Handling Gametes and Embryos: Quality Control for Culture Conditions

2014 ◽  
pp. 39-58 ◽  
Author(s):  
Jason Swain
Keyword(s):  
Quality Control ◽  
Culture Conditions

The Use of Real-Time Cell Analyzer Technology in Drug Discovery: Defining Optimal Cell Culture Conditions and Assay Reproducibility with Different Adherent Cellular Models

2011 ◽  
Vol 16 (6) ◽  
pp. 575-587 ◽  
Author(s):  
Franck A. Atienzar ◽  
Karen Tilmant ◽  
Helga H. Gerets ◽  
Gaelle Toussaint ◽  
Sebastien Speeckaert ◽  
...  
Keyword(s):  
Quality Control ◽  
Drug Discovery ◽  
Real Time ◽  
Culture Conditions ◽  
Label Free ◽  
Kinetic Measurements ◽  
Cell Index ◽  
Cellular Models ◽  
Index Data ◽  
Cell Analyzer

The use of impedance-based label-free technology applied to drug discovery is nowadays receiving more and more attention. Indeed, such a simple and noninvasive assay that interferes minimally with cell morphology and function allows one to perform kinetic measurements and to obtain information on proliferation, migration, cytotoxicity, and receptor-mediated signaling. The objective of the study was to further assess the usefulness of a real-time cell analyzer (RTCA) platform based on impedance in the context of quality control and data reproducibility. The data indicate that this technology is useful to determine the best coating and cellular density conditions for different adherent cellular models including hepatocytes, cardiomyocytes, fibroblasts, and hybrid neuroblastoma/neuronal cells. Based on 31 independent experiments, the reproducibility of cell index data generated from HepG2 cells exposed to DMSO and to Triton X-100 was satisfactory, with a coefficient of variation close to 10%. Cell index data were also well reproduced when cardiomyocytes and fibroblasts were exposed to 21 compounds three times (correlation >0.91, p < 0.0001). The data also show that a cell index decrease is not always associated with cytotoxicity effects and that there are some confounding factors that can affect the analysis. Finally, another drawback is that the correlation analysis between cellular impedance measurements and classical toxicity endpoints has been performed on a limited number of compounds. Overall, despite some limitations, the RTCA technology appears to be a powerful and reliable tool in drug discovery because of the reasonable throughput, rapid and efficient performance, technical optimization, and cell quality control.

scholarly journals Microbiological Control of Cellular Products: The Relevance of the Cellular Matrix, Incubation Temperature, and Atmosphere for the Detection Performance of Automated Culture Systems

2019 ◽  
Vol 47 (3) ◽  
pp. 254-263
Author(s):  
Susanne-Katharina Günther ◽  
Celina Geiss ◽  
Stefan J. Kaiser ◽  
Nico T. Mutters ◽  
Frank Günther
Keyword(s):  
Quality Control ◽  
Microbial Contamination ◽  
Incubation Temperature ◽  
Culture Conditions ◽  
Detection Performance ◽  
Negative Effects ◽  
Cell Matrix ◽  
Microbiological Control ◽  
Incubation Temperatures ◽  
Method Suitability

Background: The microbiological control of cellular products sometimes causes significant procedural issues for quality control laboratories. According to the European Pharmacopoeia (EP), the microbiological control of cellular products requires a 7- to 14-day incubation period at two different incubation temperatures using aerobic and anaerobic growth media. However, the suitability of these test conditions for efficient quality control can be influenced by many conditions, such as the expected microbial spectrum of contamination or the texture and composition of the cellular product. Because of interference, direct inoculation and membrane filtration as reference methods of pharmacopoeia are largely unsuitable for the microbiological control of cellular products; therefore, alternative and, above all, automated methods are the focus of interest. Objective: The aim of our study was to evaluate the method suitability and possible effects of cell matrix, incubation temperature, and oxygen pressure on the detection performance of automated culture systems. Methods: The BacT/ALERT® 3DTM Dual T system (bioMérieux, Nürtingen, Germany) was used to evaluate the factors influencing automated microbiological control of cellular products. The tests were performed using microbial strains recommended by the EP for microbiological method suitability testing and additional relevant possible contaminants of human-derived stem-cell products under varying culture and cell matrix conditions. Results: All contaminants were detected by the system in the required period of 2–5 days. Low incubation temperatures (22°C) had overall negative effects on the detection kinetics of each type of microbial contamination. The adverse effects of the accompanying cell matrix on the detection properties of the system could be compensated in our study by incubation at 32°C in both the aerobic and the anaerobic culture conditions. Conclusion: Automated culture techniques represent a sufficient approach for the microbiological control of cellular products. The negative effects of the cell matrix and microbial contamination on the detection performance can be compensated by the application of variable culture conditions in the automated culture system.

