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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 identification of molecular targets for diseases associated with pathological mechanotransduction^6,7. One limiting factor in projecting mechanobiology studies onto the related physiological processes is using individual cells with conventional culture dishes instead of intact multicellular organisms. Model organisms, such as Drosophila melanogaster (fruit fly), have contributed greatly to understanding the genes, signaling pathways, and proteins involved in animal development^8,9,10. Nevertheless, using Drosophila and other multicellular model organisms in mechanobiology research has been hindered by challenges with experimental tools. Conventional techniques for preparing, sorting, imaging, or applying various stimuli require mostly manual manipulation; these approaches are time-consuming, require expertise, introduce variability, and limit the experimental design and sample size¹¹. Recent microtechnological advancements are a great resource for enabling novel biological assays with very high throughput and highly controlled experimental parameters^12,13,14.

This article describes the development of an enhanced microfluidic device to align, immobilize, and precisely apply mechanical stimulation in the form of uniaxial compression to hundreds of whole Drosophila embryos¹⁵ (Figure 1). Integration of the microfluidic system with a glass coverslip allowed high-resolution confocal imaging of the samples during the stimulation. The microfluidic device also enabled fast collection of the embryos after the stimulation for running -omics assays (Figure 2). Explanations of the design considerations of this device, as well as the fabrication using soft lithography and experimental characterization, are described herein. Since making a silicon wafer mold of such a device requires a uniform coating of thick photoresist (thickness >200 µm) over large areas with high aspect ratio (AR) trenches (AR >5), this method considerably modified the traditional photolithographic mold fabrication protocol. In this way, this method facilitated the handling, adhesion, coating, patterning, and development of the photoresist. Additionally, potential pitfalls and their solutions are discussed. Lastly, the versatility of this design and fabrication strategy was demonstrated using other multicellular systems such as Drosophila egg chambers and brain organoids¹⁶.

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Protokół

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 Table of Materials) in a 60 °C oven for 15 min to reduce its viscosity.
   NOTE: Upon heating in the oven, the viscosity of the photoresist decreases. Photoresists with lowered viscosity can be handled more easily and can be poured more accurately on top of the wafer.
5. Place the silicon wafer on a 60 °C hot plate and pour 1 mL of the heated photoresist for each inch of the wafer until the photoresist covers most of the surface (Figure 3C).
6. Transfer the photoresist-coated silicon wafer to a spin coater (see Table of Materials).
7. Apply pre-spin first at 250 rpm for 30 s and then at 350 rpm for another 30 s, both with 100 rpm/s acceleration (Figure 3D).
8. Remove the excess photoresist from the edges of the silicon wafer using a cleanroom swab.
9. Apply a spin first at 500 rpm for 15 s with 100 rpm/s acceleration and then at 1450 rpm for 30 s with 300 rpm/s acceleration (Figure 3E).
10. Remove the edge bead with a cleanroom swab.
11. Place the silicon wafer on a 50 °C hot plate and spray acetone on the wafer to remove imperfections and promote uniform coating (Figure 3F).
12. Slowly ramp up the temperature of the hot plate to 95 °C at the rate of 2°C/min.
13. Soft bake the silicon wafer at 95 °C for 50 min (Figure 3G).
14. Slowly cool down the hot plate to room temperature at a rate of 2°C/min.
15. Place the silicon wafer on a mask aligner (see Table of Materials) and place the photomask on top of it (please refer to Supplementary Figure 1 for the photomask geometry).
16. Expose the silicon wafer to 350 mJ/cm² UV light (35 mW/cm² for 10 s) through the photomask using the contact mask aligner (Figure 3H).
17. Apply consecutive post-exposure bakes on the silicon wafer by placing the wafer on a hot plate at 50 °C for 5 min, at 65 °C for an additional 5 min, and finally at 80 °C for another 20 min (Figure 3I).
18. Slowly cool down the silicon wafer to room temperature at the rate of 2 °C/min.
19. Place a magnetic stirrer with a slightly smaller diameter than the silicon wafer in a beaker. Turn the silicon wafer upside-down and place it on top of the beaker.
20. Place the beaker inside another larger beaker and fill the larger beaker with a fresh developer solution (see Table of Materials). Leave the silicon wafer submerged in the developer for 30 min with the stirrer turned on (Figure 3J).
21. Transfer the silicon wafer into an ultrasonic bath sonicator filled with the fresh developer for 1 h at 40 kHz (Figure 3K).
22. Thoroughly wash the silicon wafer with an IPA solution to obtain the final silicon wafer mold (Figure 3L).

