Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

Summary

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

Abstract

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

Introduction

Living systems constantly experience and respond to various mechanical inputs throughout their lifetimes¹. Mechanotransduction has been linked to many diseases, including developmental disorders, muscle and bone loss, and neuropathologies through signaling pathways directly or indirectly affected by the mechanical environment². However, the genes and proteins that are regulated by mechanical stimulation³ in the mechanosensitive signaling pathways⁴ remain largely unknown⁵, preventing the elucidation of the mechanical regulation mechanisms and the iden....

Protocol

1. Preparation of the silicon wafer mold

1. Clean the silicon wafer (see Table of Materials) first with acetone and then with isopropyl alcohol (IPA).
2. Place the silicon wafer on a 250 °C hot plate for 30 min for dehydration bake (Figure 3A).
3. Coat the silicon wafer with hexamethyldisilazane (HDMS) in a vapor prime oven (see Table of Materials) (process temperature: 150 °C, process pressure: 2 Torr, process time: 5 min, HDMS volume: 5 µL) (Figure 3B).
4. Place a bottle of SU-8 2100 photoresist (see Tabl....

Results

The microfluidic system is divided into two sub-compartments separated by deformable PDMS sidewalls. The first compartment is the liquid system where Drosophila embryos are introduced, automatically aligned, lined up, and compressed. The second compartment is a gas system where the gas pressure at either side of the compression channels is controlled via dead-end microchannels to precisely control the effective width of the compression channels. The microfluidic device is sealed with a glass slide at th.......

Discussion

The article describes the development of a microfluidic device to automatically align, immobilize, and precisely apply mechanical stimulation to hundreds of whole Drosophila embryos. The integration of the microfluidic system with a thin glass coverslip allowed for the imaging of embryos with high-resolution confocal microscopy during the stimulation. The microfluidic device also enabled the collection of the embryos right after the stimulation for running downstream biological assays. The design considerations,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 mL tri-cornered perforated plastic beakers with 60 mm Petri dishes	Fisher	14-955-111B	Perferate with air holes
100 mm P B <100> 0-100 500um SSP Test Grade Si Wafer	University Wafer	452
Biopsy punches	Ted Pella	15110
Bleach			Not brand specific
Blunt needle set	CML Supply	901
Contact Mask Aligner	Quintel	Q4000 MA
Cutting mat	Dahle	Vantage 10670	size: 24" x 36"
Developer	Kayaku Advance Materials	SU-8 2000
Direct Write Lithographer	Heidelberg	MLA100
Dissecting microscope			Any commericailly availble dissecting microscope with transmitted light
Glass petri dish	Fisher	FB0875713A
Glass slide	Warner Instruments	64-0710  (CS-24/60)
HMDS Vapor Prime Oven	Yes Engineering	YES-3TA
NaCl			Not brand specific
Oven	Labnet	I5110A
Paintbrush			Not brand specific
PDMS	Dow Corning	Sylgard 184
Photoresist	MicroChem	SU-8 2100
Plasma cleaner	Harrick Plasma	PDC-32G
Portable pressure source	hygger Quietest	HGD946
Pressure gauge	Cole-Parmer	EW-68950-25
Spin Coater	Laurell	WS-650-8B
Trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFOCTS)	Sigma-Aldrich	448931-10G
Triton-X 100	Fisher	AAA16046AP
Tubing	Saint-Gobain	02-587-1A
Ultrasonic Cleaner	Cole-Parmer	UX-08895-05
Vacuum Pump	Cole-Parmer	EW-07164-87