scholarly journals The Charcot Marie Tooth Disease Mutation R94Q in MFN2 Decreases ATP Production but Increases Mitochondrial Respiration under Conditions of Mild Oxidative Stress

2019 ◽  
Author(s):  
Christina Wolf ◽  
Rahel Zimmermann ◽  
Osamah Thaher ◽  
Diones Bueno ◽  
Verena Wüllner ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Quality Control ◽  
Mitochondrial Respiration ◽  
Culture Conditions ◽  
Mitochondrial Quality Control ◽  
Mitofusin 2 ◽  
Charcot Marie Tooth ◽  
Atp Production ◽  
Charcot Marie Tooth Disease ◽  
Tooth Disease

Charcot-Marie-Tooth disease is a hereditary polyneuropathy caused by mutations in Mitofusin-2 (MFN2), a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. Autosomal-dominant inheritance of a R94Q mutation in MFN2 causes the axonal subtype 2A2A which is characterized by early onset and progressive atrophy of distal muscles caused by motoneuronal degeneration. Here, we studied mitochondrial shape, respiration, cytosolic and mitochondrial ATP content as well as mitochondrial quality control in MFN2-deficient fibroblasts stably expressing wildtype or R94Q MFN2. Under normal culture conditions, R94Q cells had slightly more fragmented mitochondria but similar mitochondrial oxygen consumption, membrane potential and ATP production. Mild oxidative stress procured by 100 &micro;M hydrogen peroxide applied 24 h before analysis, however, significantly increased respiration but decreased mitochondrial ATP production only in R94Q cells. This was accompanied by increased glucose uptake and an upregulation of hexokinase 1 and pyruvate kinase M2 suggesting increased pyruvate shuttling into mitochondria. As these changes coincided with decreased levels of PINK1/Parkin-mediated mitophagy in R94Q cells, we conclude that the disease-causing R94Q mutation in MFN2 causes uncoupling of mitochondrial respiration from ATP production by a less efficient mitochondrial quality control.

scholarly journals A Conserved Mitochondrial Chaperone-Protease Complex Involved in Protein Homeostasis

2021 ◽  
Vol 8 ◽  
Author(s):  
Mauro Serricchio ◽  
Peter Bütikofer
Keyword(s):  
Quality Control ◽  
Protein Complex ◽  
Elevated Temperatures ◽  
Protein Complexes ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Protein ◽  
Culture Conditions ◽  
Inner Mitochondrial Membrane ◽  
Knock Out ◽  
Mitochondrial Functions

Mitochondria are essential organelles involved in cellular energy production. The inner mitochondrial membrane protein stomatin-like protein 2 (SLP-2) is a member of the SPFH (stomatin, prohibitin, flotilin, and HflK/C) superfamily and binds to the mitochondrial glycerophospholipid cardiolipin, forming cardiolipin-enriched membrane domains to promote the assembly and/or stabilization of protein complexes involved in oxidative phosphorylation. In addition, human SLP-2 anchors a mitochondrial processing complex required for proteolytic regulation of proteins involved in mitochondrial dynamics and quality control. We now show that deletion of the gene encoding the Trypanosoma brucei homolog TbSlp2 has no effect on respiratory protein complex stability and mitochondrial functions under normal culture conditions and is dispensable for growth of T. brucei parasites. In addition, we demonstrate that TbSlp2 binds to the metalloprotease TbYme1 and together they form a large mitochondrial protein complex. The two proteins negatively regulate each other’s expression levels by accelerating protein turnover. Furthermore, we show that TbYme1 plays a role in heat-stress resistance, as TbYme1 knock-out parasites displayed mitochondrial fragmentation and loss of viability when cultured at elevated temperatures. Unbiased interaction studies uncovered putative TbYme1 substrates, some of which were differentially affected by the absence of TbYme1. Our results support emerging evidence for the presence of mitochondrial quality control pathways in this ancient eukaryote.

