Laser Microdissection: A Technique to Isolate Invading Colorectal Cancer cells from a 3D Organotypic Culture Model

1. Organotypic Preparation:

Preparation of the fibroblast impregnated organotypic gel:

1. Prepare a fibroblast cell suspension containing 5 x 10⁵ cells per organotypic in DMEM supplemented with 10% FCS and 292 μg/ml L-Glutamine.
2. Make up the organotypic gels on ice in the following ratios:
   • •  7 volumes of Rat-Tail Collagen: Matrigel mix (1:1)
   • •  1 volume of filtered 10x DMEM
   • •  1 volume of FCS
   • •  1 volume of fibroblast cell suspension containing 5 x 10⁵ fibroblasts
     For 9 gels (1 ml per gel), prepare 10 ml and mix gently to avoid bubbles.
3. Add 1 ml to each well of a 24-well plate and incubate for 1 hr at 37 °C in a humidified atmosphere to allow gels to set. After 1 hr, add 1 ml DMEM (10% FCS)/Glut on top of the gels and return to the incubator overnight.
4. The following day, aspirate medium from the top of the gels and plate 1ml of media containing 5 x 10⁵ CRC epithelial cells.

Preparation of gel-coated nylon sheets:

5. Prepare sterile, autoclaved nylon sheets measuring 1.5 cm x 1.5 cm in advance.
6. Using sterile forceps, place the required number of nylon sheets in a 10 cm culture dish (usually 4 nylon sheets can be accommodated per dish).
7. Make up gel on ice in the following order (250 μl total volume is required per nylon sheet):
   • •  7 volumes of Rat-Tail Collagen
   • •  1 volume of filtered 10x DMEM
   • •  1 volume of FCS
   • •  1 volume of DMEM 10% FCS/Glut
8. If the solution is yellow, neutralize by adding 0.1 M NaOH in 50 μl aliquots until the solution turns pink.
9. Add 250 μl of this solution to each nylon sheet and incubate at 37 °C in a humidified atmosphere of 5% CO₂ for 30 min to allow the gel to set.
10. Makeup 1% glutaraldehyde solution in PBS. Add 10 ml to each 10 cm culture dish once the gel has set and incubate at 4 °C for 1 hr.
11. Wash nylon sheets 3x in PBS and 1x in DMEM (10% FCS)/Glu and incubate overnight in DMEM/Glu at 4 °C.

Raising organotypic gels onto steel grids for invasion:

12. Pre-prepare scaffolding grids by folding stainless steel sheets into a tripod formation. Autoclave before use.
13. Use forceps sterilized in ethanol to place a steel grid into each well of a six-well plate.
14. Place a gel-coated nylon sheet with the collagen side uppermost, onto each grid.
15. Carefully transfer the organotypic gels from the 24-well plate to the raised nylon sheet using a spatula sterilized in ethanol.
16. Fill the well with DMEM 10% FCS/Glut supplemented with 10% FCS until the nylon sheet is in contact with the medium but not submerged. Make sure the medium does not touch the organotypic gel.
17. Incubate for 14 days at 37 °C in a humidified atmosphere of 5% CO₂, replacing media every 2 days.

2. Organotypic Fixation

1. Remove whole organotypic including nylon sheet from well and place on cling film.
2. Bisect organotypic and nylon sheet using a clean disposable scalpel and fix both halves in formaldehyde for 24 hr at room temperature.
3. Replace formaldehyde with 70% ethanol after 24 hr and leave overnight before embedding in paraffin, sectioning, and staining.

3. Laser Microdissection of the Invasive Margin

1. Section to 10 μm-thickness onto membrane mounted slides.
2. Deparaffinize sections by applying Xylene for 1 min, then remove Xylene and fix 75% ethanol for a further 1 min.
3. Remove ethanol and stain with 0.125% Cresyl Violet solution for 1 min. Cresyl Violet highlights epithelial cells and permits easy discrimination from the stroma.
4. Remove Cresyl Violet and apply 100% ethanol for a further 1 min before rinsing with 100% ethanol. Allow slides to air dry for 30 min.
5. Conduct laser microdissection using the chosen platform (e.g., the Leica AS system).
6. Place the glass slide with the stained section to be microdissected face down on the microscope stage.
7. Mount a 0.5 ml microcentrifuge tube into the collection cassette and add 50 μl of cell lysis buffer (e.g., DNA, RNA, or protein lysis buffer) to the cap into which microdissected tissue will collect.
8. Under direct vision, use the joystick to identify the tissue of interest. Using the software interface, annotate the stained organic section, highlighting cells to be microdissected at the tumor invasion front.
9. Instruct the laser to fire, which should both cut out the highlighted section and propel microdissected tissue into the cap of the microcentrifuge tube.
10. Once sufficient material has been collected, eject the collection cassette, close the microcentrifuge tube and spin gently to draw the lysis buffer to the bottom of the tube.
11. Place samples on ice until microdissection is complete. Process samples by an appropriate method to extract the analyte of interest.