Human cell lines, first cultured in the 1950s 1 , are indispensable in biomedical research. Today, a wide range of cell types are available, and sophisticated advanced 'omics' and visualization techniques allow for the routine assessment of cell identity and cellular responses 2 . However, the culture methods have remained relatively unchanged. Major advances in culture systems were made over three decades ago 3,4 , yet the old standard approach of batch cell culturethe culture of cells either in suspension or as adherent monolayers of cells in standard media [5][6][7] remains the predominant method in biomedical research.Culture media provides crucial nutrients, signalling molecules (such as growth factors), and suitable osmotic conditions. The gaseous and thermal environments of cell cultures are typically controlled by the incubator. The initial media conditions are generally stabilized by adjusting them to 18.6% O2 and a standard pH of 7.4, and this adjustment is achieved by adding a given amount of HCO3 − salt (a base), and by enriching the media with CO2 to a given percentage in the air (usually 5% or 10%). However, cell metabolism involves the exchange of gasesspecifically the release of CO2 and the consumption of O2and this can affect cellular growth via the alteration of, for example, the pH and the level of dissolved O2 (dO2) in the cellular microenvironment 8 . In theory, the equilibration of the medium with the gaseous and thermal environments of the incubator provides a way to reliably mimic O2, CO2 and HCO3 − homeostasis in metazoan body fluids. Yet this doesn't take into account the fact that homeostasis in a living mammal is supported by the active exchange of gases with the atmosphere. The absence of such active gas exchange in cell cultures suggests that, over time, cellular metabolic activities might acidify and deoxygenate the cellular microenvironment 8-10 , if intermittent monitoring and (when necessary) corrective action are not carried out.To mimic a physiological environment when using cell cultures, careful control over environmental factors (such as pH, CO2 and O2) is typically needed, in particular because even small deviations of environmental parameters from physiological levels may impair cellular function. For instance, in human blood, pH values below 7.2 (acidaemic conditions) and above 7.44 (alkalaemic conditions) can be fatal [11][12][13] . In cell cultures, the optimal growth of normal cells (that is, non-cancerous cells and non-transformed cells) occurs within a specific alkaline pH range, whereas cancer cells grow in a broader pH range that is shifted towards acidic values [14][15][16][17][18] . Cells have evolved mechanisms, including the use of Na + /H + antiporters or histone deacetylation, that restore the alkaline pH of the cytoplasm when the extracellular pH deviates from physiological levels [19][20][21][22][23][24][25] . However, such regulatory mechanisms require cellular energy, and changes in the acetylation state of chromatin can alter gene transcription and reduc...

The development of a suitable strategy for eliminating remaining undifferentiated cells is indispensable for the use of human-induced pluripotent stem (iPS) cell-derived cells in regenerative medicine. Here, we show for the first time that TRPV-1 activation through transient culture at 42 °C in combination with agonists is a simple and useful strategy to eliminate iPS cells from bioengineered cardiac cell sheet tissues. When human iPS cells were cultured at 42 °C, almost all cells disappeared by 48 hours through apoptosis. However, iPS cell-derived cardiomyocytes and fibroblasts maintained transcriptional and protein expression levels, and cardiac cell sheets were fabricated after reducing the temperature. TRPV-1 expression in iPS cells was upregulated at 42 °C, and iPS cell death at 42 °C was TRPV-1-dependent. Furthermore, TRPV-1 activation through thermal or agonist treatment eliminated iPS cells in cardiac tissues for a final concentration of 0.4% iPS cell contamination. These findings suggest that the difference in tolerance to TRPV-1 activation between iPS cells and iPS cell-derived cardiac cells could be exploited to eliminate remaining iPS cells in bioengineered cell sheet tissues, which will further reduce the risk of tumour formation.