Gerhard Gstraunthaler

Optimization of chemically defined cell culture media - Replacing fetal bovine serum in mammalian in vitro methods

Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manufacturing practice (GMP) are now established standards. The biomedical field aims at an increasing reliance on the use of in vitro methods. Cell and tissue culture methods are generally fast, cheap, reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an attempt to develop a common standard for in vitro methods. The implementation of the use of chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement with an undefined and variable composition. Defined media supplements are commercially available for some cell types. However, information on the formulation by the companies is often limited and such supplements can therefore not be regarded as completely defined. The development of defined media is difficult and often takes place in isolation. A workshop was organised in 2009 in Copenhagen to discuss strategies to improve the development and use of serum-free defined media. In this report, the results from the meeting are discussed and the formulation of a basic serum-free medium is suggested. Furthermore, recommendations are provided to improve information exchange on newly developed serum-free media.

Glutathione depletion and in vitro lipid peroxidation in mercury or maleate induced acute renal failure

Nephrotoxic acute renal failure was experimentally induced in male rats by s.c. application of mercuric chloride and i.p. administration of maleate, respectively. Mercuric chloride and maleate are known to enhance the formation of free radicals and peroxides, which presumably overload the cells natural elimination mechanisms for these highly reactive intermediates. In addition, a reduction in activities of superoxide dismutase, catalase and glutathione-peroxidase, enzymes responsible for the protection of cells against peroxidative action of superoxide anions and hyperperoxides was found. In both models of acute renal failure, enhanced lipid peroxidation in kidney homogenates in vitro, monitored as malondialdehyde production, was observed. Furthermore, HgCl2 and maleate may react with free SH-groups and thus lead to a depletion of glutathione in tubular cells. Indeed, renal cortical contents of reduced and oxidized glutathione were drastically diminished. These results suggest that alterations in membrane integrity, possibly caused by peroxidative processes, can be considered the cause underlying the well-known disturbances in renal function commonly observed during the initiation phase of HgCl2 and maleate induced acute renal failure.

Epithelial Cells in Tissue Culture

In this review the characteristics of established renal and intestinal epithelial cell lines are described by summarizing the accumulated literature about specific properties retained by the cells in tissue culture. Furthermore, brief examples are given for the use of cultured epithelia as model systems to study epithelial transport and metabolic functions, epithelial cell polarity, and aspects of the differentiation and maturation of epithelia by physiological, biochemical and genetic, or cell and molecular biological approaches.

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

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 of enzyme activities, respectively.

Madin-Darby canine kidney cells

Vectorial transport of salt and water in the Madin-Darby canine kidney (MDCK) cell line is indicated by the formation of domes when a monolayer is grown on an impermeable support. We investigated aldosterone-induced dome formation and evaluated the dome as an experimental model. Transepithelial dome resistance was about 80 Ωcm2 and constant when dome size exceeded 2 · 10−4 cm2. The relative ion conductances (expressed as transference numbers) across the dome epithelium were tNa∶tCl∶tK= 0.64∶0.24∶0.06. They reflect the permeability properties of the paracellular shunt pathway tested at physiological concentrations of the individual ions. Aldosterone accelerated dome formation in serum-deprived MDCK monolayers. Prostaglandin E1 and transferrin were supportive but not essential for aldosterone-induced dome formation. After 72 h dome density was equal in monolayers cultured in serum-supplemented medium either in the presence or absence of mineralocorticoids. We conclude that aldosterone induces cell polarization in MDCK monolayers, leading to the formation of domes. The dome epithelium appears to be electrically isolated from the adjacent monolayer and can be studied by microelectrode techniques.

