Oxygen Considerations in the Design of Microfluidics for Studying Oxygen Dependent ATP Release from Erythrocytes
Authors: Sove Richard J , Goldman Daniel , Fraser Graham M , Ellis Christopher G 
Published: November 2023

In recent years, microfluidic devices have become increasingly popular for use in biological studies due to their cost efficiency, low sample consumption rates and length scales that are relevant to cellular biology. They are used in a wide variety of areas including cell signalling. Though microfluidics have proven to be useful in biological settings, it is important to be aware of the O2 levels to which the living cells are exposed since exposure to unphysiological levels may affect cellular function. Our interests are in the hemoglobin oxygen (O2) saturation dependent ATP release from erythrocytes. Following release, the intravascular ATP then binds to purinergic receptors on the endothelium, causing a conducted signal to upstream arterioles which then vasodilate, increasing flow to the network. This mechanism is believed to be a key player in the local regulation of O2 supply in the microvasculature. Further, ATP release has been shown to be impaired in multiple cardiovascular diseases including type II diabetes and sepsis, thus understanding this fundamental signalling phenomenon is of paramount importance. In a previous study (Sove PLOS ONE 2013), we designed and computationally modelled an idealized microfluidic system to measure the dynamics of O2‐dependent ATP release from erythrocytes in vitro. The objective of the design was to create a steep O2 gradient in the channel to cause a rapid change in hemoglobin O2 saturation and measure the corresponding levels of ATP released from the erythrocytes; this was verified with the model. Due to constraints in the fabrication process, the design was altered to one that could be fabricated using common soft lithography techniques. Early prototypes of this design did not sufficiently desaturate the erythrocytes, motivating the need for a computational model of the O2 levels in the microfluidic device. In the present study, a set of computational tools were developed to investigate the O2 levels in microfluidic systems. These tools include methods for generating the multi‐domain geometries of the microfluidic systems in three spatial dimensions, as well as methods for simulating mass transport using a finite element method. The mass transport model includes the O2 interaction with the hemoglobin in erythrocytes. The computational model confirms that our early prototypes were not sufficiently desaturating the erythrocytes (maximum desaturation < 2%). This has led to the design of a new device which was guided by our computational model while considering fabrication constraints. The computational model indicates that the new prototype will perform 117.8% better at desaturating the erythrocytes compared to the original design. Thus our new prototype can be easily fabricated using common soft lithography techniques and is able to sufficiently decrease erythrocyte hemoglobin O2 saturation for our needs. In sum, our computational tools have allowed us to investigate our experimental setup in order to improve our design. These tools can also be applied to other microfluidic systems to verify that appropriate levels of O2 are being supplied. The model can also be used to account for O2 consumption by living cells (e.g. endothelial cells) governed by Michaelis‐Menton kinetics ensuring they are exposed to the intended O2 levels. The next stage of this work will validate the computational model by measuring the O2 saturation in the new prototype. This new device will then be used to measure the dynamics of ATP release in vitro.

Support or Funding Information

RJ Sove is funded by a NSERC Doctoral Canada Graduate Scholarship and this project is funded by CG Ellis’ NSERC Discovery Grant