High throughput microfluidic system with multiple oxygen levels for the study of hypoxia in tumor spheroids

Abstract

Replication of physiological oxygen levels is fundamental for modeling human physiology and pathology in in vitro models. Environmental oxygen levels, applied in most in vitro models, poorly imitate the oxygen conditions cells experience in vivo, where oxygen levels average ∼5%. Most solid tumors exhibit regions of hypoxic levels, promoting tumor progression and resistance to therapy. Though this phenomenon offers a specific target for cancer therapy, appropriate in vitro platforms are still lacking. Microfluidic models offer advanced spatio-temporal control of physico-chemical parameters. However, most of the systems described to date control a single oxygen level per chip, thus offering limited experimental throughput. Here, we developed a multi-layer microfluidic device coupling the high throughput generation of 3D tumor spheroids with a linear gradient of five oxygen levels, thus enabling multiple conditions and hundreds of replicates on a single chip. We showed how the applied oxygen gradient affects the generation of reactive oxygen species (ROS) and the cytotoxicity of Doxorubicin and Tirapazamine in breast tumor spheroids. Our results aligned with previous reports of increased ROS production under hypoxia and provide new insights on drug cytotoxicity levels that are closer to previously reported in vivo findings, demonstrating the predictive potential of our system.

Hypoxia, acidification and oxidative stress in cells cultured at large distances from an oxygen source

“…In particular, to define more precisely the links between hypoxia and oxidative stress, methodologies devised to simultaneously assess the intracellular hypoxic and ROS status would constitute a useful contribution. Microfluidic systems have been used to expose cells to different levels of ambient O 2 while simultaneously imaging the fluorescence intensity of ROS sensitive fluorescent probes 20 . Alternatively, cell culture chambers that restrict gas exchange with the environment have been used to create cell-generated oxygen gradients, and to image hypoxic gradients and other relevant parameters, such as mitochondrial membrane potential 21,22 .…”

Diversity Models and Applications of 3D Breast Tumor-on-a-Chip

“…A spheroid has the geometrical property similar to solid tumor in vivo, providing the native properties of gradient oxygen situation [70]. For the study of hypoxia in tumor spheroids, Ilana Berger Fridman et al [71] developed a multi-layer microfluidic device to generate 3D breast tumor spheroids under the microenvironment of five levels of oxygen concentrations with high throughput. The author exploited the oxygen gradient model to investigate how two chemotherapeutic drugs, Doxorubicin and Tirapazamine, affect breast tumor spheroids.…”

“…Using this system, the authors monitored the migratory behaviors of MDA-MB-231 breast cancer cells in the gradient conditions of pH and/or oxygen. [71]. Open access; (B) A linear oxygen gradient from 3% to 17% was generated through the oxygen permeability of ECM.…”

“…Hypoxia model in breast tumor-on-a-chip. (A) The gradient of oxygen concentration was established in microfluidic chip by cellular oxygen consumption in the relatively closed spaces[71]. Open access; (B) A linear oxygen gradient from 3% to 17% was generated through the oxygen permeability of ECM.…”

A Machine Learning and Computer Vision Approach to Rapidly Optimize Multiscale Droplet Generation

“…Creating microspheres with a high surface area to volume ratio is beneficial for a large variety of applications such as materials generation, bio­(chemical) analysis, polymeric microcapsules, and more. − Industries like food science, biomedical, and cosmetics use microcapsules as material delivery vehicles with the ability to tune the capsule wall chemistry for slow or triggered release of the capsulated material. − For each of these unique applications, a different microdroplet volume is required. However, these droplet volumes and structures are a function of several physical parameters like the interfacial tension between the immiscible fluids, their viscosities, the microfluidic device geometry, and fluid flow rates. , …”