Multiwell capillarity-based microfluidic device for the study of 3D tumour tissue-2D endothelium interactions and drug screening in co-culture models

María Virumbrales-Muñoz,Luis J Fernandez,Séverine Le Gac,Diego De Miguel,Manuel Doblare,Luis Martínez-Lostao,Ignacio Ochoa,Rosa Monge,José María Ayuso,Marta Olave

doi:10.1038/s41598-017-12049-4

Abstract

The tumour microenvironment is very complex, and essential in tumour development and drug resistance. The endothelium is critical in the tumour microenvironment: it provides nutrients and oxygen to the tumour and is essential for systemic drug delivery. Therefore, we report a simple, user-friendly microfluidic device for co-culture of a 3D breast tumour model and a 2D endothelium model for cross-talk and drug delivery studies. First, we demonstrated the endothelium was functional, whereas the tumour model exhibited in vivo features, e.g., oxygen gradients and preferential proliferation of cells with better access to nutrients and oxygen. Next, we observed the endothelium structure lost its integrity in the co-culture. Following this, we evaluated two drug formulations of TRAIL (TNF-related apoptosis inducing ligand): soluble and anchored to a LUV (large unilamellar vesicle). Both diffused through the endothelium, LUV-TRAIL being more efficient in killing tumour cells, showing no effect on the integrity of endothelium. Overall, we have developed a simple capillary force-based microfluidic device for 2D and 3D cell co-cultures. Our device allows high-throughput approaches, patterning different cell types and generating gradients without specialised equipment. We anticipate this microfluidic device will facilitate drug screening in a relevant microenvironment thanks to its simple, effective and user-friendly operation.

Highlights

Our understanding of cancer has undergone significant changes in the last years
Spatial and temporal complexities, together with the variety of cell types and phenotype differences within the same cell type add up to compose this orchestrated environment in tumour tissues, which is known as the tumour microenvironment (TME)[2,3,4,5]
The proposed design consists of several linear arrays of microwells (Fig. 1c), in which 3D tumour models are created by embedding tumour cells in a 3D collagen matrix and, on top of which confluent Human Umbilical Vein Endothelial Cells (HUVECs) monolayers are prepared as 2D mimics of the endothelial barrier

Summary

Introduction

Our understanding of cancer has undergone significant changes in the last years. We used to think of cancer as a clonally expanded cell population originated from a single cell[1]. Of particular interest for the generation of sophisticated models are the so-called organ-on-a-chip platforms, which combine different cell types and microfabricated structures and allow mimicking the architecture of a whole organ and/or tissues[12] Such microfluidic technology shows great potential in the field of cancer research to faithfully mimic the TME13. Optimised optical inspection and easy access to the endothelium would provide an advantageous alternative and additional information on cell morphology and tight junctions in the endothelium using immunofluorescent staining In this context, we present here a simple and self-filling SU-8-based microdevice design, which exploits capillary forces, to study endothelium-tumour interactions. We have developed a simple and highly versatile microfluidic device design with a physiologically relevant tumour-endothelium co-culture model, which allows testing the penetration and efficiency of drugs on tumour tissues. Endothelium co-culture model recreated in the present study within the device; (b) Chip fabrication process flow: (1) Kapton film bonding to a pyrex wafer, (2) deposition of a 90-μm thick SU-8 layer, UV-exposure and polymerization of the microchannel and microwell structures, (3) spinning of a 90- μm thick SU-8 layer and patterning of the microchannel and microwell structures, (4) development of the two SU-8 layers, (5) Kapton film bonding to a pyrex wafer, (6) spinning of a 90-μm thick SU-8 layer and patterning by photolithography, (7) cover development, (8) SU-8 to SU-8 thermal bonding, (9) SU-8 device release. (c) Schematic representation of an open device, showing the different SU-8 layers: the bottom layer (blue) the channel layer (red), including the channels and microwells and a top layer (grey) with microwell structures to yield open microwells and closed microchannels; (d) Photography of a final microdevice consisting of 3 arrays of 5 microwells each

Methods

Results

Conclusion