Preparation of 2-dGuo-Treated Thymus Organ Cultures

Hi, I'm Will Jenkinson from the group of Graham Anderson and Eric Jenkinson at the MRC Center for Immune Regulation at the University of Birmingham. Today I'll be showing you a technique for setting up fetal thymic organ cultures. In addition, I'll also be showing you how to treat these organ cultures with two dioxazine.

And this is a substance that's selectively toxic for lymphoid cells in the organ culture. There are several applications of this technique in the lab. One such application is the production of re-aggregate organ cultures.

Other applications include the study of the different stages of cyte development and using fetal thymic organ cultures, we're able to add in reagents that either inhibit or stimulate different pathways, meaning that we can study the role of specific molecules in this process. So let's begin. First step to preparing thymus organ cultures is to harvest day 14 or 15 mouse embryos from one to two pregnant dams.

First, spray off the euthanized mouse with 70%ethanol. Then use forceps to pull upon the fur and make a V-shaped incision in the abdominal region with scissors starting from the bladder and continuing up the two lateral horns of the uterus. Remove the uterine horn and place it into a sterile 10 centimeter Petri dish.

Cut along the length of the uterine wall and remove the embryos still in their amniotic sacks. Wash them in a Petri dish containing 10 mil PBS using two pairs of fine forceps. Carefully tear open the amniotic sac and free the embryo from the sac and placenta.

Next, transfer the embryos to a fresh petro dish containing 10 mills with a 50 50 mixture of PBS with RPMI 1640. Media with 10%FCS under a dissecting microscope with the embryo submerged in medium, decapitate the embryo using forceps. Open the anterior surface of the chest wall by placing the tips of a closed pair of forceps into the chest cavity and open the forceps to reveal the internal thoracic cavity.

Remove the entire thoracic tree, heart, lungs, trache and thyme by grasping gently below the heart and place into a 35 millimeter dish containing five mils of RPMI 1640 plus 10%FCS in a day. 14 to 15 embryo. The rudimentary thymus lobes are located just above the heart on either side of the truck here.

Remove individual thymus lobes using fine forceps and also remove excess connective tissue and any adherent blood before expanding into organ culture. Now we are ready for organ culture. To make a lymphoid detox, take a Thor nine millimolar stock of two dioxazine and prepare 600 microliters into a 90 millimeter bacterial plastic Petri dish.

Add four mils of prewarm DMEM media and swirl the dish so the entire surface is covered with the media.Obviously. If you want lymphoid cultures to study T-cell development, leave the two dioxazine out using forceps. Place two sterile art wraps, sponges, precut to one centimeter squared into the dish.

Allow the media to soak into the sponge for approximately 30 seconds. Then turn the sponge over to wet both sides. This ensures that the media in the dish has access to the tissue, again, using forceps.

Place a pre sterilized nor 0.8 micron filter onto the surface of each sponge. Filters are place shiny side up onto the sponge supports to transfer the thymus loaves to the organ culture filter, we like to use a math control glass pipet to make glass pipe pets. Heat the glass tubing over a bun and burner flame and when the glass is pable, remove the glass from the flame and pull on both ends to stretch the glass to no more than 10 centimeters, allowing the glass to cool.

Then pull on each end to snap the glass. This produces two tapered glass pipe pets. The thin area of each is about five centimeters long with a tip of about nought 0.5 millimeters in diameter.

To set up the pipette apparatus, place a plastic pipee tip in the other end as a mouthpiece and place the blunt end of a glass pipet into the tubing. Carefully pick up individual thymus lobes with the mouth pipette and place them on the surface of the nor 0.8 micron filter. In organ culture, we recommend putting five to six thymus lobes on each filter for a total of 10 to 12 thymus lobes per dish.

This allows plenty of space for the organ cultures to grow and allows the two dioxazine to eliminate all of the endogenous lymphoid cells. Once the cultures are set up, assemble a sandwich box lid with a lid of a 10 centimeter Petri dish as a platform and double distilled water just below the level the platform. Place up to three dishes into the box and seal the lid to the box with tape.

The lid should have two five millimeter air holes drilled into opposite corners. Place the boxes into a 37 degree Celsius incubator with 10%CO2 for five to seven days after which time we disaggregate and then re-aggregate these cultures to study stromal thymic site interactions in a controlled three-dimensional environment. After a period of culture, typically being five to seven days, F talks are harvested by submerging filters in media and pushing the lobes off gently With forceps, you can see that a lymphoid f tos are typically smaller and cystic compared to non dioxazine treated lymphoid lobes.

So I've just shown you the technique for setting up fetal thymic organ cultures. In this protocol, we've shown you how to treat the fetal thymic organ cultures with two dioxazine and this depletes all lymphoid cells typically after five to seven days of culturing the thine. We use these organ cultures to produce re-aggregate organ cultures.

However, this is not the only technique. We also use non dioxazine treated fetal thymic organ cultures to transfer under the kidney capsule of a thymic mice. In addition, we use lymphoid fetal thymic organ cultures to study T-cell development in controlled conditions in vitro, which are free of any secondary effects that may occur in vivo.

So that's it. Thanks for watching and good luck with your experiments.