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Annals of Laboratory Medicine Nov 2022Adoptive cell therapy using umbilical cord blood (UCB)-derived allogeneic natural killer (NK) cells has shown encouraging results. However, because of the insufficient...
BACKGROUND
Adoptive cell therapy using umbilical cord blood (UCB)-derived allogeneic natural killer (NK) cells has shown encouraging results. However, because of the insufficient availability of NK cells and limited UCB volume, more effective culture methods are required. NK cell expansion and functionality are largely affected by the culture medium. While human serum is a major affecting component in culture media, the way it regulates NK cell functionality remains elusive. We elucidated the effects of different culture media and human serum supplementation on UCB NK cell expansion and functionality.
METHODS
UCB NK cells were cultured under stimulation with K562-OX40L-mbIL-18/21 feeder cells and IL-2 and IL-15 in serum-containing and serum-free culture media. The effects of the culture media and human serum supplementation on NK cell expansion and cytotoxicity were evaluated by analyzing the expansion rate, activating and inhibitory receptor levels, and the cytotoxicity of the UCB NK cells.
RESULTS
The optimal medium for NK cell expansion was Dulbecco's modified Eagle's medium/Ham's F12 with supplements and that for cytotoxicity was AIM V supplemented with Immune Cell Serum Replacement. Shifting media is an advantageous strategy for obtaining several highly functional UCB NK cells. Live cell imaging and killing time measurement revealed that human serum enhanced NK cell proliferation but delayed target recognition, resulting in reduced cytotoxicity.
CONCLUSIONS
Culture medium supplementation with human serum strongly affects UCB NK cell expansion and functionality. Thus, culture media should be carefully selected to ensure both NK cell quantity and quality for adoptive cell therapy.
Topics: Cell Proliferation; Culture Media; Humans; Killer Cells, Natural
PubMed: 35765872
DOI: 10.3343/alm.2022.42.6.638 -
Current Protocols in Molecular Biology Jan 2019We provide protocols for titering and isolating bacterial colonies from single cells by serial dilutions, for streaking agar plates, and for spreading suspensions of...
We provide protocols for titering and isolating bacterial colonies from single cells by serial dilutions, for streaking agar plates, and for spreading suspensions of cells on plates. Support protocols describe replica plating and methods for storing strains as agar stabs and frozen stocks. © 2018 by John Wiley & Sons, Inc.
Topics: Agar; Bacteriological Techniques; Colony Count, Microbial; Culture Media; Escherichia coli; Preservation, Biological
PubMed: 30414382
DOI: 10.1002/cpmb.82 -
Current Protocols in Molecular Biology Jan 2019We describe the procedure for inoculating overnight (starter) cultures of E. coli from a single colony, along with considerations for growing larger cultures. We also...
We describe the procedure for inoculating overnight (starter) cultures of E. coli from a single colony, along with considerations for growing larger cultures. We also include two methods for monitoring the number of cells per unit volume (density) of liquid cultures using a spectrophotometer and a hemacytometer or "count slide." © 2018 by John Wiley & Sons, Inc.
Topics: Colony Count, Microbial; Culture Media; Escherichia coli; Spectrophotometry
PubMed: 30412369
DOI: 10.1002/cpmb.81 -
Scientific Reports Dec 2020The aim of this study was to investigate how carbohydrates (glucose or sucrose) affect the characteristics of Enterococcus faecalis (E. faecalis) planktonic and biofilm...
The aim of this study was to investigate how carbohydrates (glucose or sucrose) affect the characteristics of Enterococcus faecalis (E. faecalis) planktonic and biofilm in vitro. For this study, E. faecalis was cultured in tryptone-yeast extract broth with 0% glucose + 0% sucrose, 0.5% glucose, 1% glucose, 0.5% sucrose, or 1% sucrose. Viability of E. faecalis was examined by colony forming unit counting assays. Biofilm formation was assessed by measuring extracellular DNA (eDNA), a component of the biofilm matrix. Quantitative real-time PCR (qRT-PCR) was performed to investigate the expression of virulence-associated genes. Field emission scanning electron microscopy analysis, confocal laser scanning microscopy analysis, and crystal violet colorimetric assay were conducted to study E. faecalis biofilms. E. faecalis showed the highest viability and eDNA levels in 1% sucrose medium in biofilms. The result of qRT-PCR showed that the virulence-associated genes expressed highest in 1% sucrose-grown biofilms and in 1% glucose-grown planktonic cultures. E. faecalis showed highly aggregated biofilms and higher bacteria and exopolysaccharide (EPS) bio-volume in sucrose than in 0% glucose + 0% sucrose or glucose. The results indicate that the production of eDNA and EPS and expression of virulence-associated genes in E. faecalis are affected by the concentration of carbohydrates in biofilm or planktonic culture.
