Name: Reaggregate Thymus Cultures (Video) | JoVE Journal
Uploaded: 2008-08-28T13:00:00.000+00:00
Duration: 5 min 47 s
Description: Learn reaggregate thymus culture methods for studying T-cell differentiation using embryonic thymic lobes. This protocol enables investigation of positive and negative selection processes in immunology research.

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5 minutes

For more information on preparing reggregate thymus cultures please visit <a href="http://www.springerprotocols.com/">Springer Protocols</a>.

The authors have nothing to disclose.

A correction was made to: <a href="http://www.jove.com/video/905/">Reaggregate Thymus Cultures.</a> A revised abstract was republished due to a publisher error. The abstract was corrected to:
Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and development of haemopoeitic cells. Importantly, the maintenance of this three-dimensional organization appears to be critical for normal stromal cell function, with two-dimensional monolayer cultures often being shown to be capable of supporting only individual fragments of lymphoid tissue function. In the thymus, complex networks of cortical and medullary epithelial cells act as a framework that controls the recruitment, proliferation, differentiation and survival of lymphoid progenitors as they undergo the multi-stage process of intrathymic T-cell development. Understanding the functional role of individual stromal compartments in the thymus is essential in determining how the thymus imposes self/non-self discrimination. Here we describe a technique in which we exploit the plasticity of fetal tissues to re-associate into intact three-dimensional structures in vitro, following their enzymatic disaggregation. The dissociation of fetal thymus lobes into heterogeneous cellular mixtures, followed by their separation into individual cellular components, is then combined with the in vitro re-association of these desired cell types into three-dimensional reaggregate structures at defined ratios, thereby providing an opportunity to investigate particular aspects of T-cell development under defined cellular conditions. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).
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In the thymus, immature CD4+8+ thymocytes expressing randomly rearranged T-cell receptor &alpha;- and b-chain genes undergo positive and negative selection events based on their ability to recognize self-peptide/major histocompatibility complex (MHC) molecules expressed by thymic stromal cells. In vivo analysis of the role of thymic stromal cells during intrathymic selection is made difficult by the cellular complexity of the thymic microenvironment in the steady-state adult thymus, and by the lack of appropriate targeting strategies to manipulate gene expression in particular thymic stromal compartments. We have shown that the thymic microenvironment can be readily manipulated in vitro through the use of reaggregate thymus organ cultures, which allow the preparation of three-dimensional thymus lobes from defined stromal and lymphoid cells. Although other in vitro systems support some aspects of T-cell development, reaggregate thymus organ culture remains the only in vitro system able to support efficient MHC class I and II-mediated thymocyte selection events, and so can be used as an effective tool to study the cellular and molecular regulation of positive and negative selection in the thymus.

A correction was made to: <a href="http://www.jove.com/video/905/">Reaggregate Thymus Cultures</a>. A revised abstract was republished due to a publisher error.

Erratum: Reaggregate Thymus Cultures

reaggregate thymus cultures

Reaggregate Thymus Cultures

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and development of haemopoeitic cells. Importantly, the maintenance of this three-dimensional organization appears to be critical for normal stromal cell function, with two-dimensional monolayer cultures often being shown to be capable of supporting only individual fragments of lymphoid tissue function. In the thymus, complex networks of cortical and medullary epithelial cells act as a framework that controls the recruitment, proliferation, differentiation and survival of lymphoid progenitors as they undergo the multi-stage process of intrathymic T-cell development. Understanding the functional role of individual stromal compartments in the thymus is essential in determining how the thymus imposes self/non-self discrimination. Here we describe a technique in which we exploit the plasticity of fetal tissues to re-associate into intact three-dimensional structures in vitro, following their enzymatic disaggregation. The dissociation of fetal thymus lobes into heterogeneous cellular mixtures, followed by their separation into individual cellular components, is then combined with the in vitro re-association of these desired cell types into three-dimensional reaggregate structures at defined ratios, thereby providing an opportunity to investigate particular aspects of T-cell development under defined cellular conditions. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

In this video the preparation of 2-dGuo-treated reaggregate thymus cultures is demonstrated.

