components of oxidative phosphorylation 9 .…”

“…Several technologies exist that enable the measurement of oxygen consumption in intact cells and isolated mitochondria [7][8][9][10] . In addition, assays have been developed for multiple cell types and tissues, and in a variety of species that allow for the measurement of mitochondrial pathways and respiratory control 9,11,12 .…”

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

“…In addition, methods are provided to establish the quality of the mitochondrial isolation preparation and the integrity of the mitochondrial membranes. Given that skeletal muscle mitochondria are involved in many pathological conditions, the measurement of O 2 consumption in mechanistically driven studies is becoming more prevalent in biomedical research 9,10 .…”