Under-Oil Autonomously Regulated Oxygen Microenvironments: A Goldilocks Principle-Based Approach For Microscale Cell Culture

Abstract

Oxygen levels in vivo are autonomously regulated by a supply-demand balance, which can be altered in disease states. However, the oxygen levels of in vitro cell culture systems, particularly microscale cell culture, are typically dominated by either supply or demand. Further, the oxygen microenvironment in these systems are rarely monitored or reported. Here, we present a method to establish and dynamically monitor autonomously regulated oxygen microenvironments (AROM) using an oil overlay in an open microscale cell culture system. Using this method, the oxygen microenvironment is dynamically regulated via a supply-demand balance of the system. We simulate the kinetics of oxygen diffusion in multiliquid-phase microsystems on COMSOL Multiphysics and experimentally validate the method using a variety of cell types including mammalian, fungal and bacterial cells. Finally, we demonstrate the utility of this method to establish a co-culture between primary intestinal epithelial cells and a highly prevalent human gut species Bacteroides uniformis.

Under-oil open microfluidic systems for rapid phenotypic antimicrobial susceptibility testing

“…Specifically in the under-oil approach, we use fluorinated oil and/or silicone oil as the oil barrier, which minimizes oil extraction of lipophilic molecules [see the ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS) characterization in our previous publication]. 31 The volume of oil required in UOMS-AST is small as long as enough to cover the sessile droplets on a device. For example, to cover the whole surface and sessile droplets in the 1-well chambered coverglass, we only need 1 to 2 mL of the oil.…”

“…5, and see details in our previous publications), 25,29 and iii) autonomously regulated oxygen microenvironments (AROM), where cells spontaneously set up, regulate, and respond to the oxygen kinetics via a supply-demand balance as seen in vivo (see details in our previous publication). 31 Studies have shown the broad impact of hypoxia on microbial infection and pathogenesis. 43,44 The double-oil conditions (i.e., one oil plus another oil) showed the flexibility of adjusting the oil overlay by combining the properties of two oil types, e.g., different diffusion coefficients of vital gases (e.g., O 2 and/or CO 2 ), 31 or under-oil media evaporation/loss rate.…”

“…31 Studies have shown the broad impact of hypoxia on microbial infection and pathogenesis. 43,44 The double-oil conditions (i.e., one oil plus another oil) showed the flexibility of adjusting the oil overlay by combining the properties of two oil types, e.g., different diffusion coefficients of vital gases (e.g., O 2 and/or CO 2 ), 31 or under-oil media evaporation/loss rate. 23 Specifically, SO20 allows smooth and robust under-oil sweep distribution due to its low viscosity and SO10000 can significantly reduce under-oil media evaporation/loss rate due to its ultra-high viscosity.…”

“…In microfluidics, there has been renewed interest in the development of a fundamental branch in multi-liquid-phase open microfluidics, named UOMS. [21][22][23][24][25][26][27][28][29][30][31][32][33][34] Different from closedchamber/-channel microfluidics or single-liquid phase open microfluidics, cell culture in UMOS is implemented with the culture media and cells contained under an oil overlay, separating the cell culture/detection microenvironment from the ambient with an immiscible liquid (i.e., oil) rather than the closed chambers/channels of traditional microfluidic devices (that inflicts limited physical access to the systems) or open chambers/channels directly exposed to air (that causes media loss via evaporation, airborne sample contamination). Uniquely, UOMS allows: i) free physical access to the system with minimized evaporation and sample contamination, ii) highresolution optical access with various microscopic techniques (e.g., bright-field, [22][23][24][25]27 epifluorescence, 23,25,29 confocal, 31 multiphoton 30 ), and iii) low adoption barrier (i.e., ease to make/use, cost efficiency).…”

“…[21][22][23][24][25][26][27][28][29][30][31][32][33][34] Different from closedchamber/-channel microfluidics or single-liquid phase open microfluidics, cell culture in UMOS is implemented with the culture media and cells contained under an oil overlay, separating the cell culture/detection microenvironment from the ambient with an immiscible liquid (i.e., oil) rather than the closed chambers/channels of traditional microfluidic devices (that inflicts limited physical access to the systems) or open chambers/channels directly exposed to air (that causes media loss via evaporation, airborne sample contamination). Uniquely, UOMS allows: i) free physical access to the system with minimized evaporation and sample contamination, ii) highresolution optical access with various microscopic techniques (e.g., bright-field, [22][23][24][25]27 epifluorescence, 23,25,29 confocal, 31 multiphoton 30 ), and iii) low adoption barrier (i.e., ease to make/use, cost efficiency). When combined with automated data analytics and reporting, UOMS provides an open microfluidics-based solution for rapid phenotypic AST (i.e., UOMS-AST) with advantages over the current standard AST methods (Table 1) and a natural alignment with application toward standard clinical laboratory settings where open-fluid handling (e.g., pipetting) and optical microscopy are typically adopted.…”