scholarly journals The Charcot–Marie Tooth Disease Mutation R94Q in MFN2 Decreases ATP Production but Increases Mitochondrial Respiration under Conditions of Mild Oxidative Stress

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1289 ◽  
Author(s):  
Christina Wolf ◽  
Rahel Zimmermann ◽  
Osamah Thaher ◽  
Diones Bueno ◽  
Verena Wüllner ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Quality Control ◽  
Culture Conditions ◽  
Mitochondrial Quality Control ◽  
Mitochondrial Oxygen Consumption ◽  
Mitofusin 2 ◽  
Charcot Marie Tooth ◽  
Atp Production ◽  
Charcot Marie Tooth Disease ◽  
Tooth Disease

Charcot–Marie tooth disease is a hereditary polyneuropathy caused by mutations in Mitofusin-2 (MFN2), a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. Autosomal-dominant inheritance of a R94Q mutation in MFN2 causes the axonal subtype 2A2A which is characterized by early onset and progressive atrophy of distal muscles caused by motoneuronal degeneration. Here, we studied mitochondrial shape, respiration, cytosolic, and mitochondrial ATP content as well as mitochondrial quality control in MFN2-deficient fibroblasts stably expressing wildtype or R94Q MFN2. Under normal culture conditions, R94Q cells had slightly more fragmented mitochondria but a similar mitochondrial oxygen consumption, membrane potential, and ATP production as wildtype cells. However, when inducing mild oxidative stress 24 h before analysis using 100 µM hydrogen peroxide, R94Q cells exhibited significantly increased respiration but decreased mitochondrial ATP production. This was accompanied by increased glucose uptake and an up-regulation of hexokinase 1 and pyruvate kinase M2, suggesting increased pyruvate shuttling into mitochondria. Interestingly, these changes coincided with decreased levels of PINK1/Parkin-mediated mitophagy in R94Q cells. We conclude that mitochondria harboring the disease-causing R94Q mutation in MFN2 are more susceptible to oxidative stress, which causes uncoupling of respiration and ATP production possibly by a less efficient mitochondrial quality control.

scholarly journals The Charcot Marie Tooth Disease Mutation R94Q in MFN2 Decreases ATP Production but Increases Mitochondrial Respiration under Conditions of Mild Oxidative Stress

2019 ◽  
Author(s):  
Christina Wolf ◽  
Rahel Zimmermann ◽  
Osamah Thaher ◽  
Diones Bueno ◽  
Verena Wüllner ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Quality Control ◽  
Mitochondrial Respiration ◽  
Culture Conditions ◽  
Mitochondrial Quality Control ◽  
Mitofusin 2 ◽  
Charcot Marie Tooth ◽  
Atp Production ◽  
Charcot Marie Tooth Disease ◽  
Tooth Disease

Charcot-Marie-Tooth disease is a hereditary polyneuropathy caused by mutations in Mitofusin-2 (MFN2), a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. Autosomal-dominant inheritance of a R94Q mutation in MFN2 causes the axonal subtype 2A2A which is characterized by early onset and progressive atrophy of distal muscles caused by motoneuronal degeneration. Here, we studied mitochondrial shape, respiration, cytosolic and mitochondrial ATP content as well as mitochondrial quality control in MFN2-deficient fibroblasts stably expressing wildtype or R94Q MFN2. Under normal culture conditions, R94Q cells had slightly more fragmented mitochondria but a similar mitochondrial oxygen consumption, membrane potential and ATP production as wildtype cells. However, when inducing mild oxidative stress 24 h before analysis using 100 &micro;M hydrogen peroxide, R94Q cells exhibited significantly increased respiration but decreased mitochondrial ATP production. This was accompanied by increased glucose uptake and an upregulation of hexokinase 1 and pyruvate kinase M2 suggesting increased pyruvate shuttling into mitochondria. As these changes coincided with decreased levels of PINK1/Parkin-mediated mitophagy in R94Q cells, we conclude that the disease-causing R94Q mutation in MFN2 causes uncoupling of mitochondrial respiration from ATP production by a less efficient mitochondrial quality control triggered by oxidative stress.