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and ...

Hi, I'm Andrea White and I'm from Graham Anderson and Eric Jenkinson's lab at the MRC Center for Immune Regulation at the University of Birmingham. Today I present a protocol for deriving re-aggregate thymic organ cultures, which can be used to study complex cellular interactions which occur during T-cell differentiation. Prior to the start of this procedure, embryonic thymic lobes were isolated and treated to remove all lymphocytes.This can be seen on a separate video. The alienoid lobes are then disaggregated to produce a stromal cell preparation. And finally, the cells are aggregated to form a re-aggregate fmic organ culture.Let's begin. After five to seven days in culture, the LO should be ready to disaggregate for shoal cell preparations submerge the 0.8 micrometer filters in a Petri dish containing RPMI 1640 plus 10%FCS Using forceps gently push off any fiber slopes that do not float off. Use a one mil per pet to transfer the lobes to a 1.5 mil eend orph tube.We recommend no more than 50 lobes per eend orph tube. Allow the lobes to settle to the bottom of the tube under unit gravity and remove the medium. Wash the lobes with one mil PBS and then let the lobes sink to the bottom again.Repeat this for a total of three PBS washes, remove the last PBS wash and add 600 microliters of a 0.25%trips in solution. Place the einor tube in a 37 degree incubator for 25 minutes. Gently tritrate the lobes using a one mil blue tip on a perpet for 10 seconds, and then place back in the incubator after 20 minutes, pipet the lobes up and down to completely disaggregate them.It should take about 30 seconds To achieve a single cell suspension, add 400 microliters ice cold, RPMI 1640 plus 10%FCS to neutralize the trypsin imp pellet the cells by centrifugation at a thousand RPM, 10 minutes at four degrees C, remove the sup natant and resuspend the pellet in one mil. RPMI 1640 plus 10%FCS if desired. Count cells.Typically we would expect around 50, 000 cells per dioxazine lobe, starting from an E 15 fibrous. Now we are ready to form the aggregate fmic Corgan cultures in a one and a half mil eend orph tube. Mixed, freshly prepared fine stromal cells.Together with pre-selected CD four positive CD eight positive fibrocytes. We recommend about a one-to-one ratio of stromal cells to fibrocytes. The number of cells you will use will depend on the number of cells available and the experiments to be performed.We recommend at least a hundred thousand cells in total and no more than a 1.5 million cells. You want the V aggregates to be large enough to be reproducible, but not so large that the center will die pellet cells by centrifugation at a thousand RPM for 10 minutes. At four degrees C, assemble the organ culture filter carefully.Remove all a SUP natin in stage with a P 200 per pet. The vortex cell pellet for 10 seconds to form a slurry. The volume should be around one to two microliters mouth.Pet the slurry into a finely drawn pet, keeping the suspension of cells at the tip of the pet. Under our dissecting microscope, carefully mouth PET the cell slurry onto the center of a 0.8 micrometer filter. Ideally, cells should be placed as a standing drop in a circle of one to two millimeters in diameter.If the slurry begins to spread out, allow the fluid in the slurry to drain through the pos before depositing the remaining cells. Place dish in a plastic sandwich box and seal. As for thymus organ cultures, and place a box in an incubator, we aggregate cultures form into intact structures within 18 hours of culture.After four to five days of culture, they will generate both CD four positive eight negative and CD four negative eight positive mature cyte subsets. Starting with 50 dioxazine treated lobes should give enough stromal cells to prepare three to five reaggregate cultures. These cultures typically appear as coherent mounds of cells that can be pushed off the filters using forceps without disrupting them.We have just described the method in which to study positive selection by F epithelial cells. This method can also be used to study dendritic cell mediated negative selection. Finally, by using TCR transgenic FIBROCYTES or MHC Class one deficient or MHC Class two deficient stromal cells.This system allows separate investigations into CD four positive or CD eight positive T-cell selection. Thanks for tuning in.

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Learn reaggregate thymus culture methods for studying T-cell differentiation using embryonic thymic lobes. This protocol enables investigation of positive and negative selection processes in immunology research.

Reaggregate Thymus Cultures

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