Impact of serum in cell culture media on in vitro lactate dehydrogenase (LDH) release determination

Abstract The hematopoietic stem cell niche presents a localised environment supporting the balanced maintenance, self-renewal and occasional expansion of the stem cell pool. These options are widely assumed to be regulated exclusively by signalling from specific combinations of stroma-bound or soluble ligands. However, a consideration of the rare conditions under which absolute numbers of stem cells increase in vivo as well as the selective pressures acting on regenerative systems during evolution has led us to propose a metabolic component to the stem cell niche which serves to limit cumulative damage, to avoid the selection of potentially oncogenic mutations and to tie symmetric division to slow proliferation. This would mean that traditional cell culture media based on “systemic” substrates such as glucose and glutamine may actively prevent the symmetric amplification of high quality stem cells, offering a possible explanation for the limited success in this area to date. To investigate this possibility, we have examined the effects of range of carbon and energy sources on the proliferation and maintenance of stem and progenitor cells. Our strategy is to screen a wide variety of culture conditions using murine FDCPmix cells, which are non-tumorigenic but have an innate tendency to amplify symmetrically in the presence of IL-3, and then to test key observations in human UCB CD133+ cells provided with SCF, TPO and FLT-3L. In both cell systems, we do indeed find an unusually low requirement for the systemic substrates glucose and glutamine normally included as major energy and carbon sources in cell culture media. Reducing glucose reduces the yield of committed cells from CD133+ cultures without affecting the accumulation of CD133+CD34+cKit+ progenitors. When provided with alternative substrates more likely to reflect a “niche” type environment, FDCPmix cells can be maintained for long periods in media containing only the trace levels of glucose or glutamine derived from dialysed serum, and show improved self-renewal under these conditions. We have also found that raising osmolarity reduces glucose dependence and simultaneously favours the maintenance both of self-renewing CFU (FDCPmix culture) and of CAFCweek13 (CD133+ culture). In parallel, the use of NMR and mass spectrometry techniques to profile intracellular metabolites in self-renewing and differentiating FDCPmix cells reveals a shift in the metabolite balance indicating reduced glycolysis in the early cells. Taken together, these results suggest that hematopoietic stem cells do indeed have remarkable metabolic characteristics consistent with adaptation to a metabolically limiting niche environment. It may therefore be necessary to identify niche substrates and to combine these with the relevant signalling environment in vitro in order to effectively increase stem cell numbers for research, stem cell transplantation and tissue engineering applications.

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Surpassing light-induced cell damage in vitro with novel cell culture media

Scientific Reports ◽

10.1038/s41598-017-00829-x ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 21

Author(s):

John H. Stockley ◽

Kimberley Evans ◽

Moritz Matthey ◽

Katrin Volbracht ◽

Sylvia Agathou ◽

...

Keyword(s):

Cell Culture ◽

Culture Media ◽

Cell Damage ◽

Cell Culture Media

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In Vitro Bioavailability of the Hydrocarbon Fractions of Dimethyl Sulfoxide Extracts of Petroleum Substances

Toxicological Sciences ◽

10.1093/toxsci/kfaa007 ◽

2020 ◽

Vol 174 (2) ◽

pp. 168-177 ◽

Cited By ~ 3

Author(s):

Yu-Syuan Luo ◽

Kyle C Ferguson ◽

Ivan Rusyn ◽

Weihsueh A Chiu

Keyword(s):