Topics: Culture Media; Enterococcus faecalis; Extracellular Polymeric Substance Matrix; Microbial Viability; Virulence
PubMed: 33318537
DOI: 10.1038/s41598-020-78998-5 -
Journal of Bacteriology Dec 2007Luria-Bertani broth supports Escherichia coli growth to an optical density at 600 nm (OD(600)) of 7. Surprisingly, however, steady-state growth ceases at an OD(600) of...
Luria-Bertani broth supports Escherichia coli growth to an optical density at 600 nm (OD(600)) of 7. Surprisingly, however, steady-state growth ceases at an OD(600) of 0.3, when the growth rate slows down and cell mass decreases. Growth stops for lack of a utilizable carbon source. The carbon sources for E. coli in Luria-Bertani broth are catabolizable amino acids, not sugars.
Topics: Amino Acids; Carbohydrates; Carbon; Cell Proliferation; Culture Media; Escherichia coli; Fermentation
PubMed: 17905994
DOI: 10.1128/JB.01368-07 -
Toxicology in Vitro : An International... Sep 2022Scientists are using in vitro methods to answer important research questions and implementing strategies to maximize the reliability and human relevance of these...
Scientists are using in vitro methods to answer important research questions and implementing strategies to maximize the reliability and human relevance of these methods. One strategy is to replace the use of fetal bovine serum (FBS)-an undefined and variable mixture of biomolecules-in cell culture media with chemically defined or xeno-free medium. In this study, A549 cells, a human lung alveolar-like cell line commonly used in respiratory research, were transitioned from a culture medium containing FBS to media without FBS. A successful transition was determined based on analysis of cell morphology and functionality. Following transition to commercially available CnT-Prime Airway (CELLnTEC) or X-VIVO™ 10 (Lonza) medium, the cells were characterized by microscopic evaluation and calculation of doubling time. Their genotype, morphology, and functionality were assessed by monitoring the expression of gene markers for lung cell types, surfactant production, cytokine release, the presence of multilamellar bodies, and cell viability following sodium dodecyl sulphate exposure. Our results showed that A549 cells successfully transitioned to FBS-free media under submerged and air-liquid-interface conditions. Cells grown in X-VIVO™ 10 medium mimicked cellular characteristics of FBS-supplemented media while those grown in CnT-Prime Airway medium demonstrated characteristics possibly more reflective of normal human alveolar epithelial cells.
Topics: A549 Cells; Cell Culture Techniques; Cells, Cultured; Culture Media; Culture Media, Serum-Free; Humans; Reproducibility of Results; Serum Albumin, Bovine
PubMed: 35753526
DOI: 10.1016/j.tiv.2022.105423 -
Trends in Cell Biology Nov 2019Developed decades ago, traditional culture media were not intended to resemble the metabolic composition of human blood, and indeed poorly do so. Yet, despite what is... (Review)
Review
Developed decades ago, traditional culture media were not intended to resemble the metabolic composition of human blood, and indeed poorly do so. Yet, despite what is now a clear recognition that environmental factors influence metabolism, such media remain standard to in vitro studies across virtually all areas of biological research. The recent development of physiologic media, like other efforts designed to address the modeling capacity of cell culture, holds immense potential to improve understanding and interpretation of diverse biological and pharmacological studies.
Topics: Animals; Cell Culture Techniques; Cell Line; Culture Media; Humans; Mice; Plasma
PubMed: 31623927
DOI: 10.1016/j.tcb.2019.08.009 -
Biotechnology and Bioengineering Jun 2019Nowadays, chemically defined cell culture media (CCM) have replaced serum- and hydrolysate-based media that rely on complex ingredients, such as yeast extracts or... (Review)
Review
Nowadays, chemically defined cell culture media (CCM) have replaced serum- and hydrolysate-based media that rely on complex ingredients, such as yeast extracts or peptones. Benefits include a significantly lower lot-to-lot variability, more efficient manufacturing by reduction to essential components, and the ability to exclude components that may negatively influence growth, viability, or productivity. Even though current chemically defined CCMs provide an excellent basis for various mammalian biotechnological processes, vitamin instabilities are known to be a key factor contributing to the variabilities still present in liquid CCM as well as to short storage times. In this review, the chemical degradation pathways and products for the most relevant vitamins for CCM will be discussed, with a focus on the effects of light, oxygen, heat, and other CCM compounds. Different approaches to stabilize vitamins in solution, such as replacement with analogs, encapsulation, or the addition of stabilizing compounds will also be reviewed. While these vitamins and vitamin stabilization approaches are presented here as particular for CCM, the application of these concepts can also be considered relevant for pharmaceutical, medical, and food supplement purposes. More precise knowledge regarding vitamin instabilities will contribute to stabilize future formulations and thus decrease residual lot-to-lot variability.
