Guidance document on Good Cell and Tissue Culture Practice 2.0 (GCCP 2.0).

Good Cell and Tissue Culture Practice (GCCP) 2.0 is an updated guidance document from GCCP 1.0 (published by ECVAM in 2005), which was developed for practical use in the laboratory to assure the reproducibility of in vitro (cell-based) work. The update in the guidance was essential as cell models have advanced dramatically to more complex culture systems and need more comprehensive quality management to ensure reproducibility and high-quality scientific data. This document describes six main principles to consider when performing cell culture including characterization and maintenance of essential characteristics, quality management, documentation and reporting, safety, education and training, and ethics. The document does not intend to impose detailed procedures but to describe potential quality issues. It is foreseen that the document will require further updates as the science and technologies evolve over time.

Generation and characterization of iPSC-derived renal proximal tubule-like cells with extended stability.

The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.

A Protocol for One-Step Differentiation of Human Induced Pluripotent Stem Cells into Mature Podocytes.

Within the glomerulus, podocytes are highly specialized visceral epithelial cells that are part of the glomerular filtration barrier. Human podocyte cell culture is rather challenging for primary or immortalized cells, due to the nonproliferative state of the cells. In addition, rapid dedifferentiation is often observed. Hence, iPSC-derived podocytes offer an exciting alternative to culture podocyte-like cells from different donors over prolonged time. Here we report a simple and rapid one-step protocol that drives iPSC into podocyte-like cells in 10 days.

Major changes of cell function and toxicant sensitivity in cultured cells undergoing mild, quasi-natural genetic drift.

Genomic drift affects the functional properties of cell lines, and the reproducibility of data from in vitro studies. While chromosomal aberrations and mutations in single pivotal genes are well explored, little is known about effects of minor, possibly pleiotropic, genome changes. We addressed this question for the human dopaminergic neuronal precursor cell line LUHMES by comparing two subpopulations (SP) maintained either at the American-Type-Culture-Collection (ATCC) or by the original provider (UKN). Drastic differences in susceptibility towards the specific dopaminergic toxicant 1-methyl-4-phenylpyridinium (MPP+) were observed. Whole-genome sequencing was performed to identify underlying genetic differences. While both SP had normal chromosome structures, they displayed about 70 differences on the level of amino acid changing events. Some of these differences were confirmed biochemically, but none offered a direct explanation for the altered toxicant sensitivity pattern. As second approach, markers known to be relevant for the intended use of the cells were specifically tested. The "ATCC" cells rapidly down-regulated the dopamine-transporter and tyrosine-hydroxylase after differentiation, while "UKN" cells maintained functional levels. As the respective genes were not altered themselves, we conclude that polygenic complex upstream changes can have drastic effects on biochemical features and toxicological responses of relatively similar SP of cells.

Differentiation of human iPSCs into functional podocytes.

Podocytes play a critical role in glomerular barrier function, both in health and disease. However, in vivo terminally differentiated podocytes are difficult to be maintained in in vitro culture. Induced pluripotent stem cells (iPSCs) offer the unique possibility for directed differentiation into mature podocytes. The current differentiation protocol to generate iPSC-derived podocyte-like cells provides a robust and reproducible method to obtain podocyte-like cells after 10 days that can be employed in in vitro research and biomedical engineering. Previous published protocols were improved by testing varying differentiation media, growth factors, seeding densities, and time course conditions. Modifications were made to optimize and simplify the one-step differentiation procedure. In contrast to earlier protocols, adherent cells for differentiation were used, the use of fetal bovine serum (FBS) was reduced to a minimum, and thus ß-mercaptoethanol could be omitted. The plating densities of iPSC stocks as well as the seeding densities for differentiation cultures turned out to be a crucial parameter for differentiation results. Conditionally immortalized human podocytes served as reference controls. iPSC-derived podocyte-like cells showed a typical podocyte-specific morphology and distinct expression of podocyte markers synaptopodin, podocin, nephrin and WT-1 after 10 days of differentiation as assessed by immunofluorescence staining or Western blot analysis. qPCR results showed a downregulation of pluripotency markers Oct4 and Sox-2 and a 9-fold upregulation of the podocyte marker synaptopodin during the time course of differentiation. Cultured podocytes exhibited endocytotic uptake of albumin. In toxicological assays, matured podocytes clearly responded to doxorubicin (Adriamycin™) with morphological alterations and a reduction in cell viability after 48 h of incubation.

Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems.

A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.

Fetal Bovine Serum (FBS): Past - Present - Future.

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as "fetal calf serum") is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations has gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where regulatory aspects, the serum dilemma, alternatives to FBS, case-studies of serum-free in vitro applications, and the establishment of serum-free databases were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with the above-mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically-defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.

Towards optimisation of induced pluripotent cell culture: Extracellular acidification results in growth arrest of iPSC prior to nutrient exhaustion.

Human induced pluripotent stem cells (iPSC) have the potential to radically reduce the number of animals used in both toxicological science and disease elucidation. One initial obstacle culturing iPSC is that they require daily medium exchange. This study attempts to clarify why and propose some practical solutions. Two iPSC lineages were fed at different intervals in a full growth area (FGA) or a restricted growth area (RGA). The FGA consisted of a well coated with Matrigel™ and the RGA consisted of a coated coverslip placed in a well. Glucose, lactate, extracellular pH and cell cycle phases were quantified. Without daily feeding, FGA cultured iPSC had significantly reduced growth rates by day 2 and began to die by day 3. In contrast, RGA cultured cells grew to confluence over 3days. Surprisingly, glucose was not exhausted under any condition. However, extracellular pH reached 6.8 after 72h in FGA cultures. Artificially reducing medium pH to 6.8 also inhibited glycolysis and initiated an increase in G0/G1 phase of the cell cycle, while adding an additional 10mM bicarbonate to the medium increased glycolysis rates. This study demonstrates that iPSC are highly sensitive to extracellular acidification, a likely limiting factor in maintenance of proliferative and pluripotent status. Culturing iPSC in RGA prevents rapid extracellular acidification, while still maintaining pluripotency and allowing longer feeding cycles.

Impairment of human neural crest cell migration by prolonged exposure to interferon-beta.

Human cell-based toxicological assays have been used successfully to detect known toxicants, and to distinguish them from negative controls. However, there is at present little experience on how to deal with hits from screens of compounds with yet unknown hazard. As a case study to this issue, we characterized human interferon-beta (IFNß) as potential developmental toxicant affecting neural crest cells (NCC). The protein was identified as a hit during a screen of clinically used drugs in the 'migration inhibition of neural crest' (MINC) assay. Concentration-response studies in the MINC combined with immunocytochemistry and mRNA quantification of cellular markers showed that IFNß inhibited NCC migration at concentrations as low as 20 pM. The effective concentrations found here correspond to levels found in human plasma, and they were neither cytostatic nor cytotoxic nor did they did they affect the differentiation state and overall phenotype of NCC. Data from two other migration assays confirmed that picomolar concentration of IFNß reduced the motility of NCC, while other interferons were less potent. The activation of JAK kinase by IFNß, as suggested by bioinformatics analysis of the transcriptome changes, was confirmed by biochemical methods. The degree and duration of pathway activation correlated with the extent of migration inhibition, and pharmacological block of this signaling pathway before, or up to 6 h after exposure to the cytokine prevented the effects of IFNß on migration. Thus, the reduction of vital functions of human NCC is a hitherto unknown potential hazard of endogenous or pharmacologically applied interferons.

Serotonin improves glucose metabolism by Serotonylation of the small GTPase Rab4 in L6 skeletal muscle cells.

BACKGROUND: Serotonin (5-HT) improves insulin sensitivity and glucose metabolism, however, the underlying molecular mechanism has remained elusive. Previous studies suggest that 5-HT can activate intracellular small GTPases directly by covalent binding, a process termed serotonylation. Activated small GTPases have been associated with increased GLUT4 translocation to the cell membrane. Therefore, we investigated whether serotonylation of small GTPases may be involved in improving Insulin sensitivity and glucose metabolism. METHODS: Using fully differentiated L6 rat skeletal muscle cells, we studied the effect of 5-HT in the absence or presence of insulin on glycogen synthesis, glucose uptake and GLUT4 translocation. To prove our L6 model we additionally performed preliminary experiments in C2C12 murine skeletal muscle cells. RESULTS: Incubation with 5-HT led to an increase in deoxyglucose uptake in a concentration-dependent fashion. Accordingly, GLUT4 translocation to the cell membrane and glycogen content were increased. These effects of 5-HT on Glucose metabolism could be augmented by co-incubation with insulin and blunted by co incubation of 5-HT with monodansylcadaverine, an inhibitor of protein serotonylation. In accordance with this observation, incubation with 5-HT resulted in serotonylation of a protein with a molecular weight of approximately 25 kDa. We identified this protein as the small GTPase Rab4, the activity of which has been shown to be stimulated by both insulin signalling and serotonylation. CONCLUSION: Our data suggest that 5-HT elicits its beneficial effects on Glucose metabolism through serotonylation of Rab4, which likely represents the converging point between the insulin and the 5-HT signalling cascades.