Ultrastructure of the effect of T-514 obtained from Karwinskia humboldtiana on assembly and integrity of yeast's peroxisomes

1991 ◽  
Vol 49 ◽  
pp. 136-137
Author(s):  
J. Sepulveda-Saavedra ◽  
I. Vander-Klei ◽  
M. Venhuis ◽  
Y. Piñeyro-Lopez
Keyword(s):  
Culture Conditions ◽  
Toxic Effects ◽  
Candida Boidinii ◽  
Central Mexico ◽  
Poisonous Plant ◽  
Biological Behaviour ◽  
Enzyme Content ◽  
Fat Deposits ◽  
Yeast Model

Karwinskia humboldtiana is a poisonous plant that grows in semi desertic areas in north and central México. It produces several substances with different toxic effects. One of them designated T-514 damages severely the lung, kidney and liver, producing in the hepatoeyte large intracellular fat deposits and necrosis. Preliminary observations demonstrated that three is a decrease in the amount of peroxisomes in the hepatocytes of experimentally intoxicated rats and monkeys. To study the effect exerted by the T-514 on peroxisomes, a yeast model was selected, thus, three species: Saccha romices cerevisiae, Ilansenula polymorpha and Candida boidinii were used, because there is information concerning their peroxisome's morphology, enzyme content, biological behaviour under different culture conditions and biogenesis.

Scanning Microscopy of Human Intestinal Explants in Organ Culture

1972 ◽  
Vol 30 ◽  
pp. 396-397
Author(s):  
Bruce Wetzel ◽  
Robert Buscho ◽  
Raphael Dolin
Keyword(s):  
Electron Microscopy ◽  
Room Temperature ◽  
Culture Conditions ◽  
Human Fetuses ◽  
Enteric Disease ◽  
Organ Cultures ◽  
Growth Of A Variety ◽  
Normal Human ◽  
Fetal Intestine ◽  
Scanning Electron

It has been reported that explants of human fetal intestine can be maintained in culture for up to 21 days in a viable condition and that these organ cultures support the growth of a variety of known viral agents responsible for enteric disease. Scanning electron microscopy (SEM) has been undertaken on several series of these explants to determine their appearance under routine culture conditions.Fresh specimens of jejunum obtained from normal human fetuses were washed, dissected into l-4mm pieces, and cultured in modified Leibowitz L-15 medium at 34° C as previously described. Serial specimens were fixed each day in 3% glutaraldehyde for 90 minutes at room temperature, rinsed, dehydrated, and dried by the CO2 critical point method in a Denton DCP-1 device. Specimens were attached to aluminum stubs with 3M transfer tape No. 465, and one sample on each stub was carefully rolled along the adhesive such that villi were broken off to expose their interiors.

Lectin Binding Studies on RNA Tumor Virus-Infected Cells

1975 ◽  
Vol 33 ◽  
pp. 326-327
Author(s):  
D. C. Hixson
Keyword(s):  
Binding Sites ◽  
Lectin Binding ◽  
Culture Conditions ◽  
3T3 Cells ◽  
Binding Studies ◽  
Con A ◽  
Microscopic Studies ◽  
Tumor Virus ◽  
Infected Cells ◽  
Cell Culture Conditions

The abilities of plant lectins to preferentially agglutinate malignant cells and to bind to specific monosaccharide or oligosaccharide sequences of glycoproteins and glycolipids make them a new and important biochemical probe for investigating alterations in plasma membrane structure which may result from malignant transformation. Electron and light microscopic studies have demonstrated clustered binding sites on surfaces of SV40-infected or tryp- sinized 3T3 cells when labeled with concanavalin A (con A). No clustering of con A binding sites was observed in normal 3T3 cells. It has been proposed that topological rearrangement of lectin binding sites into clusters enables con A to agglutinate SV40-infected or trypsinized 3T3 cells (1). However, observations by other investigators have not been consistent with this proposal (2) perhaps due to differences in reagents used, cell culture conditions, or labeling techniques. The present work was undertaken to study the lectin binding properties of normal and RNA tumor virus-infected cells and their associated viruses using lectins and ferritin-conjugated lectins of five different specificities.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

Sign in / Sign up

Export Citation Format

Share Document