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 is rarely monitored or reported. Here, a method to establish and dynamically monitor autonomously regulated oxygen microenvironments (AROM) using an oil overlay in an open microscale cell culture system is presented. Using this method, the oxygen microenvironment is dynamically regulated via the supply-demand balance of the system. Numerical simulation and experimental validation of oxygen transport within multi-liquid-phase, microscale culture systems involving a variety of cell types, including mammalian, fungal, and bacterial cells are presented. Finally, AROM is applied to establish a coculture between cells with disparate oxygen demands-primary intestinal epithelial cells (oxygen consuming) and Bacteroides uniformis (an anaerobic species prevalent in the human gut).

Application of 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 22 ]. The testing spots (i.e., plasma-treated areas) are hydrophilic [with an under-oil (fluorinated oil) water contact angle (CA) θ = 6.2 o ] and the untreated background hydrophobic (with an under-oil water CA θ = 139.7 o ).…”

“…We added silicone oil to this test because it provides three unique functions in UOMS: i) exclusive liquid repellency (ELR), where a liquid (e.g., culture media) is inherently and completely repelled by a solid surface (i.e., θ = 180°) when exposed to a secondary, immiscible liquid (e.g., oil) (see details in our previous publications 14,16,20 ), ii) under-oil sweep distribution, where thousands of microdrops with a volume ranging from microliter to picoliter can be arrayed using automated or manual pipetting in a minute by dragging (or the so-called sweeping) a hanging drop of culture media (+ cells and/or drugs) across a patterned surface with double-ELR (i.e., under-oil water ELR + under-water oil ELR) (see details in our previous publications 16,20 ), 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 22 ). The double-oil conditions (i.e., one oil plus another oil) show the flexibility of adjusting the oil overlay by combining the properties of two oil types, e.g., different diffusion coeficcients of vital gases (e.g., O 2 and/or CO 2 ) 22 , or under-oil media evaporation/loss rate 14 . 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 32 .…”

“…UOMS-AST, further combined with the three unique functions - ELR 14,16,20 , under-oil sweep distribution 16,20 , and AROM 22 as described above in Results, provides a “less-is-more” strategy that leads to a next-generation phenotypic AST. The low adoption/implementation barriers give easy access to a significantly broadened group of end users, especially if commercialized.…”

“…In addition, access to these automated systems and platforms can be limited for new antibiotics and settings 11 with low laboratory resources such as research labs, outpatient clinics, point of care, and middle-and low-income countries. Recently, there has been renewed interest in the development of multi-liquid-phase microfluidics, named UOMS [12][13][14][15][16][17][18][19][20][21][22][23][24][25] . In UOMS, cell culture 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.…”

“…In UOMS, cell culture 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. Uniquely, UOMS allows: i) free physical access to the system with minimized evaporation and sample contamination, and ii) high-resolution optical access with various microscopic techniques (e.g., bright-field [13][14][15][16]18 , epifluorescence 14,16,20 , confocal 22 , multi-photon 21 ). When combined with improved data analytics and reporting, UOMS has promising capabilities as a next-generation AST platform with significant advantages over standard and microfluidic AST methods (Table 1).…”