This non-conventional protocol presents a facile and robust system to culture. Human epithelial cells into organotypic 3D models that recapitulate the human in vivo tissue. First, epithelial cells are cultured to a confluent monolayer in a tissue culture flask harvested, and then combined with micro carrier beads in the STLV bioreactor, rotate the STLV on the platform to create a constant free fall environment of low fluid shear, allowing the cells to attach and grow on the beads and form cellular aggregates after 96 hours and approximately every day thereafter.
Replenish the media in the SDLV to accommodate for cellular metabolism upon aggregate formation image samples by light microscopy weekly to monitor developmental progression once complete aggregate formation and cellular differentiation has occurred. Seed the aggregates for downstream experimental analysis and assays. Ultimately, this system can generate in vivo like organotypic model systems for a multitude of scientific investigations.
The main advantage of this technique over existing methods like conventional cell culture, is that the epithelial cells will differentiate and polarize into tissue like aggregates, therefore behaving morphologically and functionally similar to the authentic human tissue. This 3D model system provides further insight into epithelial cell biology, host pathogen interactions, and the complex interplay among cell types that may be introduced into the system. Dr.Andrea Radkey.
A postdoc in my laboratory, will now demonstrate this procedure To detoxify the slow turning lateral vessel bioreactor. Cover open ports with lure caps. Fill the STLV with 95%ethanol and equilibrate for 24 hours.
Remove the ethanol, then fill with sterilized distilled water and incubate for 24 hours using tools provided by the vendor, loosen all screws and remove the center plug from the STLV. Place them in a sterilization pouch and autoclave at 110 degrees Celsius for 20 minutes. Reassemble the STLV and repeat the detoxification to ensure sterility.
Next, tighten the screws of the STLV. Attach the pre sterilized one-way stopcock to each port. Now remove the plungers from a 10 milliliter and five milliliter lure lock syringe and releve the plungers in the wrappers.
To maintain sterility, screw the syringes onto the stop stopcock. Proceed to add DPBS to the 10 milliliter syringe until the STLV is full and the five milliliter syringe begins to fill. Then replace the sterile plungers into each syringe and remove any residual bubbles in the bioreactor by alternating between driving plungers of the syringes.
Close the stop cocks and attach the STLV to the rotating vertical platform. Rotate for a minimum of 24 hours to monitor for leaks in a 50 milliliter conical tube. Add 15 milliliters of DPBS to 250 milligrams cyto dex microcar beads and autoclave after cooling.
Remove the DPBS wash three times with culture media for the epithelial cells. Swirling the tube to resuspend the beads. Then resuspend the beads in 15 milliliters of culture media.
First culture one times 10 to the seventh, epithelial cells as monolayers in tissue culture. Flasks harvest the cells and enumerate and determine cellular viability by trian blue exclusion. Staining, add the cells to the prepared beads.
Rinse the conical tube to ensure all cells are transferred. Next, remove DPBS and syringes from the STLV. Also, remove the plug from the center port using a 10 milliliter serological pipette.
Load the epithelial cell bead suspension into the STLV. Rinse the conical tube once to transfer all cells and beads. Then replace the center port plug.
Next, remove the plungers from a five milliliter and a 10 milliliter syringe and releve the plungers in the wrappers. To maintain sterility, screw the syringe's inch ports with open stopcocks. Fill the STLV with media.
Replace the plungers and close the stop.Cox. Now equilibrate the newly seeded STLV culture in a 37 degree Celsius incubator for 30 minutes with no rotation. After removing the STLV from the incubator, open the stop cock.
Remove any bubbles using the plungers and close the stop cock. Then set the STLV to continuous rotation at 20 RPM in the incubator. After 96 to 120 hours, change the media by removing the syringes and opening both stopcocks tilled the STLV to allow the beads and cells to settle.
Once settled, pour off about 75%of the media from the side port using the syringes to perform the feeding protocol. Again, continue to replenish media as needed based on cellular metabolism. Also monitor the growth and viability of aggregates every five to seven days.
To avoid aggregate shearing, use a 1000 microliter pipette tip that has been cut about two centimeters from the point and sterilized Carefully remove 200 microliters of aggregates from the center port and aliquot into two 1.5 milliliter tubes. Use one aliquot for monitoring cellular viability. After sizing cells off the beads, evaluate viability by trian blue exclusion.
Staining for imaging cellular aggregates. Add 0.5 to one milliliter of media to the second 1.5 milliliter Aggregate aliquot and transfer to a small Petri dish using a cutoff. 1000 microliter pipette tip image aggregates.
Using an inverted light microscope for some epithelial cell models, it is necessary to transfer the aggregates to a disposable halve to increase culture aeration approximately one week prior to harvesting aggregates. For analysis, remove the syringes from the STLV and pour off about 50%of the media from the side port. Remove the center plug and carefully pour all contents into a 50 milliliter conical tube from the central port.
Rinse the STLV twice with five milliliters media and combine rinses with harvested aggregates. Next, transfer the aggregates from the 50 milliliter conical tube to the Harv. After one week, carefully harvest aggregates for potential assay formats, assays and analysis differentiated human epithelial aggregates were grown in the STLV bioreactor system.
The SEM and TEM images are collected from human vaginal cells grown in the STLV for 39 days. Note the 3D aggregates display ultra structural features similar to tissues, the micro ridges, invaginations, intracellular secretory vesicles and microvilli on the apical surface confocal immunofluorescence microscopy images of 3D vaginal cells indicate expression of mucin and markers specific for epithelial cells and terminal differentiation. Physiologically relevant phenotypes also serve as an excellent platform for evaluating toxicity of compounds.
The MTT assay, a commonly used assay to measure cell viability, metabolism, or proliferation can be used to measure toxicity to various chemicals and compounds. Alternatively, measure toxicity following treatment with test compounds by trian blue exclusion. In this example, following treatment with the NONOXINOL nine, the vaginal aggregates demonstrated a toxicity response similar to that of cervical X explan models, but a different profile compared to mono layers.
The 3D aggregates are functionally responsive, for example, upon stimulation with molecules derived from microbes that are recognized by the toll-like receptors. The aggregates respond and secrete pro-inflammatory cytokines, including IL six protein and transcript expression of the progesterone receptor. A receptor that responds to hormone stimulation can also be observed in the vaginal cells.
The 3D aggregates not only have the capability to respond to pathogen and hormone molecules, but are also able to functionally support a herpes simplex virus Type two infection. A confocal microscopy image of HSV two infected 3D vaginal aggregates demonstrates infection. The physical and functional properties of individual cells within the aggregates can also be quantified by flow cytometry, for example, of flow cytometry assay to measure muck one's surface expression on individual vaginal cells.
The 3D vaginal aggregates expressed a lower percentage of muck one on their surface compared to monolayers After its development. This technique allowed researchers in the fields of tissue biology and infectious diseases to study structure and physiological responses to stimuli. Using these organotypic models, the better we capitulate human biology.
