Fabrication of a versatile polydimethylsiloxane chip for cardiac and lung tissue in vitro modeling

Introduction

Organ-on-a-chips are defined as physiological organ biomimetic systems built on microfluidic devices. By combining cell biology, engineering and biomaterial technology, the microenvironment of the chip is able to simulate tissue interfaces and mechanical stimulation of the native organ. They are in the list of top 10 emerging technologies and highly demanded in the market, especially by the pharmaceutical industry.

Polydimethylsiloxane (PDMS) is one of the most employed materials to mold microfluidic devices. It is biocompatible, optically transparent and has low autofluorescence, thus enabling high-resolution live cell imaging. Thin layers of PDMS are also highly elastic and gas permeable, allowing the fabrication of stretchable and gas permeable membranes that are physically relevant in in vitro tissue modeling. This method describes the fabrication of a versatile PDMS chip that can be employed as cardiac and lung tissue biomimetic systems, since it allows for precisely controlled exposures of cultured cells to high-frequency hypoxia/re-oxygenation cycles and cyclic stretch.

Materials

1. Sylgard 184 Silicone Elastomer kit
2. Standard silicone Gel-Film
3. 3 and 1.3 mm ID polytetrafluoroethylene tubes
4. 1, 2 and 4 mm diameter punches
5. 35 mm glass bottom culture dish
6. 150 cm² plastic culture dishes
7. 1 and 2 mm drill bits
8. Plastic square weight boat
9. Scalpel
10. Tweezers
11. Plastic Pasteur pipettes
12. Tips for micropipettes

Equipment required

1. Jar vacuum desiccator
2. Portable corona treater
3. Precision balance
4. Hot plate
5. Oven
6. Drill
7. Servocontrolled gas blender
8. Two-way solenoid valve system

Design of the chip

The chip consists of a PDMS well-covered with a thin elastic PDMS membrane. Cells are seeded onto the circular clamped membrane. An inlet tubing connects the well to a gas source providing a constant mixture of gases or cyclic changes in gas composition. A high resistance outlet tubing increases well-pressure deflecting upwards the membrane. A low resistance venting tubing connects the well to the atmosphere through a cyclically activated two-way solenoid valve to produce cyclic membrane stretch.

 

Experimental procedure

Mixing and degassing of PDMS for well molding

1. Place an empty plastic weight boat on a precision balance and pour silicone elastomer base and curing agent in a 10:1 (base:curing agent) weight proportion. Prepare enough amount (~130 g) to produce a PDMS block with the desired thickness (~8 mm) in a 150 cm² plastic culture dish.
2. Mix vigorously (~1 min) base and curing agent with a plastic Pasteur pipette.
3. Pour 120 g of the mixture into a 150 cm² plastic culture dish and leave the remaining PDMS in the dispensable weigh boat for the ulterior chip assembly.
4. Place the open dish and the weigh boat containing the PDMS in a vacuum dessicator.
5. Apply vacuum for 45 min to remove air bubbles from PDMS.

Fabrication of the PDMS well

6. Remove the culture dish containing the uncured PDMS from the bell jar dessicator.
7. Cover the culture dish with the lid and place it in an oven at 65⁰C for 3 h to cure PDMS. The dimensions of the obtained block result sufficient to produce ~100 chips.
8. With a scalpel, cut the cured PDMS into rectangular (~10 mm x ~10 mm) blocks.
9. Perforate the block from top to bottom with a 4 mm ID punch to produce a central well.
10. Make a lateral perforation in two opposite sides of the well using a 1 mm ID punch. These lateral perforations will be used to connect inlet and outlet tubes to the well as described in steps 21 and 22.
11. Perforate a third lateral side of the block using a 2 mm ID punch, connecting to the well. This hole will be used to connect the well to the venting tube as described in step 23.

Chip assembly

12. Activate the PDMS well by applying oxygen plasma at close proximity (~ 5 mm) at the highest voltage using the portable corona treater for 1 min.
13. Peel off the plastic layer protecting the silicone membrane. Activate the surface of the membrane applying plasma (~ 5 mm) with the corona treater for 30 s.
14. Immediately after plasma activation, place in contact the activated surfaces and apply pressure.
15. Dispense uncured PDMS with a 10 µl pipette tip at the edges to facilitate the ulterior handling.
16. Cure on the hot plate at 95⁰C for 20 min. Interpose a glass slide between the plastic layer supporting the membrane and the hot plate to avoid plastic melting.
17. Cut at the edges using a scalpel and peel off the block containing the membrane from the slide very carefully.
18. To improve cell culture imaging in an inverted microscope, adhere the base of the device to the lid of a 35 mm glass bottom culture dish by dispensing PDMS around its edges using a micropipette tip.
19. Place in the oven at 65⁰C for 2 h to achieve the complete adhesion of the chip to the dish.
20. Once the chip is completely adhered, drill the culture dish to allow the passage of inlet, outlet and venting tubes for gas inflow and outflow.
21. Insert a 1 m long 0.3 mm ID in one of the 1 mm holes performed in step 10 (inlet tube).
22. Insert a 10 cm long 0.3 mm ID in the other 1 mm hole performed in step 10 (outlet tube).
23. Insert a ~ 80 cm long 1.3 mm ID tube into the 2 mm hole performed in step 11 (venting tube).
24. Seal all possible gas and liquid leakages by dispensing PDMS in areas at risk (tube passage).
25. Cure PDMS in the oven at 65⁰C for 1 h.
26. Examine the fabricated chip in an optical microscope and discard those presenting membrane cracks or liquid leaks.
27. The inlet tubing of 0.3 mm ID (~1 m long) is connected to a servocontrolled gas blender able to provide fast changes in gas composition.
28. Cyclic strain of the membrane is produced by increasing the pressure inside the well by a high resistance outlet tubing of 0.3 mm ID and periodic closure of the low resistance venting tubing 1.3 mm ID (~80 cm long) with a two-way solenoid valve. The length of the outlet tube has been adjusted to provide the desired level of strain.
29. Calibrate the chip to ensure its correct operation prior to its use.

For a visual of the protocol, please refer to the supplementary video of Campillo et al., Front Physiol. 2016.

References

Campillo N, Jorba I, Schaedel L, Casals B, Gozal D, Farré R, Almendros I, Navajas D. A novel chip for cyclic stretch and intermittent hypoxia cell exposures mimicking obstructive sleep apnea. Front Physiol, 2016; 7:319.

Number	Category	Product	Amount
1	-	Sylgard 184 Silicone Elastomer kit	1
2	-	Standard silicone Gel-Film	1
3	-	0.3 and 1.3 mm ID polytetrafluoroethylene tubes	1
4	-	1, 2 and 4 mm diameter punches	1
5	-	35 mm glass bottom culture dish	1
6	-	150 cm2 plastic culture dishes	1
7	-	1 and 2 mm drill bits	1
8	-	Plastic square weight boat	1
9	-	Scalpel	1
10	-	Tweezers	1
11	-	Plastic Pasteur pipettes	1
12	-	Tips for micropipettes	1

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