Delineation of the Key Aspects in the Regulation of Epithelial Monolayer Formation

ABSTRACT The formation, maintenance, and repair of epithelial barriers are of critical importance for whole-body homeostasis. However, the molecular events involved in epithelial tissue maturation are not fully established. To this end, we investigated the molecular processes involved in renal epithelial proximal-tubule monolayer maturation utilizing transcriptomic, metabolomic, and functional parameters. We uncovered profound dynamic alterations in transcriptional regulation, energy metabolism, and nutrient utilization over the maturation process. Proliferating cells exhibited high glycolytic rates and high transcript levels for fatty acid synthesis genes (FASN), whereas matured cells had low glycolytic rates, increased oxidative capacity, and preferentially expressed genes for beta oxidation. There were dynamic alterations in the expression and localization of several adherens (CDH1, -4, and -16) and tight junction (TJP3 and CLDN2 and -10) proteins. Genes involved in differentiated proximal-tubule function, cilium biogenesis (BBS1), and transport (ATP1A1 and ATP1B1) exhibited increased expression during epithelial maturation. Using TransAM transcription factor activity assays, we could demonstrate that p53 and FOXO1 were highly active in matured cells, whereas HIF1A and c-MYC were highly active in proliferating cells. The data presented here will be invaluable in the further delineation of the complex dynamic cellular processes involved in epithelial cell regulation.

Zell- und Gewebekultur

Dieses Lehrbuch iiber die Zellund Gewebekulturtechniken, das nun bereits in der funften Auflage vorliegt, beansprucht zu Recht inzwischen den Ruf eines Klassikers im deutschsprachigen Raum, der mit Sicherheit viel dazu beigetragen hat, dass die Einarbeitung in diese Thematik fur viele Einsteiger wesentlich unkomplizierter wurde als zunachst befurchtet. Die Tendenz aller friiheren Auflagen sowohl in der Zunahrne des Buchurnfanges als auch in der Zunahrne der Anzahl der behandelten Themenbereiche als Folge der immer spezialisierteren Anwendungsbereiche der in vitro Forschung setzt sich fort. Dass dabei die grundlegenden Techniken der Zellkultivierung nahezu unverandert geblieben sind, zeigt sich in den Kapiteln zu den Bereichen raumliche und apparative Voraussetzungen fur ein Zellkulturlabor, fur Kulturgefalse, Steriltechnik, Herstellung von Zellkulturmedien u.a .. Die verschiedenen Methoden und Techniken der Zellund Gewebekultur werden erneut anschaulich und vor allem fur den Praktiker leicht nachvollziehbar beschrieben. Von den Basistechniken wie Sterilisation der Geratschaften bis hin zu komplizierten Farbetechniken und Organpraparationen wird die gesamte Bandbreite der Zellkultivierung abgedeckt. Der Zunahme von immer spezialisierteren und verfeinerten Methoden wie z.B. der Kultivierung von ausdifferenzierten organspezifischen Zellen oder

A plea to reduce or replace fetal bovine serum in cell culture media

To the Editor,Fetal bovine serum (FBS) is a universal growthsupplement of cell and tissue culture media. FBS is anatural cocktail of most of the factors required for cellattachment, growth, and proliferation, effective formost types of human and animal (including insect)cells. Although in use for more than 50 years, FBS hasnever been fully characterized. Recent proteomic andmetabolomic studies revealed approx. 1,800 proteins(Anderson and Anderson 2002; Anderson et al. 2004)and more than 4,000 metabolites (Psychogios et al.2011) present in serum. However, the use of serum incell culture also bears a number of disadvantages.These disadvantages can either be seen from: (a) ascientific, cell biological point of view, since serum ingeneral is an ill-defined mixture of components inculture media, with qualitative and quantitative,geographical and seasonal batch-to-batch variations,(b) from biosafety aspects, since FBS may containadverse factors, like endotoxin, mycoplasma, viralcontaminants or prion proteins, (c) from ethicalperspectives in terms of animal protection argumentsregarding the harvest and collection of FBS frombovine fetuses, and (d) in terms of recent concernsabout the global supply versus demand of FBS. As aconsequence, a number of strategies were developedto reduce or replace the requirement for FBS in cellculture media.FBS is a by-product of the beef packing industry.Thus, the supply is dictated by many factors, includingbeef consumption, dairy product consumption, feedprices, environmental factors such as drought, cattleimport and export, governmental farm policies (Sha-iler and Corrin 1999), and the outbreak of diseases(foot and mouth disease, BSE) (Asher 1999; Dormont1999; Even et al. 2006; Wessmann and Levings ).The availability of FBS has changed dramatically overthe past few years (Fujimoto 2002). Therefore, allefforts and attemptsshouldbe undertaken toovercomethe expected shortfall in FBS supply. In the case ofFBS, supply versus demand models do not followtypical economic principles. Normally, supply can beadjusted to meet the demand. However, in case ofFBS, supply and demand operate independently ofeach other. In addition, there is a severe geographicalmismatch between the supply of and the demand forFBS. Demand is highest in US and Europe, while themajor sources of FBS are far away—in Brasil,Argentina, South Africa, Australia, New Zealand,and Central America, since in those countries huge

