Preparation and Applications of Organotypic Thymic Slice Cultures

Summary

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Abstract

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

Introduction

T cells differentiate through a series of developmental intermediates in the thymus during which time they encounter several checkpoints that ensure the generation of a functional, self-tolerant T cell repertoire^1-3. Positive selection promotes the survival of thymocytes with T cell receptors (TCR) capable of recognizing, with low to moderate affinity, peptide presented by major histocompatibility complex molecules (MHC) on cortical thymic epithelial cells (cTEC)^2,3. Negative selection and regulatory T (T_reg) cell development contribute to the establishment of self-tolerance via the elimination or diversion of thymocytes that respond strongly to self-peptide presented by MHC^2,4. Immature CD4⁺CD8⁺ double positive (DP) thymocytes expressing TCRs that pass the selection process differentiate into mature T cell subpopulations, the majority of which are MHC class I-restricted CD8⁺ cytotoxic or MHC class II-restricted CD4⁺ helper single positive (SP) T cells, before exiting the thymus to perform effector functions in the secondary lymphoid organs^1-3.

Adding to the complexity of T cell development is the dynamic migration and cellular encounters of developing thymocytes throughout the stromal cell network^5-9. These stromal cells play distinct roles in thymocyte development and are differentially distributed between the thymic cortical and medullary regions where positive and negative selection occur¹⁰. Although positive selection takes place primarily in the cortex, there is accumulating evidence that DP thymocytes migrate to the medulla and continue to require TCR signals before they differentiate into mature T cells suggesting that the medulla may provide additional signals necessary for completion of positive selection and lineage differentiation^11,12. Further, despite the presence of specialized medullary thymic epithelial cells (mTEC) that express and present tissue-restricted antigens facilitating deletion of autoreactive thymocytes^13,14, a large proportion of negative selection occurs in the cortex in response to ubiquitously expressed self-peptide presented by dendritic cells^15,16. Thus, accurate models of T cell development must provide a highly organized thymic microenvironment, with intact cortical and medullary regions, that facilitates interaction between thymocytes and stromal cells, and supports thymocyte migration as these cells undergo positive and negative selection.

To complement ex vivo analyses of thymocytes as a means of studying positive and negative selection, a number of in vitro, in situ, and in vivo models of T cell development have been developed^17-22. It has been notoriously difficult to recapitulate positive selection in vitro, but coculture of stem cell populations or T cell precursors with stromal cells expressing Notch ligand, notably OP9-DL1/4 cells, has the capability to support T lineage commitment and limited positive selection making it an invaluable in vitro model to study T cell development^23-25. Limitations of this system, however, include the fact that these cells lack the unique peptide processing machinery found in thymic stromal cells and the three-dimensional thymic microenvironment.

Though more technically cumbersome, in situ and in vivo models of thymic selection can overcome some of the barriers related to in vitro systems. Reaggregate thymic organ cultures (RTOC) contain defined mixtures of thymocytes and thymic stromal cells^18,26,27. These thymic epithelial cell reaggregates maintain MHC class I and II expression and can support development of both conventional T cell subsets, yet still lack defined cortical and medullary structures. Fetal thymic organ culture (FTOC) is a popular model of T cell development that can be seeded with thymocytes via hanging-drop culture of lymphodepleted thymic lobes or via injection of thymocytes into lymphoreplete thymic lobes and support efficient development of CD4⁺ and CD8⁺ T cells over time in culture^18,28-31. At the initiation of culture of fetal thymic lobes there is a paucity of mTECs, but defined cortical and medullary structures may develop over time depending on conditions. An important consideration is that this model may preferentially support fetal versus adult T cell development. Finally, intrathymic injection of defined thymic precursors in adult mice is technically challenging but clearly provides an environment to support T cell development in vivo. These in situ and in vivo models are excellent tools to study T cell development and their use should be considered on an experiment-by-experiment basis.

Thymic slices, however, have recently emerged as a versatile, complementary model to study thymic selection in situ with the possibility to accommodate unique, complex, and generally higher throughput experiments. Thymic slices maintain the integrity of the cortical and medullary regions and provide a framework of stromal cells that supports thymocyte migration during development as well as efficient positive and negative selection^11,32-39. Thymocyte subsets added atop thymic slices migrate into the tissue and to their appropriate microenvironmental niche^34,37. The overlaid thymocytes can be distinguished from thymic slice endogenous cells via congenic markers or fluorescent labels and can be maintained in culture for several days. Thymic slice organotypic cultures can be used to study various aspects of T cell development including thymic selection, thymocyte behavior (migration and cellular interactions), and thymocyte localization, among others. Given the ability to generate ~20 thymic slices per mouse, the scalability of experiments is generally greater than other in situ models of thymic selection. Although the preparation of thymic slices requires specialized equipment, such as the vibratome, and the life time of thymic slices in culture is limited owing to loss of cells over time via cell death and the lack of an encapsulating membrane, thymic slices provide an excellent model for analysis of thymic selection of synchronized populations of thymocytes within a mature thymic microenvironment. Here we describe the preparation of thymic slices (including harvesting the thymus, agarose embedding of thymus lobes and vibratome sectioning of the embedded tissue), isolation and overlaying of thymocytes, and dissociation of thymic slices for flow cytometric analysis.