Cell Culture ◽

Protein Binding ◽

Aromatic Compounds ◽

Culture Media ◽

In Vitro Testing ◽

Cell Culture Media ◽

Chemical Profiles ◽

In Vivo Extrapolation

Abstract Determining the in vitro bioavailable concentration is a critical, yet unmet need to refine in vitro-to-in vivo extrapolation for unknown or variable composition, complex reaction product or biological material (UVCB) substances. UVCBs such as petroleum substances are commonly subjected to dimethyl sulfoxide (DMSO) extraction in order to retrieve the bioactive polycyclic aromatic compound (PAC) portion for in vitro testing. In addition to DMSO extraction, protein binding in cell culture media and dilution can all influence in vitro bioavailable concentrations of aliphatic and aromatic compounds in petroleum substances. However, these in vitro factors have not been fully characterized. In this study, we aimed to fill in these data gaps by characterizing the effects of these processes using both a defined mixture of analytical standards containing aliphatic and aromatic hydrocarbons, as well as 4 refined petroleum products as prototypical examples of UVCBs. Each substance was extracted with DMSO, and the protein binding in cell culture media was measured by using solid-phase microextraction. Semiquantitative analysis for aliphatic and aromatic compounds was achieved via gas chromatography-mass spectrometry. Our results showed that DMSO selectively extracted PACs from test substances, and that chemical profiles of PACs across molecular classes remained consistent after extraction. With respect to protein binding, chemical profiles were retained at a lower dilution (higher concentration), but a greater dilution factor (ie, lower concentration) resulted in higher protein binding in cell medium, which in turn altered the ultimate chemical profile of bioavailable PACs. Overall, this case study demonstrates that extraction procedures, protein binding in cell culture media, and dilution factors prior to in vitro testing can all contribute to determining the final bioavailable concentrations of bioactive constituents of UVCBs in vitro. Thus, in vitro-to-in vivo extrapolation for UVCBs may require greater attention to the concentration-dependent and compound-specific differences in recovery and bioavailability.

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In vitro cytocompatibility testing of oxidative degradation products

Journal of Bioactive and Compatible Polymers ◽

10.1177/08839115211003115 ◽

2021 ◽

pp. 088391152110031

Author(s):

Scott M Herting ◽

Mary Beth B Monroe ◽

Andrew C Weems ◽

Sam T Briggs ◽

Grace K Fletcher ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Cell Culture ◽

Culture Media ◽

Degradation Products ◽

Oxidative Degradation ◽

Cell Culture Media ◽

Novel Approach ◽

Gene And Protein Expression

Implantable medical devices must undergo thorough evaluation to ensure safety and efficacy before use in humans. If a device is designed to degrade, it is critical to understand the rate of degradation and the degradation products that will be released. Oxidative degradation is typically modeled in vitro by immersing materials or devices in hydrogen peroxide, which can limit further analysis of degradation products in many cases. Here we demonstrate a novel approach for testing the cytocompatibility of degradation products for oxidatively-degradable biomaterials where the materials are exposed to hydrogen peroxide, and then catalase enzyme is used to convert the hydrogen peroxide to water and oxygen so that the resulting aqueous solution can be added to cell culture media. To validate our results, expected degradation products are also synthesized then added to cell culture media. We used these methods to evaluate the cytocompatibility of degradation products from an oxidatively-degradable shape memory polyurethane designed in our lab and found that the degradation of these polymers is unlikely to cause a cytotoxic response in vivo based on the guidance provided by ISO 10993-5. These methods may also be applicable to other biocompatibility tests such as tests for mutagenicity or systemic toxicity, and evaluations of cell proliferation, migration, or gene and protein expression.

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Degradation profile of synthetic coral scaffold in cell culture media

Digital Press Health Sciences ◽

10.29037/digitalpress.31223 ◽

2018 ◽

Vol 1 ◽

pp. 00002

Author(s):

Erlina Sih Mahanani ◽

Dwi Rizki Lestari

Keyword(s):

Cell Culture ◽

Incubation Time ◽

Culture Media ◽

In Vitro Study ◽

Ph Measurement ◽

Phenol Red ◽

Cell Culture Media ◽

Significant Difference ◽

Coral Scaffold

The scaffold is one of the factors in tissue engineering that determine the success of bone regeneration. The important characteristic of the scaffold is able to degrade gradually. In vitro study using cells, the scaffold will be exposed to culture media. Therefore, degradation profile for scaffold needs to be examined. This study aims to investigate the degradation profile of synthetic coral scaffold in cell culture media using pH measurement. The method used the synthetic coral scaffolds were prepared from denaturalized collagen (gelatin) and calcium carbonate (calcite) with a concentration of 5:5 and 4:6 weight % in aqua dest. The scaffolds were fabricated in membrane thick film which was then physically crosslinked.  The 10 % of gelatin scaffold was used as a control.  The scaffolds were incubated in cell culture media (non-phenol red Dulbecco’s Modified Eagle Medium) for 1 until 8 days, and pH changes of the medium were measured. As the result, Profile of degradation on day 1 to day 4 showed the 5:5 scaffold had the smallest degradation. The results indicated the significant different between scaffold concentration in day 1 (p=0.005), 5th(p=0.03), and 6th day (p=0.011). At the end of incubated days, the pH changed but not significantly different. LSD showed the significant differences between scaffold (5:5 and 4:6) with control and no significant difference between 2 concentrations of the scaffold. The conclusion of this study is the synthetic coral scaffold degraded gradually until the end incubation time and between concentration had different degradation profile in the early incubation time using pH measurement.

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