The overall goal of this procedure is to describe the development of a multi-cellular 3-D organotypic model of the human intestinal mucosa cultured under micro-gravity provided by rotating wall vessel bioreactors. This method has a wide range potential as a tool for discovery in both health and disease. Including interactions with pathogens, antigen trafficking and the inflammatory processes.
The main advantages of this technique are the mimicking of the phenotypic diversity of the cat and the use of the D-12 vessel bioreactors that allow efficient diffusion of nutrients and oxygen. Start by unscrewing the cap from the fill port of the culture vessel and adding 50 milliliters of our PMI. Once the vessel is full, replace the cap and wipe any spilled medium with 70%ethanol.
Allow the vessel to incubate for at least 15 minutes to overnight at room temperature. When ready to continue, use the fill port to discard the culture medium and add 30 milliliters of the 3-D culture medium to the vessel. Replace the cap on the fill port and again, wipe up any spilled medium with 70%ethanol.
Remove near confluent flasks containing human umbilical vein endothelial cells and human colonic fibroblasts from the incubator and wash them twice with PBS. Then, detach the cells by covering them using a 0.25%trypsin solution with 0.05%trypsin EDTA. Once the cells detach, collect them in a 50 milliliter tube preloaded with 30 milliliters of DMEM containing 30%FBS.
Centrifuge the tube for 10 minutes to pellet the cells. Then, re-suspend the cells in 30 milliliters of DMEM containing 30%FBS. Next, dilute 20 microliters of the re-suspended cells with 20 micro-liters of trypan blue dye.
Load the hemocytometer with the cell mixture and count the concentration of viable cells. Then, re-suspend each cell type at 50 to 80 million cells per milliliter in DMEM containing 30%FBS. First, place the appropriate number of culture inserts into the wells of a six well plate.
Next, prepare the collagen mixture by first adding 10x DMEM, 10x Reconstitution Media, 200 millimolar L-glutamine and heat inactivated FBS to a 50 milliliter conical tube. Then add bovine collagen type one, laminin, collagen type four, fibronectin, heparin sulfate proteoglycan and finally add sodium hydroxide to neutralize the solution's pH. If at any time the mixture develops a yellowish colored acidic appearance, add a few drops of sterile 1 normal sodium hydroxide to neutralize the mixture.
Close the tube and mix the solution by inverting it several times while avoiding bubble formation. When not mixing, keep the solution on ice to prevent it from polymerizing. Once mixed, add the cell suspensions to the collagen preparation and mix the tubes by gently inverting the tubes several times to avoid bubble formation.
Then place them back on the ice. Add four to five milliliters of the cell containing collagen solution into each insert and allow the collagen to gel in the hood with little or no movement for one hour. After one hour, transfer the six well plate to an incubator for one to two additional hours.
Next, return the plate to the tissue culture hood and use a pair of sterile forceps and a scalpel to aseptically cut the membrane from the insert. Detach the cell containing gel from the membrane and then cut the detached gel into small squares. Add the small cell containing collagen squares into one of the pre-filled 50 milliliter vessels using the fill port.
Next prepare a suspension of HCT-8 human epithelial cells as described in the accompanying text protocol. Re-suspend the cells at a concentration of 10 million cells per milliliter in DMEM containing 30%FBS. Then, add one milliliter of the HCT-8 cell suspension into the 50 milliliter vessel using its fill port.
After adding the cells, add an additional 20 milliliters of 3-D culture medium into the vessel so that it is nearly full. Replace the cap and wet the lip of the fill port with 70%ethanol. Next, place a five milliliter syringe containing three to four milliliters of 3-D culture medium into one port of the vessel and an empty five milliliter syringe in the other port.
Remove any visible bubbles by pulling into the empty syringe while approximately the same volume of medium is injected through the other syringe into the vessel. With all of the air now removed, place the vessels in the bioreactor, turn on the power and set the speed at 13 to 14 rotations per minute. Adjust the speed as necessary to prevent the cells from contacting the vessel walls.
Culture the cells under micro-gravity and change the culture medium every three to four days. To change the media, use the dual syringe method to replace approximately 30 milliliters of the used medium with fresh 3-D culture medium. After three to five days in culture, isolate peripheral blood mononuclear cells as described in the accompanying text protocol.
Then remove the vessel from the bioreactor and add approximately 20 million cells consisting of lymphocytes and monocytes to the vessels through the fill port. Place the vessels back in the bioreactor and culture for an additional four to six days. Around day nine of the culture, add an additional 20 million cells consisting of lymphocytes and monocytes into the vessel and continue the cultures for up to 12 additional days.
At the end of the experiment, use a transfer pipette to harvest each small gel fragment, now called a construct, and place them into the wells of a six well plate containing 10 milliliters of 10%formalin buffer. Fix the constructs for three hours at room temperature and then proceed with standard histological embedding, sectioning and staining protocols. The methods presented in this video produce a multi-cellular 3-D organotypic model of the human intestinal mucosa.
In micro-gravity culture, cells become well differentiated and form ordered, villus-like features by day 20. Once mastered, this technique can be done in six hours if it is performed properly. While attempting this procedure, it's important to remember to adhere to good cell culture guidelines.
It is crucial to systematically control cells for viability, mycoplasma contamination and changes in cell growth behavior. Following this procedure, other methods like immunohistochemistry can be performed in order to identify markers of lineage and cell differentiation. After watching this video, you should have a good understanding of how to develop a multi-cellular 3-D organotypic model of the human intestinal mucosa cultured under micro-gravity.