Protocol

Protocols for all animal studies were approved by the Animal Care Committee at the Centre de recherche - Hôpital Maisonneuve-Rosemont.

1. Harvesting Mouse Thymus for Preparation of Thymic Slices and Single Cell Suspensions

1. Euthanize the mouse with CO₂ followed by cervical dislocation.
2. In a laminar flow hood, pin the mouse ventral side up to a dissection board. Spray the mouse with 70% ethanol. Remove any excess alcohol by dabbing with gauze to prevent ethanol from entering the thoracic cavity and damaging the tissue.
3. Lift the skin at the base of the sternum with a pair of forceps and make a cut through the skin. Extend the cut of the skin upwards to each forelimb.
4. Make an additional cut at the base of the sternum to separate the diaphragm from the rib cage. Then cut each side of the rib cage towards the clavicle. Flip the rib cage over towards the head exposing the thoracic cavity. The two lobes of the thymus lie on top of the heart.
5. Remove the connective tissue around the thymic lobes using forceps, micro-dissection scissors, and/or sharp curved scissors. Use a pair of fine tip curved forceps to lift the individual lobes from underneath or via remaining connective tissue.
   Note: Do not grasp the thymus directly.
6. Place the thymic lobes in a 15 ml conical tube containing PBS and set aside on ice until needed.

2. Agarose Embedding and Vibratome Slicing of Thymic Lobes

1. Prepare 4% agarose solution for embedding by dissolving 2 g low-melting point agarose in 50 ml sterile PBS and microwaving on a low setting until the agarose has completely dissolved. Cover flask with aluminum foil and place in a water bath at 55 °C until ready for use.
2. In a tissue culture hood, prepare complete RPMI-1640 media that contains 10% fetal bovine serum (FBS), 4 mM L glutamine, 1x penicillin/streptomycin, and 10 µM 2-mercaptoethanol.
3. Add 1.5 ml complete RPMI-1640 media per well to a 6-well cell culture plate and place a cell culture insert in each well. Set the plate aside in an incubator at 37 °C until needed.
4. Prepare a slushy ice water bath with water and ice in an ice bucket.
5. Carefully remove all remaining connective tissue surrounding each thymic lobe using fine tip forceps. Do this while the thymic lobe is submerged in PBS in a tissue culture dish, after transfer onto a tissue wipe soaked with PBS, or under a dissecting microscope.
6. Allow the agarose to cool below 40 °C, when the flask is just warm to the touch, to avoid overheating the tissue. Pour the cooled 4% agarose into a tissue mold to a height of ~1 cm.
7. Use a pair of forceps to carefully transfer the thymic lobe to a tissue wipe and roll it gently to dry it without damaging the tissue. Ensure that the tissue dries completely or it will slide out of the agarose during slicing.
8. Carefully insert the lobe into the agarose and position it either horizontally (to increase surface area of each slice) or vertically (to increase number of slices) at the bottom of the mold. Place the mold in ice water for 5-10 min to allow the agarose to solidify.
9. During this time, prepare the vibratome for slicing by mounting the buffer tray, assembling the specimen disc, and inserting the vibratome blade. Place a piece of laboratory tape on the specimen disc. Sterilize the clean, assembled workspace with 70% ethanol.
10. Once the agarose solidifies, invert the mold and press gently at its center to release the agarose embedded lobe.
11. Use a sharp blade to trim the excess agarose surrounding the lobe leaving ~2 mm of agarose on each side and ~0.5 cm at the bottom.
12. Secure each agarose block with a drop of tissue glue to the piece of tape on the specimen disc. Multiple blocks can be glued on the tape.
13. Align the vibratome blade with the top of the agarose block. Fill the buffer tray with sterile PBS until the blade and agarose block(s) are fully immersed.
14. Section the agarose embedded tissue to obtain slices with a thickness of 400-500 µm. Set the vibratome to 0.225 mm/sec speed, 100 Hz frequency, and 5° angle.
15. Use a bent spatula to collect the thymic slices into a tissue culture plate containing sterile PBS as they are cut. Discard the first and last slice.
16. Examine the slices under a light microscope at 4X magnification. Choose the slices with intact thymic tissue and surrounding agarose (Figure 1A).
17. Transfer the slices to the plate prepared in step 2.3 by using a pipette tip to gently slide the thymic slice from the bent spatula onto the cell culture insert. Each insert can accommodate ~3 slices. Make sure the slices do not touch each other or the cell culture insert walls.
18. Maintain the plate at 37 °C until needed.

