Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

We are trying to study the mechanisms responsible for self-tolerance, autoimmunity, especially the role played by air in the expression of self antigens presented by thymic epithelial cells to developing thymocytes. Our research has applications for cell therapy in the context of autoimmune diseases, organ transplants, and cancer. Our system produces thymic organoids, which are formed and seeded in a way that reproduces crucial biological and mechanical cues, which are essential for the maintenance and maturation of thymic epithelial cells.

The which in turn are essential to support thymobiosis, can be maintained for up to six weeks in the system. Our findings contribute to developing better techniques for in vitro t-lymphocyte production with many potential applications for research and cell therapy. As our research progresses, we hope to contribute to a better understanding of the mechanisms underlying self-tolerance and autoimmunity.

We are currently focusing on characterizing our organoids further in terms of cellular composition and organization, and our goal is to explore the role played by the r-gene in regulating cell antigen expression in the thymus and to establish a study model for the autoimmune disease episode. To begin thaw of thrombin and aprotinin on ice, and place a fibrinogen vial in a 37 degree Celsius water bath. Using a pipette, homogenize them under the hood.

Then using sterile pliers, place the inserts into the culture wells, leaving at least one column or row empty on the plate. To a 0.25 milliliter tube first add the required volumes of fibrinogen and aprotinin. Next, add the thrombin into the tube and quickly flush two times without creating bubbles.

Draw the entire content of the tube. Position the pipette vertically above the center of the insert and gently flush a reagent mix without generating bubbles. Incubate for at least one hour at 37 degrees Celsius for solidification until the mix turns opaque white.

For organoid seeding, confirm under a microscope that the micro masses forms spheric cell masses with a compact chorus surrounded by a lower density halo of early thymic progenitors. Cut the tip of a P 200 cone and wash it with an anti-adherent solution. Tilt the plate to a nearly vertical position and aspirate the cell masses from the bottom of the well.

Delicately deposit the mass at the top of the hydrogels without touching the gel. Check under the microscope that no organoid is left in the plate wells. Then slowly add a quarter of the volume of culture medium at the top of the hydrogel without touching it, and add the remaining 3/4s at the bottom of the well.

Add one milliliter of PBS to the empty culture wells to maintain humidity in the plate and incubate it at 37 degrees Celsius with 5%carbon dioxide. The organoids form spheric to oblong structures and occasionally merge to form larger structures in the hydrogels.