Gstraunthaler 1999 Cell Physiol Biochem

Gstraunthaler G, Seppi T, Pfaller W (1999) Impact of culture conditions, culture media volumes, and glucose content on metabolic properties of renal epithelial cell cultures. Are renal cells in tissue culture hypoxic? https://doi.org/10.1159/000016312

» Cell Physiol Biochem 9:150-72 PMID: 10494029 Open Access

Gstraunthaler G, Seppi T, Pfaller W (1999) Cell Physiol Biochem

Abstract: When renal proximal tubular cells are brought into tissue culture, they revert from oxidative metabolism and gluconeogenesis to high rates of glycolysis. Among the factors possibly responsible for this metabolic conversion, limited oxygen availability and/or substrate supply are discussed. In order to study the role of these factors on long-term cultures, the impact of growth conditions, culture media volume, and glucose content on carbohydrate metabolism of the continuous renal cell lines LLC-PK(1) (porcine kidney) and OK (opossum kidney) was investigated. The impact of culture media volumes and glucose content, respectively, was determined by overlaying confluent monolayer cultures of LLC-PK(1) and OK cells (i) with increasing volumes of culture medium and thus increasing amounts of glucose, and (ii) with increasing culture medium volumes at constant absolute amounts of glucose by adding glucose-free medium, in order to increase volume at a constant glucose supply. Alternatively, and in order to improve cell oxygenation, LLC-PK(1) cells were also cultured in roller bottles. Cell carbohydrate metabolism was assessed by measuring rates of glucose consumption and lactate production, respectively, and by determination of specific activities of the key glycolytic enzymes hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH). Mitochondrial phosphate-dependent glutaminase (PDG) was assayed as marker enzyme of oxidative metabolism of glutamine. In LLC-PK(1) and OK cells, rates of glucose consumption were independent of the initial glucose concentrations and/or the culture media volumes used. Glucose was quantitatively converted to lactate, which accumulated in a 1:2 molar ratio. Lactate in culture media reached a maximum content after 24 h, and was reutilized by the cell lines thereafter. Interestingly, the rates of lactate reuptake strictly depended on culture medium volume, indicating a volume-induced stimulation of oxidative lactate metabolism. Marked changes were found for the specific activities of glycolytic enzymes. In LLC-PK(1) cells, increased glucose supply caused increases in HK, PFK, PK and LDH activities, which were superimposed to the stimulatory effects of increased media volumes. Enzyme activity showed a biphasic response, indicating that both glucose supply and culture media volumes covering the cell monolayer are factors determining glycolytic rates of LLC-PK(1) renal cells. Conversely, in OK cells glycolytic enzyme activities decreased with increasing culture media volumes at constant glucose levels. As expected, under conditions of enhanced oxygenation of LLC-PK(1) cells in roller bottle culture, glycolytic enzyme activities decreased, whereas PDG activity increased, which was paralleled by increased rates of ammonia generation. Thus, changes in nutrient supply and oxygenation of renal epithelial cell cultures by altered culture media volumes dramatically influence metabolic rates and levels of enzyme activities, respectively.

• Bioblast editor: Gnaiger E

Selected quotes

The oxygen concentration in culture media and thus the oxygen supply of cultured cells are essential elements in in vitro tissue culture. .. Values of p_O2 measured in tissue range from 30 to <5 mm Hg [86–88]. Thus, in growth media equilibrated with water-saturated air/CO₂ incubator atmosphere, p_O2 levels are well within physiologic or normoxic blood gas values seen in vivo.

In the present study, LLC-PK 1 and OK epithelial cultures were maintained in 10-cm culture dishes at a constant growth area of 55 cm² with 7, 14, and 21 ml of culture medium. Thus, based on the above described assumptions [42, 90], OTR might be decreased with increasing volume.

It has been calculated that in conventional culture techniques using medium depths of 1.5–5.0 mm, monolayers of normal liver cells are severely hypoxic. Thus, in order to attain a physiologically high O₂ tension (140 mm Hg) in stationary monolayers of liver cells incubated in an air-CO₂ atmosphere, there must be the fluid overlay not greater than 0.34 mm in thickness [92], a situation which can only be met by the culture medium film in roller cultures.

It was demonstrated that epithelial cell proliferation requires a p_O2 140 mm Hg. Below this threshold, glycolysis continues with maximal rates of lactate accumulation at hypoxic culture conditions, a situation at which oxidative lactate metabolism may cease (see fig. 5).

Thus, one can speculate that in static monolayer cultures maintained in tissue culture dishes at ambient p_O2 of 140 mm Hg, a disbalance between culture medium oxygenation by surface diffusion and the rate of O₂ consumption of cultured cells may exist [43], leading to temporary hypoxia [91, 92].

Based on the results of the present study we therefore recommend for routine monolayer cultures of epithelial cells the use of physiological glucose concentrations in culture media (i.e. 5.5 mM), and the lowest possible culture media volumes of about 0.2 mL/cm² [41].

Labels:

Stress:Hypoxia 

Tissue;cell: Kidney, Endothelial;epithelial;mesothelial cell  Preparation: Intact cells  Enzyme: Marker enzyme, TCA cycle and matrix dehydrogenases  Regulation: Aerobic glycolysis 

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