3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

The overall goal of this procedure is to culture thymic, medullary, epithelial cells, or mtech in a three dimensional organotypic culture or OTC system. This is accomplished by first placing the fibrous scaffold material into a 12 well filter insert, followed by the layering of a freshly prepared fibrin gel containing human skin fibroblasts. After four to five days of pre culture, the MTS are seated onto the scaffold and cultured under submerged culture conditions.

Ultimately, immunohistochemistry, RNA isolation and facts can be performed on the co cultures to assess the development of the CED Mtech. The main advantage of the 3D Organotypic co-culture systems versus the existing 2D culture systems is that they are far superior in recapitulating mtech differentiation, proliferation, and the maintenance of promiscuous gene expression. Today demonstrating this technique will be Iris Martin, a technician from our collaboration laboratory.

When the human dermal fibroblasts reach co fluency, prepare the scaffold fibers by boiling, drying, and ironing the 0.4 to 0.6 millimeter thick pieces of visco non-woven fibrous material. Use a metal hole punch to cut them into 11 millimeter circles. Place the cut fibrous material between two slides, wrap the slides in foil and autoclave them.

Once sterilized, place the inserts into wells of a sterile 12 well plate, and then each scaffold into a corresponding insert. Next mix, 2.7 times 10 to the fifth fibroblasts in 100 microliters of fetal calf serum into 100 microliters of freshly diluted thrombin per scaffold. Dispense the thrombin fibroblast mixture onto each of the scaffold, followed by 200 microliters of freshly diluted fibrinogen.

Then mix the cell mixture within each well distributing the solution evenly over the whole scaffold with gentle pipetting. The image shows the growth of the human dermal fibroblasts with the fibrin gel at day one and day four of culture. Prepare a test gel in a control well without a scaffold to track the time and formation of the clots at this time as well.

Assess the clot formation using a pipette tip or by inverting the plate after the fibroblasts, have clotted submerge the organ cultures in medium, supplemented with la sorbic acid and TGF beta one for four to five days on the day of the mtech seeding, replace this medium with RFAD medium containing 500 units per milliliter of protein in to prevent precocious fibrinolysis. Seven to eight hours later, seed 2.5 times 10 to the fifth MT E suspended in 100 microliters of RFAD onto the top of each scaffold, and to incubate the co cultures for 24 hours. The next day, replace the medium with fresh medium containing 250 units per milliliter of a proin for four to seven more days of culture.

To assess the development of the m Texs by immunohistochemistry at the end of the culture, use a pair of forceps to separate the scaffold dermal equivalent from the filter of each. Well insert and embed the OTCs in OCT compound. Freeze the OTCs in the gaseous phase over liquid nitrogen to isolate the O-T-C-R-N-A.

After separating the scaffold tissue from the filters, use a scalpel to cut the OTCs into pieces and transfer the pieces into a two milliliter RNA free tube containing one milliliter of denaturing solution. Using a fast prep instrument mechanically shred the OTCs twice at a speed of 6.0 for 30 seconds each time with a two minute rest period on ice between each shredding. Then isolate the RNA using acid guad dium thiocyanate phenyl chloroform extraction as described by the manufacturer.

To analyze the OTCs by flow cytometry, remove the membrane and separate the scaffold from the fibrin fibroblast mtech gel. And then finally, cut the gel with a scalpel as just demonstrated. Transfer the tissue into a fax tube.

Continuing two milliliters of collagenase dis paste. Then place the tube in 37 degrees Celsius in a water bath with magnetic stirring for 20 minutes. Agitating bypass your pipette once every five minutes when the tissue is completely digested.

Filter the resulting cell suspension through a 70 micrometer strainer and stain the cells using the appropriate antibodies as illustrated. The MTS can be identified by their keratin 14 expression, making them easily distinguished from the menton positive fibroblasts. Interestingly, the immature and mature M Texs exhibit different but highly reproducible growing patterns in culture.

The immature CD 80 D low M Texs, for example, typically form bilayers in close contact with the fibroblasts. While the CD 80 D high MTS tend to form compact cell aggregates delimited by the fibroblasts, the mtech subsets do not just survive but proliferate under the demonstrated 3D OTC conditions as evidenced by their EDU incorporation. Interestingly, the CD 80 low MT techs proliferate at a higher rate in the presence of rankl.

While the reverse is true for CD 80 high MT techs, After watching this video, you should have a good understanding of how to culture MT.Techs using 3D OTCs. This is a method far superior to using 2D culture systems in terms of tech differentiation and PGE maintenance and induction with the potential for studying or manipulating several take parameters.