Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

1. Preparation of Tools, Enzyme Solutions, and Buffers

Perform the following preparative steps prior to beginning the protocol.

1. Tools
   Clean, dry and autoclave the following tools and keep them in sterile packaging until use.
   1. Small sharp scissors with either curved or straight tips for cutting the thymus tissue. Small curved forceps with serrated tips for handling the tissue.
   2. 50 ml Oak Ridge Centrifuge Tubes, PC, for the Percoll-density centrifugation step.
2. Enzyme solutions
   Prepare the Collagenase/DNase I enzyme mix (2 mg Collagenase/ml and 0.1 mg DNase I/ml) as follows:
   1. Dissolve 500 mg of lyophilized Collagenase A (Roche) in 250 ml of RPMI 1640 plain (no FCS) to obtain a 2 mg/ml Collagenase solution. Collagenase is quite hard to dissolve, so it is recommended to leave it some time on a roller-shaker at RT.
   2. Dissolve 100 mg DNase I (Roche) in 10 ml of sterile distilled water. 2.5 ml aliquots can be stored at -20 °C. Add 2.5 ml of the 10 mg DNase I solution in the 2 mg Collagenase A solution.
   3. Sterile filter the Collagenase/DNase mix using a Stericup filter unit (0.22 μm). Store in 10-20 ml aliquots at -20 °C until use.
3. Buffers and Solutions
   1. 1.5 M NaCl stock solution: Dissolve NaCl in distilled sterile water to a final concentration of 1.5 M. Filter the solution through a 0.22 μm filter and store at RT.
   2. 10x MACS buffer: 1x PBS (Ca²⁺/Mg²⁺-free) containing 5% BSA and 20 mM EDTA. Sterile filter the solution with a 0.22 μm filter and store at 4 °C. Prior to use prepare a 1x solution by diluting the 10x stock to 1x in cold sterile 1x PBS.
   3. FACS Buffer: 1x PBS (Ca²⁺/Mg²⁺-free) containing 1% BSA and 0.02% NaN₃.
   4. FACS Buffer for cell sorting: 1x PBS (Ca²⁺/Mg²⁺-free) containing 2% BSA. Sterilize using a 0.22 μm syringe filter and store at 4 °C.

2. Preparation of the Tissue

The processing of the tissue should be performed using sterile reagents and work in a laminar flow cabinet. Before the procedure begins, prepare the following reagents and equipment:

1. 1. Warm the following to RT: RPMI 1640 complete medium (RPMI 1640, 10% Fetal Calf Serum, 1% pen/strep), RPMI 1640 plain, Ca²⁺/Mg²⁺-free PBS and 2x Collagenase/DNase enzyme solution.
   2. Warm thermal incubator with rotation unit to 37 °C and cool fixed angle rotor centrifuge to 4 °C.
   3. Prepare a box with ice.

Note: the duration of this step depends on the condition and size of the tissue. Approximately 20-30 min is reasonable time needed for a medium size piece of tissue in good condition (~5 cm in width).

2. Place tissue in a Petri dish containing sterile PBS and rinse off any residual blood.
   1. Add fresh PBS to prevent tissue from drying and using forceps and scissors clean the tissue from blood clots, connective and fat tissue and any part that does not look healthy.
   2. Take care to remove necrotic tissue that will increase the amount of debris.
   3. After cleaning, weigh tissue for reference.
   4. Cut the tissue in approximately 1 cm³ pieces/blocks. Add enough PBS to cover the tissue.

Note: For reproducibility of results, tissue should be cut into pieces of uniform size.

3. Using the back of a sterile syringe (10-20 ml) apply pressure (not too vigorous or prolonged) onto the thymic tissue pieces. This procedure will remove the bulk of thymocytes, thus reducing the tissue volume to be digested later. Perform this step on ice and work fast.
4. The solution will become visibly cloudy as thymocytes are released. Stir the dish gently and then with the help of a 5 ml pipette or a glass aspirator remove the supernatant containing the thymocytes taking care not to accidentally aspirate the tissue pieces.

Tip: A sterile cell culture scraper can be used to concentrate all tissue pieces at one side of the dish then tilt the dish (45° angle) and aspirate the supernatant. Replace with fresh PBS and repeat this procedure until the supernatant is relatively transparent. Perform last wash with RPMI plain.

