Generation of Cell Lines for Detection of Transcriptional Changes under Long‐term Exposure to Low pH Conditions Characteristic of Chronic Inflammation

Abstract

Unresolved chronic inflammation, whether pathogenic or non‐pathogenic, increases chances of malignant transformation. Because inflammation is characterized by acidification due to disrupted circulation and impaired CO2 removal, chronic acidic pH becomes a stress factor. When cells are stressed they mutate to better adapt to the stress. Epigenetic effects of long‐term exposure of cells to chronically acidic pH have not yet been studied. We set to identify genes that are affected by a long exposure, for months and years, to chronically acidic pH as found in sites of chronic inflammation.Jurkat T‐cells clone E6‐1 was independently propagated into four clones, clones were maintained at normal pH7.4 (5% CO2/Air) (lines JTL7.4L and JTL7.4R), and at pH7.2 (10% CO2/Air) (lines JTL7.2L and JTL7.2R). Cells were counted using trypan blue exclusion with an aliquot of the suspension diluted with fresh medium to the density N0 = 0.1 million/mL live cells in the volume of 2 mL. The remains of the grown suspensions were either used for analyses to detect differences between the clones or frozen in liquid nitrogen for further analyses. Cells were allowed to proliferate for variable number of days before next cell count, dilution to N0 = 0.1 million/mL, analysis and freezing in liquid nitrogen. Paired t‐test showed that after eighty five dilutions, there was no difference between JTL7.4L and JTL7.4R (p=0.37), or JTL7.2L and JTL7.2R (p=0.87) cell counts. Using the logistic population growth model dN/dt=r*N0*(1‐K/N) that slows down as the cell count N approaches the carrying capacity K with the growth rate r, we found that K = 3.9±0.2 and r = 2.5±0.1, K = 4.3±0.1 and r = 2.7±0.1, K = 4.3±0.1 and r = 1.59±0.05, K = 3.7±0.2 and r = 1.84±0.07 for JTL7.4L, JTL7.4R, JTL7.2L, JTL7.2R, respectively. Combined together, K = 4.1±0.1 and r = 2.59±0.07 for JTL7.4 (R2 = 0.80), K = 4.0 ± 0.1 and r = 1.71±0.05 for JTL7.2 (R2 = 0.80) clones, respectively. R2 values, residual and visual analyses show that the growth behavior is more complex than the logistic population growth model. The model satisfactorily demonstrates that JTL grow faster at lower density and reach K faster at pH 7.4 than at pH 7.2 during first four days of proliferation. The model fails to demonstrate that JTL7.4 cell counts are greater than JTL7.2 between first four days of proliferation, while this reverses after 4 days of proliferation where JTL7.2 cell counts become greater up to twenty‐four days of proliferation. After 20 days of proliferation, viability dropped to less than 90% in two dilutions, so JTL were never kept proliferating for more than 20 days except in these two experiments. Now, with two independent control JTL clones propagating at pH 7.4, two independent clones propagating in chronically acidic pH 7.2 and samples frozen at liquid nitrogen after almost each dilution, we are planning to investigate changes in gene and protein expression caused by long‐term chronic acidic stress.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.