Influence of the Hypercapnic Tumor Microenvironment on the Viability of Hela Cells Screened by a CO₂-Gradient-Generating Device

Abstract

Carbon dioxide (CO2) levels outside of the physiological range are frequently encountered in the tumor microenvironment and laparoscopic pneumoperitoneum during clinical cancer therapy. Controversies exist regarding the biological effects of hypercapnia on tumor proliferation and metastasis concerning time frame, CO2 concentration, and cell type. Traditional control of gaseous microenvironments for cell growth is conducted using culture chambers that allow for a single gas concentration at a time. In the present paper, Hela cells were studied for their response to varying levels of CO2 in an aerogel-based gas gradient-generating apparatus capable of delivering a stable and quantitative linear CO2 profile in spatial and temporal domains. Cells cultured in the standard 96-well plate sandwiched in between the device were interfaced with the gas gradient generator, and the cells in each row were exposed to a known level of CO2 accordingly. Both the ratiometric pH indicator and theoretical modeling have confirmed the efficient mass transport of CO2 through the air-permeable aerogel monolith in a short period of time. Tumor cell behaviors in various hypercapnic microenvironments with gradient CO2 concentrations ranging from 12 to 89% were determined in terms of viability, morphology, and mitochondrial metabolism under acute exposure for 3 h and over a longer cultivation period for up to 72 h. A significant reduction in cell viability was noticed with increasing CO2 concentration and incubation time, which was closely associated with intracellular acidification and elevated cellular level of reactive oxygen species. Our modular device demonstrated full adaptability to the standard culture systems and high-throughput instruments, which provide the potential for simultaneously screening the responses of cells under tunable gaseous microenvironments.

A CO2-Responsive Imidazole-Functionalized Fluorescent Material Mediates Cancer Chemotherapy

“…CO 2 is a vital physiological gas in cellular environments, and its concentrations dramatically affect physiological homeostasis and the pH of cells [ 13 , 14 ]. Excessive CO 2 production is one of the main reasons for acidification of tumour tissues; the levels of CO 2 are much higher in cancer cells (59–84 mmHg) than normal cells (35–37 mmHg) [ 15 , 16 ]. Helmlinger et al described the relationship between the levels of CO 2 and pH of solid tumours, and stated that production of CO 2 may play a significant role in the acidification of the tumour environment due to penetration of CO 2 into the cellular matrix on reaching a state of carbonic acid equilibrium [ 17 ].…”

An illumination system for screening the light and oxygen dependence in photodynamic therapy

“…We previously developed an aerogel-based gas-gradient-generating apparatus capable of forming a stable and quantitative gaseous microenvironment for in vitro cell culture. 6 In this study, our contactless headspace O2 delivery method provides a convenient way to elucidate the role of O2 in affecting PDT efficacy under different irradiation conditions with varying light doses and wavelengths. From our findings, cells showed less sensitivity to PDT as O2 levels decreased.…”

“…The design of the aerogel-based gas-supplying apparatus was described previously in our earlier study. 6 Briefly, an aerogel monolith was sandwiched between an acrylic layer with two engraved gas channels and an acrylic backplate. The source and sink channels were supplied by feed gases with known concentrations.…”

Assays on 3D tumor spheroids for exploring the light dosimetry of photodynamic effects under different gaseous conditions

“…The design of multiparametric PDT testing device was described in our previous studies in detail [5,13,15]. As illustrated in Figure 1B, briefly, gas conditions within the device are regulated utilizing an aerogel-based apparatus, which interfaces with a 96-well plate loaded with tumor spheroids.…”

“…A dual-wavelength light-emitting diode (LED) array was engineered in a 12 Â 8 matrix to complement standard 96-well plate geometries for PDT irradiation [13]. Recognizing that precise regulation of the oxygen environment has been historically lacking from in vitro models for PDT assessment [14], we introduced an aerogel-based gas generation system capable of modulation of oxygen levels over cell culture landscapes [5,15]. Cell behavior in 2D cultures and 3D spheroids was assessed to evaluate the wavelength-dependent sensitivity of PDT dose metrics under normoxic and hypoxic conditions, utilizing the multiparametric apparatus.…”