Topics: Animals; Biotechnology; Cell Culture Techniques; Culture Media; Drug Stability; Excipients; Hot Temperature; Humans; Light; Oxygen; Vitamins
PubMed: 30793282
DOI: 10.1002/bit.26942 -
Stem Cell Research & Therapy Jun 2022Human-induced pluripotent stem cells (hiPSCs) are considered an ideal resource for regenerative medicine because of their ease of access and infinite expansion ability....
BACKGROUND
Human-induced pluripotent stem cells (hiPSCs) are considered an ideal resource for regenerative medicine because of their ease of access and infinite expansion ability. To satisfy the sizable requirement for clinical applications of hiPSCs, large-scale, expansion-oriented, xeno-free, and cost-effective media are critical. Although several xeno-free media for hiPSCs have been generated over the past decades, few of them are suitable for scalable expansion of cultured hiPSCs because of their modest potential for proliferation and high cost.
METHODS
In this study, we developed a xeno-free ON2/AscleStem PSC medium (ON2) and cultured 253G1 hiPSCs on different matrices, including iMatrix-511 and gelatin nanofiber (GNF) in ON2. Over 20 passages, we evaluated cell proliferation by doubling times; pluripotency by flow cytometry, immunofluorescence staining and qRT-PCR; and differentiation ability by three germ layer differentiation in vitro and teratoma formation in severe combined immunodeficiency mice, followed by histological analysis. In addition, we compared the maintenance effect of ON2 on hiPSCs with StemFit® AK02 (AK02N) and Essential 8™ (E8). Besides 253G1 hiPSCs, we cultivated different hiPSC lines, including Ff-l01 hiPSCs, ATCC® ACS-1020™ hiPSCs, and Down's syndrome patient-specific ATCC® ACS-1003™ hiPSCs in ON2.
RESULTS
We found that 253G1 hiPSCs in ON2 demonstrated normal morphology and karyotype and high self-renewal and differentiation abilities on the tested matrices for over 20 passages. Moreover, 253G1 hiPSCs kept on GNF showed higher growth and stemness, as verified by the shorter doubling time and higher expression levels of pluripotent markers. Compared to AK02N and E8 media, 253G1 hiPSCs grown in ON2 showed higher pluripotency, as demonstrated by the increased expression level of pluripotent factors. In addition, all hiPSC lines cultivated in ON2 were able to grow for at least 10 passages with compact clonal morphology and were positive for all detected pluripotent markers.
CONCLUSIONS
Our xeno-free ON2 was compatible with various matrices and ideal for long-term expansion and maintenance of not only healthy-derived hiPSCs but also patient-specific hiPSCs. This highly efficient medium enabled the rapid expansion of hiPSCs in a reliable and cost-effective manner and could act as a promising tool for disease modeling and large-scale production for regenerative medicine in the future.
Topics: Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Culture Media; Humans; Induced Pluripotent Stem Cells; Mice; Regenerative Medicine
PubMed: 35658933
DOI: 10.1186/s13287-022-02879-z -
Nucleic Acids Research Jan 2023We present MediaDive (https://mediadive.dsmz.de), a comprehensive and expert-curated cultivation media database, which comprises recipes, instructions and molecular...
We present MediaDive (https://mediadive.dsmz.de), a comprehensive and expert-curated cultivation media database, which comprises recipes, instructions and molecular compositions of >3200 standardized cultivation media for >40 000 microbial strains from all domains of life. MediaDive is designed to enable broad range applications from every-day-use in research and diagnostic laboratories to knowledge-driven support of new media design and artificial intelligence-driven data mining. It offers a number of intuitive search functions and comparison tools, for example to identify media for related taxonomic groups and to integrate strain-specific modifications. Besides classical PDF archiving and printing, the state-of-the-art website allows paperless use of media recipes on mobile devices for convenient wet-lab use. In addition, data can be retrieved using a RESTful web service for large-scale data analyses. An internal editor interface ensures continuous extension and curation of media by cultivation experts from the Leibniz Institute DSMZ, which is interlinked with the growing microbial collections at DSMZ. External user engagement is covered by a dedicated media builder tool. The standardized and programmatically accessible data will foster new approaches for the design of cultivation media to target the vast majority of uncultured microorganisms.
Topics: Artificial Intelligence; Data Mining; Databases, Factual; Culture Media
PubMed: 36134710
DOI: 10.1093/nar/gkac803