2. Fabrication of the microfluidic chip

1. Place the silicon wafer mold in a desiccator together with 10 drops (~500 µL) of Trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFOCTS, see Table of Materials) in a small weigh boat nearby.
2. Connect the desiccator to a vacuum pump at approximately 200 Torr for 30 min.
3. Turn off the desiccator valve and disconnect the vacuum pump overnight for PFOCTS coating.
4. Prepare the pre-cured polydimethylsiloxane (PDMS) solution by mixing the PDMS base with the curing agent (see Table of Materials) at a 10:1 ratio.
5. Degas the mixture by placing it into a centrifuge (500 x g for 5 min at room temperature).
   NOTE: This centrifugation allows bubbles to migrate to the top surface and consequently be removed in a short period of time.
6. Pour the degassed PDMS solution onto the silicon wafer mold.
7. Degas it again to remove the air bubbles trapped on the mold surface.
8. Cure the PDMS in a 60 °C oven for 1 h and 50 min (Figure 3M).
9. Use a scalpel to cut the borders of the cured PDMS region corresponding to the microfluidic chip geometry (Figure 3N).
10. Peel the PDMS part and place it upside-down on a cutting mat.
11. Use a razor to cut the PDMS part into its final shape (Figure 3O).
12. Punch the inlet and outlet holes on PDMS using a biopsy punch (see Table of Materials) or a needle with a blunt tip (Figure 3P).
    1. For the embryo inlet hole, use a 4 mm diameter punch.
    2. For the embryo outlet holes, use a 1.3 mm diameter punch.
    3. For the gas inlet hole, use a 2 mm punch.
13. Use a piece of scotch tape to remove any particulate that might remain on the patterned surface of the PDMS.
14. Clean a 24 mm x 60 mm rectangular glass slide first with acetone and then with IPA.
15. Dry the glass surface with an air gun connected to a filtered air source.
16. Apply a dehydration bake to the glass slide by placing it on a 250 °C hot plate for 2 h (Figure 3Q).
17. Cover the glass slide with a beaker to prevent surface contamination.
18. Place the PDMS, with its patterned side up, and the dehydrated glass slide into a plasma cleaner (see Table of Materials).
19. Treat the PDMS and the glass slide with oxygen plasma at 18 W for 30 s.
20. Place the PDMS on the glass slide with its patterned surface facing toward the glass slide to seal the microchannels via covalent bonding (Figure 3R).
21. Use tweezers to gently push the PDMS part against the glass slide to ensure full conformational contact.
22. Store the completed microfluidic chip at room temperature overnight to allow the bonding to reach its final strength.

3. Preparation of the fruit fly embryos

1. Allow Oregon-R adult flies to lay eggs on apple juice agar plates (1.5% agar, 25% apple juice, 2.5% sucrose) and collect the plates at the desired developmental time after egg laying for the given experiment.
   NOTE: For the present experiments, the plates were collected at 140 min to prepare and sort for embryos at the cellularization stage¹⁷.
2. Flood the agar with embryo egg wash (0.12 M NaCl, 0.04% Triton-X 100) and gently agitate the embryos with a paintbrush to dislodge them from the agar.
3. Transfer the embryos to a 50% bleach solution for 90 s, stirring occasionally. Strain the embryos through a tissue sieve and thoroughly wash away the bleach solution with water.
4. Transfer the embryos to a 90 mm glass Petri dish with enough embryo egg wash to fully cover the embryos.
5. Examine the embryos with transillumination on a dissecting microscope and select embryos of the desired development stage for loading into the microfluidic device.
   ​NOTE: In this application, embryos at the cellularization stage were selected. The detailed descriptions of how to ensure proper developmental stage selection can be found in the laboratory handbook for Drosophila¹⁷.

4. Applying mechanical stimulation to fruit fly embryos using the microfluidic chip

1. Prime all seven embryo microchannels by filling them with 0.4 µm filtered IPA through the main embryo inlet port.
2. Replace the IPA with 0.4 µm filtered deionized (DI) water.
3. Replace the DI water with embryo egg wash solution.
4. Collect approximately 100 preselected embryos from the glass Petri dish using a glass pipette.
5. Pipette the embryos into the embryo inlet port (Figure 4A).
6. Apply an approximately 3 PSI negative pressure (i.e., vacuum) to the gas inlet using a portable vacuum pump to open up the embryo microchannels.
7. Tilt the microfluidic chip downward for the embryos to automatically align and settle into the embryo microchannels (Figure 4B).
8. If the embryo microchannel inlets get clogged by multiple embryos entering simultaneously, tilt the microfluidic chip upward and then downward again to clear the clogging.
9. Based on the required throughput, introduce as many as 300 embryos into the embryo microchannels.
10. Once the embryo loading is completed, remove the vacuum to immobilize the embryos.
11. Tilt the microfluidic chip back to the horizontal position (Figure 4C).
12. Connect a portable positive pressure source (see Table of Materials) with a pressure gauge to the gas inlet to apply 3 PSI compression (Figure 4D).
13. Continuously check the pressure gauge to ensure a consistent compression level is applied.
14. If live imaging experiments will be conducted on the mechanically stimulated embryos, place the microfluidic chip on a standard microscope stage glass slide holder with the gas inlet connected to the pressure source.
15. Once the compression experiment is completed, the embryos can be collected for downstream analysis. In order to do this, first, apply the vacuum to the gas inlet to free the embryos.
16. Then, tilt the microfluidic chip upward for the embryos to move toward the embryo introduction port (Figure 4E).
17. Collect the embryos from the microfluidic chip using a glass pipette.
    NOTE: Upon collection, the effects of compression on embryonic development and viability can be investigated by growing the fruit flies into adulthood. The high-throughput processing capability of the microfluidic device also enables embryos to be used in downstream omics-based assays that require a large number of samples (Figure 2).

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Wyniki

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...

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Dyskusje

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,...

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Materiały

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