Chemical Identity and Mechanism of Action and Formation of a Cell Growth Inhibitory Compound from Polycarbonate Flasks.

This paper reports the chemical identity and mechanism of action and formation of a cell growth inhibitory compound leached from some single-use Erlenmeyer polycarbonate shaker flasks under routine cell culture conditions. Single-use cell culture vessels have been increasingly used for the production of biopharmaceuticals; however, they often suffer from issues associated with leachables that may interfere with cell growth and protein stability. Here, high-performance liquid-chromatography preparations and cell proliferation assays led to identification of a compound from the water extracts of some polycarbonate flasks, which exhibited subline- and seeding density-dependent growth inhibition of CHO cells in suspension culture. Mass spectroscopy, nuclear magnetic resonance spectroscopy, and chemical synthesis confirmed that this compound is 3,5-dinitro-bisphenol A. Cell cycle analysis suggests that 3,5-dinitro-bisphenol A arrests CHO-S cells at the G1/Go phase. Dynamic mass redistribution assays showed that 3,5-dinitro-bisphenol A is a weak GPR35 agonist. Analysis of the flask manufacturing process suggests that 3,5-dinitro-bisphenol A is formed via the combination of molding process with γ-sterilization. This is the first report of a cell culture/assay interfering leachable compound that is formed through γ-irradiation-mediated nitric oxide free radical reaction.

Synthetic surface for expansion of human mesenchymal stem cells in xeno-free, chemically defined culture conditions.

Human mesenchymal stem cells (HMSCS) possess three properties of great interest for the development of cell therapies and tissue engineering: multilineage differentiation, immunomodulation, and production of trophic factors. Efficient ex vivo expansion of hMSCs is a challenging requirement for large scale production of clinical grade cells. Low-cost, robust, scalable culture methods using chemically defined materials need to be developed to address this need. This study describes the use of a xeno-free synthetic peptide acrylate surface, the Corning® Synthemax® Surface, for culture of hMSCs in serum-free, defined medium. Cell performance on the Corning Synthemax Surface was compared to cells cultured on biological extracellular matrix (ECM) coatings in xeno-free defined medium and in traditional conditions on tissue culture treated (TCT) plastic in fetal bovine serum (FBS) supplemented medium. Our results show successful maintenance of hMSCs on Corning Synthemax Surface for eight passages, with cell expansion rate comparable to cells cultured on ECM and significantly higher than for cells in TCT/FBS condition. Importantly, on the Corning Synthemax Surface, cells maintained elongated, spindle-like morphology, typical hMSC marker profile and in vitro multilineage differentiation potential. We believe the Corning Synthemax Surface, in combination with defined media, provides a complete synthetic, xeno-free, cell culture system for scalable production of hMSCs.

Improved cell sensitivity and longevity in a rapid impedance-based toxicity sensor.

A number of toxicity sensors for testing field water using a range of eukaryotic cell types have been proposed, but it has been difficult to identify sensors with both appropriate sensitivity to toxicants and the potential for long-term viability. Assessment of bovine pulmonary artery endothelial cell (BPAEC) monolayer electrical impedance with electric cell-substrate impedance sensing (ECIS) showed promise in a previous systematic evaluation of toxicity sensor technologies. The goal of the study reported here was to improve toxicant responsiveness and field portability of this cell-based toxicity sensor. A variety of human cells, non-human mammalian cells, and non-mammalian vertebrate cells were screened for sensitivity to 12 waterborne industrial chemicals. The results of this assessment show that bovine lung microvessel endothelial cell (BLMVEC) monolayers and iguana heart (IgH-2) cell monolayers could detect nine out of the 12 waterborne industrial chemicals, an improvement over the seven chemicals previously detected using BPAEC monolayers. Both the BLMVEC and IgH-2 cell monolayers were tested for their ability for long-term survival on the ECIS test chips in a laboratory environment. Both cell lines were able to maintain high impedance readings on the ECIS electrodes for 37 days, a key trait in developing a field-portable toxicity sensor for water. Cell line optimization has greatly contributed to the on-going development of a field-portable cell-based biosensor that detects with sensitivity a wide range of waterborne toxicants.