This is our lab protocol for growing organotypic intestinal cultures using Caco-2 cells and the Slow Turning Lateral Vessels (STLV) bioreactor set-up from Synthecon.Edit me
- Use sterile tissue culture technique throughout all aspects of this protocol.
- Do not use bleach on the STLV machine or parts at any point Use of bleach may damage the center membrane of the STLV and/or leave residues that affect cell viability.
- Do not use general stock glassware, even if it has been autoclaved. Use only sterile packaged plasticware. We have had numerous situations where the general use glassware has residual detergents that affect cell viability.
Materials and supplies
- RCCS4H 3D Cell Culture System from Synthecon.
- four autoclavable 55 ml slow-turning lateral vessels (STLV)
- CO2 incubator at 37C.
- Inverted light microscope.
- 70% ethanol
- Sterile water
- Dulbecco’s Phosphate Buffered Saline (DPBS)
- Heat inactivated Fetal Bovine Serum (HI-FBS)
- D10 media: DMEM + 10% HI-FBS + 0.05% antibiotic (Pen/Strep)
- Cytodex 3 microcarrier beads (Sigma, cat# C3275).
Preparing the STLVs
- Day 1: Get a clean STLV, tighten the central screw and fill the vessel with 70% ethanol. Be careful when closing it. Leave overnight on the bench.
- Day 2: Discard ethanol of the vessel and replace it with sterilized water. Leave overnight on the benchtop.
- Day 3: Discard the contents and replace with another aliquot of sterilized water. Leave it overnight.
- Day 4: Loosen the screws halfway, take out the center port, remove the luer caps and autoclave the open vessel and the center port, inside an autoclave pouch (15 min, gravity cycle, 121C). Remove from autoclave immediately after the cycle is done. Let it cool and use it immediately or store it for later use, as needed in a clean, dry area.
- Day before use: tighten the screw, fill the STLV with sterile DPBS, close the center port. Screw one-way stopcocks to the other two openings and plug one luer-lock syringes (5 and 10ml) in each side. Let it rotate overnight in the STLV, 20 rpm and monitor for leaks.
Preparing culture beads
- Add 250 μl of Cytodex bead solution to a 50 ml conical tube contain 15 ml of DPBS.
- Autoclave it in liquid cycle, 30 min at 121C. Keep the cap loose, and keep tube in a beaker or heat resistant rack.
- Allow the the beads to cool and settle. Remove the supernatant and discard.
- Wash beads with 15 ml of D10 media. Agitate manually, let the beads settle and discard supernatant. Repeat 3x.
Note: Sterile beads can be stored at 4C for weeks for later use. Do an extra wash with fresh D10 on the day of experiment and bring the final volume to ~15 ml
Caco-2 cell culture
- Pull cells from liquid nitrogen storage. Thaw rapidly in 37C water bath.
- transfer thawed contents to a T25 flask with 8-10 ml of prewarmed D10 media.
- Incubate at 37C and wait for confluent growth.
- Expand cells to four T75 flasks.
- Keep working cultures of the cells by subculturing them once or twice a week, as needed.
Seeding the STLVs
- Remove STLV that was rotating with DPBS overnight. Remove the syringes and the luer caps and drain the DPBS. Replace with new stopcocks and syringes (without the plungers; keep them sterile for later use).
- Trypsinize one T75 flask and respend in 10 ml of DMEM. Each flask is used to seed a single reactor
- Mix 10 ml bead suspension with cells to give 20 ml of volume.
- Transfer the cell-bead suspension carefully to the STLV vessel through the center port.
- Recover residual cell-bead mix from tube with D10 media and transfer to the STLV.
- Close the center port and q.s. volume in STLV vessel to 50 ml with D10 media (until liquid starts overflow back into the syring).
- Take out as many as bubbles as you can by carefully tilting the STLV and using the syringe. Be careful to control pressure to not expel the center port.
- Incubate the STLV for 30 minutes at 37C, 5% CO2 without movement to allow the cells to attach to the beads.
- Examine the STLV and remove any remaining air bubbles
- Set STLV to rotate at 20 rpm at 37C, 5% CO2 for 21-28 days,
Feeding the STLV
- Exchanging the culture medium every other day.
- Remove STLV from incubator, remove syringes, let the beads settle
- Hold the STLV inclined, tapping the center port for the beads to stay in the bottom side of the reactor. Carefully remove ~80% of the spent culture medium through the stopcocks,
- Replace the syringe and fill the vessel with fresh D10. Return the STLV immediately for rotation.
- to monitor cell growth during the culture period, a small volume of bead suspension may be removed via syringe.
Important: use a 1 ml cut pippete tip to prevent shear and protect the 3D structures.
- After 3-4 weeks of continuous culture, transfer 3D culture beads to a 50 mL conical tube.
- adjusted to 15 ml and use as needed for microscopic or RNA Seq experiments.
- Day 1: fill empty vessel with 70% ethanol through the center port. Close all the openings. Be careful when closing it. It may spill. Wear goggle. Leave overnight on bench.
- Day 2: Drain the contents through the center port and refill the vessel with a new aliquot of 70% ethanol.
- Unscrew and take apart all the components. Wash by hand carefully. To wash parts with beads adhered, it is OK to use a toothbrush
Warning: the central membrane is very delicate and is easily damaged- it should only be washed with gentle rubbing…no brushes of any kind
- Let STLV dry on an absorbent paper in clean area (bench is fine).
- to reuse, repeat the ‘preparing the STLV’ above