5. Mince tissue pieces using sharp scissors (as finely as possible, fragments should be at least 2-4 mm). The fragments should be small enough to enter a 5-ml pipette.

3. Preparation of Single Cell Suspension from Thymus Tissue

Here, two alternative approaches for this step are described. The first approach describes a typical enzymatic tissue digestion (section 3.1) while the second involves mechanical and enzymatic tissue treatment assisted by a tissue dissociator (section 3.2).

This protocol is optimized for the processing of tissue samples weighing 5 g. For larger or smaller tissue samples, adjust enzyme volumes accordingly.

3.1 Typical enzymatic tissue digestion to obtain a single cell suspension

1. Place mashed tissue into 50 ml tubes with 10 ml collagenase/DNase solution per 5 g of tissue. Add RPMI plain to give a total volume of 20 ml.

Note: in a 50 ml tube digest up to 10 g of tissue. Adjust enzyme volume accordingly.

2. Incubate the tissue suspension for 40 min at 37 °C under slow rotation in a thermal incubator.

Note: the incubation steps can be performed either in a thermal incubator with a rotation unit, or in a 37 °C bacterial incubator with a shaker.

3. The enzyme solution will become cloudy as cells are released into it. At the end of the digestion, centrifuge tube at 110 x g for 2 min to sediment tissue fragments.
4. Collect supernatant from this digestion round. Pellet the cell suspension for 10 min at 400 x g. Aspirate supernatant and resuspend cells in 10-50 ml of RPMI complete depending on the size of the cell pellet.
5. Keep cell suspension in 37 °C incubator with the cap loose.
6. If larger cell numbers are desired, the digestion step can be repeated with the remaining tissue fragments and the first and the second digest pooled. Also, if TEC should be isolated, two rounds of digestions should be performed.
7. Dilute an aliquot of the cell suspension in trypan blue (1:10-1:100) and count viable cells using a hemocytometer.
8. Keep cells at 37 °C in the incubator until ready to proceed to the Percoll density centrifugation (section 4). Subsequent isolation of DC subsets (section 5) will be performed from these cells.

Note: thymic tissues can vary in their composition, especially with age, which affects the digestion efficiency and the generation of a single-cell suspension.

9. For the subsequent isolation of thymic epithelial cells (TEC) a third round of enzymatic digestion is required. Resuspend tissue remnants in 10 ml of fresh Collagenase/DNase solution plus 10 ml of RPMI plain and add Trypsin/EDTA (1:50 from 2.5% stock) in the solution.
10. Incubate at 37 °C for 40 min with gentle rotation. During the last 10 – 15 min of the incubation add FCS (1:5) to neutralize the activity of Trypsin.
11. Centrifuge at 110 x g for 2 min at RT to sediment the tissue fragments.

Note: The tissue may not be completely digested after these three rounds of digestions. Age and overall condition of the tissue are two parameters that influence the dissociation efficiency of the thymus tissue.

12. Collect the cell supernatant and discard the sedimented tissue fragments.
13. Centrifuge the cell supernatant at 400 x g for 10 min. Aspirate supernatant and resuspend cell pellet in RPMI complete.

Tip: Cell pellets are quite loose, so take care when discarding/aspirating supernatant.

14. Count viable cells using a hemocytometer. Place the cells in a 37 °C incubator until proceeding to Percoll separation (section 4). Subsequent pre-enrichment of TEC cells by depletion of CD45^hi cells will be performed from these cells (section 6).

3.2 Mechanical and enzymatic tissue treatment assisted by a tissue dissociator

Using the tissue dissociator approach the tissue digestion steps can be reduced to half of the time needed with the approach described above (section 3.1). The following protocol is a modified version of that used to dissociate mouse thymus tissue for the isolation of TEC^7,8.

