Primary culture, cellular stress and differentiated function

Wolffe, Alan P.; Tata, Jamshed R.

doi:10.1016/0014-5793(84)80902-3

Cited by 41 publications

(12 citation statements)

References 110 publications

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“…These data are in line with previous observations, showing that cell treatment with trypsin/ EDTA causes an increase in the permeability of nuclei, causing a release of nuclear factors in the cytoplasm. 6 Taken together our data indicate that our method, unlike trypsin treatment, leaves membrane proteins and nuclear organization intact.…”

Section: Fly 243supporting

confidence: 55%

“…5 Although, the enzymatic dissociation operated by trypsin is very effective and works over a wide range of biochemical conditions, it causes a lot of stress to live cells. 6 It is well documented that trypsin/EDTA treatment of tissues can remove membrane-associated protein markers, and causes an increase in the permeability of the plasma membrane with cellular proteins, RNA, and nuclear DNA being released, 6 often compromising the use of dissociated cells for immuno-fluorescence, cell sorting and other molecular analyses.…”

Section: Introductionmentioning

confidence: 99%

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Flow Cytometry and Karyotype Analysis ofD. melanogasterEye Disc Cells

Collesano

Corona²

2007

Fly

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Section: Fly 243supporting

confidence: 55%

Section: Introductionmentioning

confidence: 99%

Flow Cytometry and Karyotype Analysis ofD. melanogasterEye Disc Cells

Collesano

Corona²

2007

Fly

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“…It is a consistent observation that renal proximal tubular epithelial cells, when brought into primary tissue culture, revert from oxidative metabolism and gluconeogenesis to high rates of glycolysis [15-24, 31, 55]. These phenotypic changes, also termed 'culture shock' [56], show close similarities with effects of other stresses such as heat shock [57][58][59] or hypoxia [14,60,61], which point to common biochemical mechanism(s) being involved.…”

Section: General Aspectsmentioning

confidence: 86%

Impact of Culture Conditions, Culture Media Volumes, and Glucose Content on Metabolic Properties of Renal Epithelial Cell Cultures

Gstraunthaler¹,

Seppi²,

Pfaller³

1999

Cell Physiol Biochem

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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-PK₁ (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-PK₁ 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-PK₁ 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-PK₁ 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-PK₁ 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-PK₁ 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-PK₁ 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 o...

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“…The derivation of other primary cells can be challenging as isolation protocol development can be arduous. A further limitation of differentiated primary cells is that extended culture leads to a loss of phenotype or cell death [Wolffe and Tata, 1984;Gudjonsson et al, 2004]. This temporal constraint can be devastating to tissue engineering applications, especially for autologous strategies in humans, as it is often infeasible to obtain and expand sufficient quantities of cells for construction implantable products within a small number of population doublings [Vondran et al, 2008;Adam Young et al, 2010].…”

Section: Differentiated Primary Cellsmentioning

confidence: 99%

Methods of Cell Purification: A Critical Juncture for Laboratory Research and Translational Science

Amos¹,

Çağavı²,

Qyang³

2011

Cells Tissues Organs

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Research in cell biology and the development of translational technologies are driven by competition, public expectations, and regulatory oversight, putting these fields at a critical juncture. Success in these fields is quickly becoming dependent on the ability of researchers to identify and isolate specific cell populations from heterogeneous mixtures accurately and efficiently. Many methods for cell purification have been developed, and each has advantages and disadvantages that must be considered in light of the intended application. Current cell separation strategies make use of surface proteins, genetic expression, and physics to isolate specific cells by phenotypic traits. Cell purification is also dependent on the cellular reagents available for use and the intended application, as these factors may preclude certain mechanisms used in the processes of labeling and sorting cells.

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Primary culture, cellular stress and differentiated function

Cited by 41 publications

References 110 publications

Flow Cytometry and Karyotype Analysis ofD. melanogasterEye Disc Cells

Flow Cytometry and Karyotype Analysis ofD. melanogasterEye Disc Cells

Impact of Culture Conditions, Culture Media Volumes, and Glucose Content on Metabolic Properties of Renal Epithelial Cell Cultures

Methods of Cell Purification: A Critical Juncture for Laboratory Research and Translational Science

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