3. Preparation of Thymocytes for Overlaying

1. Isolate thymic lobes as described in section 1. Dissociate the lobes manually to make a single cell suspension using a sterilized 15 ml tissue grinder filled with 5 ml of sterile PBS containing 2% FBS. Transfer the cells to a 15 ml conical tube.
2. Centrifuge the cells at 545 x g for 5 min at 4 °C. Discard the supernatant and resuspend the cells in 1 ml of 1x ACK lysis buffer (0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA) at room temperature for 3 min to lyse red blood cells.
3. Fill the tube to 15 ml with PBS containing 2% FBS. Centrifuge the cells at 545 x g for 5 min at 4 °C.
4. Resuspend the cells in 10 ml of PBS containing 2% FBS and pass cells through a 255 µm mesh filter.
5. Count cells using a hemocytometer. Centrifuge the cells at 545 x g for 5 min at 4 °C, and resuspend the cell pellet in PBS containing 2% FBS at a concentration of 1 x 10⁷ cells per ml.
6. Label the cells with cellular dyes such as carboxyfluorescein succinimidyl ester (CFSE) according to manufacturer protocols if not using thymocytes from mice expressing congenic markers or genetically encoded fluorescent reporters to distinguish from slice endogenous cells.
7. After the final wash of the labeled cells, resuspend the cell pellet in complete RPMI-1640 media at 1-3 x 10⁶ cells per 15 µl.

4. Overlaying Thymocytes onto the Thymic Slices

1. Use a pipette to aspirate any liquid surrounding the thymic slices on the insert.
   Note: Take care to avoid damaging the agarose. If the agarose is damaged, the overlaid thymocytes will not attach to the slice surface due to loss of surface tension.
2. Without touching the slice with the pipette tip, overlay 15 µl of thymocytes prepared in section 3 onto each thymic slice.
3. Incubate the plate at 37 °C for 2 hr to allow the cells to migrate into the thymic slices.
4. After 2 hr, rinse the thymic slices 3 times with 1 ml PBS to remove excess overlaid thymocytes that have not yet migrated into the tissue.
5. Incubate the plate at 37 °C until the thymic slices are ready to be harvested, typically 1-72 hr depending on the experiment.

5. Dissociation of Thymic Slices for Flow Cytometry

1. Prepare a microcentrifuge tube for each slice by adding 150 µl of PBS containing 2% FBS.
2. Add 1 ml of PBS containing 2% FBS to the tissue culture insert with the thymic slices and gently stir with a bent spatula to detach the slices from the surface of the cell culture insert.
3. Transfer the slice from the insert to the microcentrifuge tube using a bent spatula.
4. Manually disrupt the slice using a microcentrifuge tube sample pestle. Add 150 µl of PBS containing 2% FBS to a final volume 300 µl.
5. Filter the dissociated tissue through a 40 µm filter into a new microcentrifuge tube. The filtered cells are ready to be stained for flow cytometric analysis⁴⁰.
6. Filter cells a second time before passing them on a flow cytometer to ensure that all the agarose is removed and does not clog the flow cytometer.
7. Acquire the cells on a flow cytometer and analyze data as has been previously described⁴⁰.

Results

Thymic slices support analysis of different aspects of T cell development such as positive and negative selection. For successful experiments, the quality of the thymic slice is paramount. Thus, thymic slices should be examined to ensure the integrity of the thymic tissue and that the agarose surrounding the thymic slice is intact (Figure 1A). Surface tension can be compromised when the agarose is damaged causing a significant decrease in the number of th...

Discussion

Here we describe a protocol for the preparation of thymic slices and representative results of efficient positive and negative selection of overlaid pre-selection MHC class I-restricted TCR transgenic thymocytes by flow cytometry. This system has been used with similar success to support positive selection of MHC class II-restricted CD4⁺ T cells from pre-selection DP thymocytes³², and, in the presence of agonist antigen, negative selection and thymic T_reg development^{11,12,36,38,39,43...}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vibratome	Leica Biosystems	VT1000S
NuSieve GTG Agarose	Lonza	50080	Low melting temperature agarose
Embedding Mold (Truncated - T12)	Polyciences	18986	22 mm x 22 mm square, truncated to 12 mm x 12 mm
Double Edge Prep Blades	Personna	74-0002
Tissue Adhesive	3M	1469SB
0.4 µm Cell Culture Inserts	BD Falcon	353090	Of several brands tested, these maintained the cells atop the slices the best
Dulbecco's Phosphate-Buffered Saline	ThermoFisher	21600-010
RPMI-1640 with L-glutamine	Wisent	350-000-CL
Fetal Bovine Serum	Wisent	080-110	Heat inactivated
L-Glutamine, 200 mM	Wisent	609-065-EL
Penicillin/Streptomycin, 100x	Wisent	450-201-EL
2-Mercaptoethanol	Alfa Aesar	A15890
15 ml Tenbroeck Tissue Grinders	Wheaton	357426
Nylon Mesh Filter	Component Supply	U-CMN-255
Microcentrifuge Tube Sample Pestle	Bel-Art	F19922-0000
40 µm Nylon Cell Strainer	BD Falcon	352340
Forceps Inox Tip	Dumont	RS-5047	Fine tip curved forceps, size .17 x .10 mm
Micro Forceps	Dumont	RS-5090