1. Transfer 5 g of minced tissue pieces (step 2.5) into a C Tube containing 10 ml of Collagenase/DNase solution.
2. Adapt C tube onto the tissue dissociator and run program m_spleen_02 (10 sec). Repeat this program 4-5x (40-50 sec).
3. Incubate with gentle rotation for 20 min at 37 °C.
4. Centrifuge at 110 x g for 2 min at RT.
5. Collect supernatant and proceed as in step 3.1.4. Add 10 ml of fresh Collagenase/DNase solution.
6. Adapt C tube onto the tissue dissociator and run program m_spleen_01 (56 sec).
7. Incubate at 37 °C for 20 min with gentle rotation.
8. Centrifuge at 110 x g for 2 min at RT.
9. Collect cell supernatant and pellet the cell suspension for 10 min at 400 x g. Proceed as in step 3.1.4.
10. As described in 3.1.9 a third round of digestion is needed for the subsequent isolation of thymic epithelial cells (TEC). For this, resuspend tissue remnants in 10 ml of fresh Collagenase/DNase solution plus Trypsin/EDTA (1:50 dilution of stock 2.5%)
11. Incubate at 37 °C for another 20 min with gentle rotation. Add FCS (1:5) and incubate for further 10 min.

Note: Using this approach, after this last digestion step the tissue is usually completely dissociated and no undigested tissue fragments are observed.

12. Proceed as in steps 3.1.11-3.1.14.

4. Enrichment of LDF of Cells Using Percoll-density Separation

After enzymatic digestion of the tissue, total thymic single cell suspensions are subjected to a single Percoll density centrifugation step to enrich for the LDF cells. The LDF of cells is highly enriched for APC. Both DC and TEC are detected in this fraction of cells.

1. Use single cell suspension obtained from the 1^st and 2^nd digests for mDC isolation (pooled cells, see step 3.1.8) or 3^rd digest (for TEC isolation) (3.1.14). Centrifuge single cell suspension obtained from each digestion step at 400 x g for 10 min.
2. Prepare a Percoll solution with a final density of 1.07 g/ml during this wash step (see example in the Table below).

   Preparation of Percoll solution to a final density of 1.07 g/ml (ρ = 1.07)
   Tube No.	1	2	3	4
   Undiluted Percoll (ρ = 1.130) (ml)	2.96	5.92	8.88	11.84
   1.5 M NaCl (ml)	0.6	1.20	1.8	2.4
   distilled H2O (ml)	2.44	4.88	7.32	9.76
   Volume of final working dilution	6 ml	12 ml	18 ml	24 ml

3. The number of Percoll tubes depends on the cell numbers determined in steps 3.1.8 and/or 3.1.14. Combine the sterile solutions and mix thoroughly. For optimal isolation, 0.6-1 x 10⁹ cells can be loaded per tube of Percoll. Combine the sterile solutions and mix thoroughly.

Tip: Percoll is light sensitive and in addition it should be kept cold. Prepare first the mixture containing the NaCl and H₂O (RT) and add undiluted Percoll last, just before resuspending the cells in the Percoll solution.

Note: Percoll density might vary between different suppliers and batches! If the density of the undiluted Percoll solution is not equal to 1.130 g/ml, use the instructions supplied by the manufacturer to calculate the exact amounts of Percoll and H₂O required to get a final density of 1.07 g/ml.

4. Resuspend up to 1 x 10⁹ cells in 6 ml of the prepared Percoll solution (ρ = 1.07), mix sufficiently to get a homogenous suspension, and transfer to a 50 ml Oak Ridge polycarbonate screw cap centrifuge tube.
5. Carefully layer 30 ml of RPMI complete per tube with a pipette on top of the Percoll solution/cell suspension. Load the medium slowly, so that it remains on the top of the suspension and take care not to disturb the layer.
6. Weigh the tubes to make sure that they have equal weights so that they are balanced during centrifugation. If they are not, carefully add more medium to the lighter tube (under sterile conditions).
7. Carefully transfer the tubes to a pre-cooled centrifuge with a fixed angle rotor, and spin at 3,500 x g for 35 min, at 4 °C with the brake off. Make sure that the temperature of the centrifuge in not higher than 4 °C.
8. Remove tubes from the centrifuge and carefully collect the enriched APC, found at the interphase between Percoll and medium (low-density fraction) from each tube using a sterile Pasteur pipette. Transfer cells into a 50 ml tube containing cold RPMI complete. Fill up the tube in order to dilute out the remaining Percoll.
9. Centrifuge the cell suspension for 10 min at 300 x g at 4 °C. Discard supernatant and repeat wash.
10. Resuspend the cell pellet in medium and determine the cell number (using trypan blue and a hemocytometer). In our experience, the percentage of the LDF cells is about 2-20% of the total single cell suspension obtained after enzyme digestion. A typical yield of low-density enriched cells (from a young thymus, range 1 day-2 years) is 1×10⁸ cells per 1 x 10⁹, representing a 10% of total single cell suspension cells.

