Novel experimental in vitro setting to reproduce different oxygenation patterns between the tumor-vessel interface

Introduction

Hypoxia is a common characteristic of many solid tumors that has been associated with tumor aggressiveness. Limited diffusion of oxygen generates a gradient of oxygen availability from the blood vessel to the interstitial space and may underlie the recruitment of macrophages fostering cancer progression. However, the available data based on the recruitment of circulating cells to the tumor microenvironment has been so far carried out by conventional co-culture systems which ignore the hypoxic gradient between the vessel to the tumor interstitium.

This labmethod describes a protocol for fabricating a custom-made co-culture system based on a thin gas permeable membrane that enables to investigate the interaction between distinct cells types simultaneously subjected to differential oxygen levels. This cell culture device provides an excellent opportunity for researchers to reproduce the in vivo hypoxic gradients in solid tumors and to study their role in recruiting circulating cells to the tumor in specific types of cancer.

Materials

1. Sylgard 184 Silicone Elastomer kit (PDMS)
2. Standard silicone Gel-Film (gas permeable membrane)
3. Polylactic acid (PLA) filament
4. 1.3 mm ID polytetrafluoroethylene tubes
5. 2 mm diameter punch
6. 100 mm diameter cell culture dish
7. 2 mm drill bits
8. Plastic square weight boat
9. Scalpel
10. Scissors
11. Tweezers
12. Spatula
13. Plastic Pasteur pipettes
14. Tips for micropipettes
15. Transwell inserts with 8 µm pore polycarbonate membrane

Equipment required

1. REGEMAT 3D bioprinter
2. Jar vacuum desiccator
3. Portable corona treater
4. Precision balance
5. Oven
6. Drill
7. Servocontrolled gas blender

Design of the device

The device consists of two polydimethylsiloxane (PDMS) well layers separated by a 37.5 μm thick membrane. The membrane constitutes a gas permeable substrate for cell culture. Oxygen concentration over the cell culture can be tightly controlled via direct diffusion through the gas permeable membrane from a gas source connected to the lower layer. More specifically, the time required for the gas stimulus to diffuse through membrane is ~5 s as previously determined. The setting was designed to allow commercially available transwell inserts to be nested in the wells of the upper layer, thus enabling the performance of a cell co-culture by seeding cells both on the upper surface of the porous membrane of the transwell and on the gas permeable membrane, which are separated by a distance of 3 mm (Fig.1). Therefore, cells cultured on the transwell insert could be exposed to either the same or to different oxygen levels to those cells growing on the chip surface, by modulating the oxygen concentration in the gas mixture provided by the gas blender and oxygen levels in the cell culture incubator.

Experimental procedure

A visual of the protocol can be found in https://www.frontiersin.org/articles/10.3389/fonc.2019.00043/full#supplementary-material

1. Download .stl files for generating the upper and lower layer negative molds and print them in polylactic acid (PLA) using the filament extruder of the REGEMAT 3D BIO V1 or REG4Life bioprinters.
2. Fabricate PDMS layers via replica molding:
   1. Prepare a 10:1 mix (w/w) of PDMS base and curing agent (total weight = 100 g)
   2. Remove air bubbles in a vacuum desiccator (45 min)
   3. Pour PDMS onto PLA molds: 19 g for lower mold and 60 g for upper mold
   4. Remove air bubbles in the vacuum desicattor (3 h)
   5. Cure PDMS in an oven at 65 °C for 3 h
   6. Peel off PDMS replicas from PLA molds with the help of a spatula
3. Assemble the device:
   1. Cut the gas permeable membrane and remove the upper protective plastic layer
   2. Activate the surface of the lower PDMS replica and the membrane with corona
   3. Adhere both surfaces by applying some pressure with the hand
   4. Seal the edges by dispensing PDMS with a micropipette tip and cure in an oven (65°C, 30 min)
   5. Carefully peel off the PDMS replica with the adhered membrane from the remaining plastic layer
   6. Activate the membrane adhered to the lower PDMS replica and the upper PDMS replica with corona
   7. Put in contact both surfaces and apply some pressure with the hand
   8. Seal the edges with PDMS and cure in an oven (65°C, 30 min)
   9. Drill the central region of the device and the lid of a 100 mm culture dish
   10. Use PDMS to adhere the base of the device to the base of the culture dish
   11. Insert a 1.3 mm ID inlet tube through the hole generated in step 9
   12. Nest transwell inserts in wells of the upper PDMS layers (six in total)
   13. Close the culture dish.

The in vitro system will be ready-to-use after UV-sterilization and surface-coating of the gas permeable membrane with proteins of the extracellular matrix. Generation of oxygen gradient requieres its connection to a servocontrolled gas blender through the inlet tube.

References:

Campillo N, Falcones B, Otero J, Colina R, Gozal D, Navajas D, Farré R,  Almendros I. Differential Oxygenation in Tumor Microenvironment Modulates Macrophage and Cancer Cell Crosstalk: Novel Experimental Setting and Proof of Concept. Front Oncol, 2019;9:43.

Number	Category	Product	Amount
1	-	Sylgard 184 Silicone Elastomer kit	1
2	-	Standard silicone Gel-Film	1
3	-	Polylactic acid (PLA) filament	1
4	-	1.3 mm ID polytetrafluoroethylene tubes	1
5	-	2 mm diameter punch	1
6	-	100 mm diameter cell culture dish	1
7	-	2 mm drill bits	1
8	-	Plastic square weight boat	1
9	-	Scalpel	1
10	-	Scissors	1
11	-	Tweezers	1
12	-	Spatula	1
13	-	Plastic Pasteur pipettes	1
14	-	Tips for micropipettes	1
15	-	Transwell inserts with 8 µm pore polycarbonate membrane	1

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