Fetal Bovine Serum (FBS) - A pain in the dish?

The use of Fetal Bovine Serum in replacement alternative methods is associated with serious animal welfare concerns, as well as worrying reproducibility issues.

Sex Differences in Renal Proximal Tubular Cell Homeostasis.

Studies in human patients and animals have revealed sex-specific differences in susceptibility to renal diseases. Because actions of female sex hormones on normal renal tissue might protect against damage, we searched for potential influences of the female hormone cycle on basic renal functions by studying excretion of urinary marker proteins in healthy human probands. We collected second morning spot urine samples of unmedicated naturally ovulating women, postmenopausal women, and men daily and determined urinary excretion of the renal tubular enzymes fructose-1,6-bisphosphatase and glutathione-S-transferase-α Additionally, we quantified urinary excretion of blood plasma proteins α1-microglobulin, albumin, and IgG. Naturally cycling women showed prominent peaks in the temporal pattern of urinary fructose-1,6-bisphosphatase and glutathione-S-transferase-α release exclusively within 7 days after ovulation or onset of menses. In contrast, postmenopausal women and men showed consistently low levels of urinary fructose-1,6-bisphosphatase excretion over comparable periods. We did not detect changes in urinary α1-microglobulin, albumin, or IgG excretion. Results of this study indicate that proximal tubular tissue architecture, representing a nonreproductive organ-derived epithelium, undergoes periodical adaptations phased by the female reproductive hormone cycle. The temporally delimited higher rate of enzymuria in ovulating women might be a sign of recurring increases of tubular cell turnover that potentially provide enhanced repair capacity and thus, higher resistance to renal damage.

Activated platelets release sphingosine 1-phosphate and induce hypersensitivity to noxious heat stimuli in vivo.

At the site of injury activated platelets release various mediators, one of which is sphingosine 1-phosphate (S1P). It was the aim of this study to explore whether activated human platelets had a pronociceptive effect in an in vivo mouse model and whether this effect was based on the release of S1P and subsequent activation of neuronal S1P receptors 1 or 3. Human platelets were prepared in different concentrations (10(5)/µl, 10(6)/µl, 10(7)/µl) and assessed in mice with different genetic backgrounds (WT, S1P1 (fl/fl), SNS-S1P1 (-/-), S1P3 (-/-)). Intracutaneous injections of activated human platelets induced a significant, dose-dependent hypersensitivity to noxious thermal stimulation. The degree of heat hypersensitivity correlated with the platelet concentration as well as the platelet S1P content and the amount of S1P released upon platelet activation as measured with LC MS/MS. Despite the significant correlations between S1P and platelet count, no difference in paw withdrawal latency (PWL) was observed in mice with a global null mutation of the S1P3 receptor or a conditional deletion of the S1P1 receptor in nociceptive primary afferents. Furthermore, neutralization of S1P with a selective anti-S1P antibody did not abolish platelet induced heat hypersensitivity. Our results suggest that activated platelets release S1P and induce heat hypersensitivity in vivo. However, the platelet induced heat hypersensitivity was caused by mediators other than S1P.

pH-responsive, gluconeogenic renal epithelial LLC-PK1-FBPase+cells: a versatile in vitro model to study renal proximal tubule metabolism and function.