Note: At this stage, you may observe that cells aggregated in the suspension (especially with samples from children of older age). Cell clumps are indication of cell death and result from the release of DNA from the dying cells that can stick cells together. In such a case, add DNase I into the sample (50 μg/ml) and incubate it for up to 20 min at RT (gently invert every 5 min) to digest the free DNA molecules.

5. Isolation of Thymic mDC

After APC enrichment (via Percoll separation), mDC can be efficiently isolated from the LDF following the first and second round of enzymatic digestions. The following protocol is a modified version of magnetic cell separation for the isolation of mDC (CD11c⁺).

1. Centrifuge APC enriched cells isolated from the single cell suspension obtained from the pooled first and second enzymatic digestion at 300 x g for 10 min at 4 °C and resuspend cell pellet in 100 μl MACS buffer per 10⁷ cells.
2. Add 5 μl of CD11c-PE antibody per 10⁷ cells.

Note: Titration experiments are recommended for optimal results.

3. Mix well and incubate for 15 min at 4 °C, protected from light.
4. Wash cells by adding 1-2 ml of MACS buffer per 10⁷ cells and centrifuge at 300 x g for 10 min.
5. Aspirate supernatant completely and resuspend up to 10⁷ cells in 80 μl of MACS buffer.
6. Add 20 μl anti-PE microbeads per 10⁷ cells.
7. Mix well (Do Not Vortex) and incubate for 15 min at 4 °C protected from light.
8. Repeat as in step 5.4.
9. Resuspend up to 10⁸ cells in 500 μl of buffer.
10. If necessary, filter cell suspension using a 40 μm cell strainer to remove debris and cell aggregates that may obstruct the flow of the column.
11. Use a LS column since thymus cell suspensions flow better through the LS columns. Prepare LS column following manufacturer's instructions. Pipette cell suspension slowly into the column avoiding generating bubbles. Use one column for every 1 x 10⁸ cells.
12. Wash column following the manufacturer's instructions. Remove column from magnet and place it in a sterile 12 ml round bottom polypropylene tube. Add 3 ml MACS buffer onto the column and collect magnetically-labeled cells by inserting and firmly pushing the plunger into the column.
13. Collect the eluted fraction representing the CD11c⁺ mDC and centrifuge at 400 x g for 6 min.
14. Determine cell number and confirm purity of the selected cell population by flow cytometric analysis. Suggested markers include CD45, CD11c, and HLA-DR.

Note: CD11c⁺mDC may also be isolated using a FACS sorter.

6. Enrichment of CD45^lo/neg Cells for Cell Sorting of TEC

For isolation of TEC, cells can be pre-enriched by depletion of CD45^hi cells using CD45 microbeads, in order to speed up their isolation through cell sorting.

Use single cell suspension obtained from the 3^rd digestion step (3.1.14) and subsequently separated by Percoll centrifugation.

1. Centrifuge cell suspension at 300 x g for 10 min at 4 °C and resuspend cell pellet in 80 μl MACS buffer per 10⁷cells.
2. Use CD45 microbeads at 1/3 of the recommended amount (6.7 μl) per 10⁷cells.

Note: By using a lower amount of the microbeads we pre-enrich cells for both CD45^lo and CD45^neg cells. Titration is recommended for optimal results.

3. Mix well by gently flicking the tube (Do Not Vortex) and incubate for 15 min at 4 °C.
4. Wash unbound microbeads by adding 10 ml of MACS buffer and centrifuge for 10 min at 300 x g at 4 °C. Aspirate supernatant completely.
5. Resuspend up to 10⁸ cells in 500 μl of buffer.
6. If necessary, filter cell suspension using a 70 μm cell strainer to remove debris and cell aggregates that may obstruct the flow of the column.
7. Use a LS column since thymus cell suspensions flow better through the LS columns. Prepare LS column following manufacturer's instructions. Pipette cell suspension slowly into the column avoiding generating bubbles. Use one column for every 10⁸ cells.
8. Collect unlabeled cells (flow-through and washes) containing the CD45^lo/neg cell fraction and wash column following manufacturer's instructions.
9. Centrifuge at 300 x g for 10 min at 4 °C and resuspend cell pellet in sterile FACS buffer in order to determine cell number and proceed to staining for cell sorting.

