Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Summary

We present a microfluidic cancer-on-chip model, the "Evolution Accelerator" technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

Abstract

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the "Evolution Accelerator," breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

Introduction

Cancer has been increasingly recognized as a complex ecosystem that depends not only on the continued dysregulation of mutated cell populations but also on vital interactions between cancer cells and the host microenvironment. In this sense, cancer evolves on an adaptive landscape manifested by a combination of factors, including a heterogeneous tumor microenvironment and crosstalk with a variety of host cells, all of which contribute selective pressures for further genetic or epigenetic changes^1,2,3. In the context of solid tumors, uneven distribution of chemotherapeutics an....

Protocol

1. Fabrication of microfluidic device

1. Generate the desired microfluidic pattern using a layout design software (see Supplemental Materials).
2. Fabricate the photomask. See Table of Materials for more details.
   1. Utilizing a laser writer, write the pattern on a soda-lime glass plate coated with 100 nm of Cr and 500 nm of photoresist AZ1518.
   2. Develop the photoresist with developer AZ300MIF for 60 s.
   3. Etch away the chromium without protection from the photoresist using Cr-7 Chromium Etchant.
   4. Strip off residual photoresist using a photoresist stripper at 70 °C for 45 min.

Results

Validation of optimum cell growth on chip
A major goal of the experiment platform is to reproduce key components and interactions in a complex tumor microenvironment in a comprehensive manner, yet simple enough to provide quantitative, reliable and reproducible data. This goal can only be achieved if we have full control of the physical and biochemical environmental factors. We must either exclude the undesired factors or figure out a way to incorporate the uncontro.......

Discussion

Conventional cell culture was developed almost a century ago and remains the most frequently used preclinical model in biomedical research, despite its proven limited ability to predict clinical results in cancer¹⁷. Animal models offer the highest physiological relevance and reasonable genetic similarity to humans, but have long been acknowledged to have significant limitations in predicting human outcomes¹⁸. Among all the existing preclinical models, microfluidic cancer-on.......

Materials

Name	Company	Catalog Number	Comments
10 mL BD Luer-Lok tip syringes	BD	14-823-16E
Antibiotic-Antimycotic	Sigma-Aldrich	A5955	1x anti-anti
AZ 300 MIF	Merck KGaA	18441123163	Photoresist developer
AZ1518	Merck KGaA	AZ1518	Photoresist
AZ4330	Merck KGaA	AZ4330	Photoresist
Cr Chromium Etchant	Sigma-Aldrich	651826
Fetal bovine serum (FBS)	Life Technologies Corporation	10437028
Heidelberg DWL 66+ laserwriter	Heidelberg Instruments	DWL66+	Writing photomask
Hexamethyldisilazane (HMDS)	Sigma-Aldrich	379212	For photoresist adhesion enhancement
Hollow steel pins	New England Small Tube	NE-1300-01	.025 OD .017 ID x .500 long / type 304 WD fullhard
ibidi Heating System, Multi-Well Plates, K-Frame	ibidi	10929	On-stage incubator
Luer-Lok 23 G dispensing needle	McMaster-Carr	75165A684	To connect syringes and tubings
Lumox dish 35	Sarstedt	94.6077.331	Gas-permeable cell culture dish
Microposit Remover 1165	Dow Electronic Materials	Microposit Remover 1165	Photoresist stripper
Microseal B Adhesive Sealer	Bio-Rad Laboratories	MSB1001	Adhesive sealer
O-Ring (for Lumox plate sealing)	McMaster-Carr	9452K114	Dash No. 27; 1-5/16" ID x 1-7/16" OD; Duro 70
O-Ring (for bottom glass window sealing)	McMaster-Carr	9452K74	Dash No. 20; 7/8" ID x 1" OD; Duro 70
Plasma-Preen Plasma Cleaning/Etching System	Plasmatic Systems, Inc	Plasma-Preen	Oxygen plasma system
RPMI 1640	Life Technologies Corporation	11875-093
Samco RIE800iPB DRIE	Samco	RIE800iPB	Deep reactive-ion etching system
Suss MA6 mask aligner	SUSS MicroTec	MA6	Mask aligner
Sylgard 184 Silicone Elastomer	Fisher Scientific	NC9285739	PDMS elastomer
TePla M4L plasma etcher	PVA TePla	M4L	Plasma etcher
Trichloro-1H,1H,2H,2H-perfluorooctyl-silane (PFOTS)	Sigma-Aldrich	448931	For silicon wafer silanization
Tygon microbore tube (0.020" x 0.060"OD)	Cole-Parmer	EW-06419-01	Tubings for media delivery