Ammoniagenesis and gluconeogenesis are prominent metabolic features of the renal proximal convoluted tubule that contribute to maintenance of systemic acid-base homeostasis. Molecular analysis of the mechanisms that mediate the coordinate regulation of the two pathways required development of a cell line that recapitulates these features in vitro. By adapting porcine renal epithelial LLC-PK1 cells to essentially glucose-free medium, a gluconeogenic subline, termed LLC-PK1-FBPase(+) cells, was isolated. LLC-PK1-FBPase(+) cells grow in the absence of hexoses and pentoses and exhibit enhanced oxidative metabolism and increased levels of phosphate-dependent glutaminase. The cells also express significant levels of the key gluconeogenic enzymes, fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK). Thus the altered phenotype of LLC-PK1-FBPase(+) cells is pleiotropic. Most importantly, when transferred to medium that mimics a pronounced metabolic acidosis (9 mM HCO3 (-), pH 6.9), the LLC-PK1-FBPase(+) cells exhibit a gradual increase in NH4 (+) ion production, accompanied by increases in glutaminase and cytosolic PEPCK mRNA levels and proteins. Therefore, the LLC-PK1-FBPase(+) cells retained in culture many of the metabolic pathways and pH-responsive adaptations characteristic of renal proximal tubules. The molecular mechanisms that mediate enhanced expression of the glutaminase and PEPCK in LLC-PK1-FBPase(+) cells have been extensively reviewed. The present review describes novel properties of this unique cell line and summarizes the molecular mechanisms that have been defined more recently using LLC-PK1-FBPase(+) cells to model the renal proximal tubule. It also identifies future studies that could be performed using these cells.

Delineation of the key aspects in the regulation of epithelial monolayer formation.

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.

Optimizing high-throughput metabolomic biomarker screening: a study of quenching solutions to freeze intracellular metabolism in CHO cells.

Metabolomics is a rapidly emerging tool for studying and optimizing both media and bioprocess development for culturing recombinant mammalian cells that are used in protein production processes. Quenching of the cells is crucial to fix their metabolic status at the time of sampling. Three precooled quenching solutions were tested for their ability to fix the metabolic activity of CHO cells: phosphate-buffered saline (PBS) (pH 7.4; 0.5°C), 60% methanol with 70 mM HEPES (pH 7.4; -20°C), and 60% methanol with 0.85% (w/v) ammonium bicarbonate (AMBIC) (pH 7.4; -20°C). The metabolic activity of the sampled CHO cells was assessed by determining the intracellular levels of ATP using a bioluminescence assay and selected metabolites with LC-MS/MS. We found the precooled PBS (pH 7.4; 0.5°C) to be the optimal quenching reagent for fixing intracellular metabolism. Importantly, the structural integrity of the cell membrane was maintained and highest yields were obtained for intracellular levels of ATP as well as for 18 out of 28 intracellular metabolites. In contrast to the previously reported studies, buffered methanol quenching was not applicable for suspension cultured CHO cells as cellular membrane integrity was affected. We recommend that the cells are quenched and washed simultaneously to keep the sampling time to a minimum and to prevent any further metabolic activity in the cells. We observed that additional washing steps are not required. Our analyses suggest that methanol as quenching solution, even in combination with a buffer substance, appears not suitable for quenching sensitive mammalian cells. The protocol we report herein is a simple cell sampling method that enables high-throughput metabolomic analyses and is suitable for a large number of samples.

Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media.

The search for alternatives to the use of fetal bovine serum (FBS) in cell and tissue culture media has become a major goal in terms of the 3R principles in order to reduce or to avoid harvesting of FBS from bovine fetuses, and, in terms of Good Manufacturing Practice (GMP), to ensure safe and animal product-free conditions for biomedical tissue engineering and (adult) stem cell therapy, respectively. In the present study, we investigated the feasibility of using platelet lysates (PL) as a substitute for FBS, based on the fact that most of the potent mitogenic factors present in serum are derived from activated thrombocytes. Platelet lysates were obtained from outdated human donor platelet concentrates. Methods were established to activate human donor platelets in order to achieve a maximum yield of platelet a-granule growth factors. Platelet lysates were successfully introduced to grow and maintain anchorage-dependent and -independent human and animal cell lines. For cell culture experiments, cells were either grown in culture media supplemented with 10% FBS, 5% PL, or under serum-free conditions. Growth experiments, viability assays, and platelet lysate-induced activation of ERK1/2 mitogen-activated protein kinase revealed platelet lysates as a valuable alternative to FBS in cell culture media.