7. Stain Cells for Fluorescence Activated Cell Sorting

1. Perform FcR blocking prior to cell staining, in order to reduce non-specific antibody binding. Incubate cell suspension with pooled human immunoglobulins solution for 15 min at RT. These reagents are commercially available (i.e. Gammunex 10% solution).
2. Wash cells by adding cold sterile FACS buffer. Centrifuge at 400 x g for 6 min at 4 °C.
3. Discard supernatant and resuspend cells in cold sterile FACS buffer. Prepare 5 samples according to the following example:
   

   Sample	Sample type	Antibody	Cell Number	Total volume
   1. unstained	control		1 x 106	50 μl
   2.scc*-Pacific Blue	control	CD45-Pacific Blue	1 x 106	50 μl
   3. scc-APC	control	CD3-APC	1 x 106	50 μl
   4.scc-Alexa 488	control	CD8-Alexa 488	1 x 106	50 μl
   5. Cell sorting	analyte	CD45-Pacific Blue EpCAM-APC CDR2-Alexa 488	10 x 106 – 50 x 106	500 μl 10 x 106 cells/100 μl

   
   *scc; single color control
   
4. Incubate cells with antibodies for 30 min, on ice in the dark.
5. Wash twice with FACs buffer by centrifuging cells at 400 x g for 6 min at 4 °C.
6. Adjust the cell concentration of the sample to be sorted to 1 x10⁷ cells/ml (or to the concentration recommended by the cell sorting facility) using sterile FACS buffer for cell sorting (i.e. without NaN₃).
7. Pass cell sample through a 70 μm cell strainer to remove any cell clumps that may clog the cytometer during sorting.
8. Resuspend control samples in 200-400 μl of buffer. Keep tubes on ice and protected from light.
9. Prepare collection tubes with medium.

Notes:

• Perform titration of the antibodies for optimal staining.
• In one sample for sorting, up to 50 x 10⁶ cells can be stained in a total reaction volume of 500 μl.
• Perform staining of the sample to be sorted in 5 ml Falcon Polystyrene round bottom tubes. The single color controls can be stained in separate wells of a 96-well plate.
• Since TEC subsets are rare populations, it is advisable to use another positive marker (of high frequency) for each fluorochrome to facilitate determination of proper FACS settings and to make sure you have proper compensation if the choice of fluorochromes requires compensation. Cells from the CD45⁺ fraction can be stained with markers such as CD45, CD3 or CD8 for the different fluorochromes and after staining unstained CD45⁺ cells can be added to the sample.

8. Isolation of TEC by Fluorescence Activated Cell Sorting

TEC subsets can be sorted from the CD45^lo/neg fraction as EpCAM^hiCDR2^– (mTEC) or EpCAM^lo CDR2⁺ (cTEC).

1. Run the sample with the unstained cells and adjust the forward and side scatter in order to place the population of interest in scale. Adjust the voltage on each detector so that the cells are visible but located in the far left hand portion of the histogram.
2. Run each single stained sample and adjust compensation for each color.
3. After adjusting the voltage and compensation for the unstained and single stained controls run the sample to be sorted and use gating tools to define the population of interest. Use a wide forward and side scatter gate to ensure inclusion of all stromal cell sizes, while excluding cell debris.
4. On the dot plot with CD45-Pacific Blue versus Forward Scatter gate on the CD45^low and CD45^neg cells.
5. Apply this gate to an EpCAM versus CDR2 dot plot and determine by gating the populations to be sorted.
6. Once gates have been determined, select the gates containing the populations of interest and start sorting.
7. Once sorted cells have been collected, centrifuge the cell suspension, and resuspend cells in medium or buffer of choice depending the intended downstream application.

Note: During sorting in order to prevent cells from sticking to the sides of the collection tube, the tube can be pre-coated by filling it up with 50% FCS in PBS and incubated at RT for 30 min. Discard the coating solution before adding collection media.