Fusion of cultured dog kidney (MDCK) cells. I: Technique, fate of plasma membranes and of cell nuclei

SummaryThe evaluation of the intracellular signal train and its regulatory function in controlling transepithelial transport with electrophysiological methods often requires intracellular measurements with microelectrodes. However, multiple impalements in epithelial cells are hampered by the small size of the cells. In an attempt to avoid these problems we fused cells of an established cell line, Madin Darby canine kidney cells, originally derived from dog kidney, to “giant” cells by applying a modified polyethylene glycol method. During trypsin-induced detachment from the ground of the petri dish, individual cells grown in a monolayer incorporate volume and mainly lose basolateral plasma membrane by extrusion. By isovolumetric cell-to-cell fusion, spherical “giant” cells are formed within 2 hr. During this process a major part of the individual cell plasma membranes is internalized. Over three weeks following cell plasma membrane fusion degradation of single cell nuclei and cell nuclear fusion occurs. We conclude that this experimental approach opens the possibility to investigate ion transport of epithelia in culture by somatic cell genetic techniques.

Morphological and Biochemical Changes of LLC-PK1Cells during Adaptation to Glucose-Free Culture Conditions

The established renal epithelial cell line LLC-PK1 retained in tissue culture several differentiated properties of renal proximal tubular cells. By adapting LLC-PK1 cells to glucose-free culture conditions, we recently succeeded in isolating a gluconeogenic strain of LLC-PK1 cells capable of growing in the absence of hexoses. In contrast to the parental wild type, the isolated strain expressed fructose-1,6-bisphosphatase activity and was, therefore, designated LLC-PK1-FBPase+. Besides the differences in glucose metabolism, the isolated gluconeogenic substrain differs form the parental wild type with respect to morphological appearance and the expression of apical membrane marker enzymes. LLC-PK1-FBPase+ cells display a drastic accumulation of autophagic vacuoles, disappearance of apical membrane alkaline phosphatase activity, and increased gamma-glutamyltranspeptidase activity. In order to find out whether or not a low alkaline phosphatase activity in combination with the enhanced formation of autophagic vacuoles is related to a change in apical membrane surface, we utilized a combined light and electron microscopic morphometric procedure to determine the absolute amount of organelle volumes and membrane surface areas. This stereologic approach shows that LLC-PK1-FBPase+ cells display a tenfold increase in the volume of autophagic vacuoles and the lysosomal compartment. Analysis of lysosomal enzyme activities, however, revealed no changes as compared to wild-type cells. The apical membrane surface of gluconeogenic cells was found to be increased by 80%. Karyotype analysis revealed that LLC-PK1 wild-type cells were diploid, whereas FBPase+ cells exhibited polyploidy with a high percentage of tetraploid nuclei. Culturing LLC-PK1-FBPase+ cells in the presence of 5 mM glucose does not abolish the morphological and biochemical changes described, indicating the stability of the FBPase+ strain.