3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

This study has been approved by the ethics committee of the Regierungspräsidium Karlsruhe. All animals were housed under specific pathogen-free conditions at the German Cancer Research Center (DKFZ). For all culture experiments mouse pups ranging from 1 to 7 days of age were used.

1. Isolation of mTECs from Thymus

NOTE: The following digestion steps were performed as described previously¹ under sterile conditions with some modifications as follows.

1. Decapitate the mouse pups and remove the thymus. Place the thymi on ice in a Petri plate containing RPMI 1640 medium (containing 5% FCS).
2. Cut the thymi into fine, small pieces and place in a round bottom tube with ~5-30 ml RPMI media and gently stir using a magnet for 10 min at RT.
3. Thereafter, decant the supernatant containing mainly thymocytes and digest the remaining tissue sequentially with one round of collagenase type IV (0.2 mg/ml and 57 U/ml final concentartion) for 15 min each at 37 °C, followed by collagenase/dispase (0.2 mg/ml and 1.2 U/ml final concentration) for 25 min each at 37 °C in a water bath with magnetic stirring until the thymi are completely digested. Use 1 ml enzyme per two to three thymi.
4. Agitate the tissue once every 7-10 min with a Pasteur pipette. Pool the collagenase/dispase fractions and filter through a 70 µm gauze.
5. Enrich the mTECs by magnetic cell sorting (MACS). Perform the purification of mTECs by magnetic cell sorting as described previously¹¹, and shown in Figure 1.
   NOTE: For magnetic sorting of mTECs we used the following antibodies: anti-CD80-PE (16-10A1, use at 1:100 dilution) and anti-EpCAM-bio (G8.8, use at 1:100 dilution)¹². Immature and mature mTECs using MACS were defined as: CD45^– EpCAM⁺ CD80^– and CD45^– CD80⁺ respectively.
6. After MACS purification (purity of immature mTECs = 83.1 ± 6.3 % and mature mTECs = 79.23 ± 3.42%), seed the mTECs onto the organotypic cultures as described below (Section 2.3).
7. Alternatively, sort mTECs by FACS (using 100 µm nozzle) after CD45 MACS depletion using the following antibodies: anti-CD45-PerCP (30-F11, use at 1:100 dilution), anti-Ly51-FITC (6C3, use at 1:100 dilution), anti-EpCAM-Alexa647 (G8.8, use at 1:500 dilution) and anti-CD80-PE (16-10A1, use at 1:100 dilution). Exclude dead cells using propidium iodide (1:5,000) (Day 4). Immature and mature mTECs using FACS were defined as: PI^– CD45^– Ly51^–EpCAM⁺ CD80^– and PI^– CD45^– Ly51^– EpCAM⁺ CD80⁺, respectively.

2. 3D Organotypic Co-cultures (OTCs)

NOTE: The 3D-dermal constructs for organotypic cultivation of keratinocytes were prepared as described previously^9,13. At all steps cells were incubated at 37 °C and 5% CO₂. The OTCs using mTECs were prepared with slight modifications as follows.

1. Preparation of Human Fibroblasts
   NOTE: The human dermal fibroblasts were obtained from explant cultures of de-epidermised dermis as described previously⁹.
   1. In brief, cut strips of human skin ( ~5 cm length) and treat with thermolysin (0.5 mg/ml in saline with 10 mM Hepes pH 7.4) O/N at 4 °C.
   2. Thereafter, separate the epidermis from the dermis using forceps.
   3. Finely cut the dermis into small pieces, place in a 10 cm Petri plate and allow to dry for 1-2 hr under a sterile airflow. Supplement the explants regularly with DMEM containing 20 % FBS.
   4. Split the out-growing fibroblasts when confluent (usually around 3 weeks) using 0.1 % trypsin making sure that the explants continue to adhere to the plate for further consecutive rounds of outgrowth.
   5. Expand the fibroblasts from the same explants for up to 3 times in DMEM with 10 % FBS and cryo-preserve them¹⁴. Use the same batch of fibroblasts for each experimental series.
      NOTE: The fibroblasts were not irradiated for the set-up of the dermal equivalents.
2. Preparation of the Scaffold
   1. Cut the 0.4-0.6 mm thick viscose, nonwoven fibrous material (product details in supporting excel sheet) into well demarcated circles using a sharp 11 mm diameter metal puncher to exactly fit into 12 well-filter inserts. Then place it into the 12- well filter insert (polyester capillary pore membrane, 3 µm pore size) as a scaffold. Place the complete filter setup into a sterile 12-well plate.
   2. Prepare the fibrin gel using a fibrin glue-kit for surgery consisting of a combination of fibrinogen and thrombin. Pre-dilute the fibrinogen- as well as the thrombin-component of the kit to 8 mg/ml and 10 units/ml, respectively. For a single well of a 12-well plate proceed as follows.
      1. Dilute 100 µl fibrinogen (8 mg/ml) with 100 µl phosphate buffered saline (PBS) without Ca₂⁺ and Mg₂⁺, pH 7.0. Dilute 100 µl thrombin (10 units/ml) with 100 µl FCS containing 270,000 fibroblasts.
      2. Dispense 200 µl of the thrombin containing fibroblasts cell-mix onto the scaffold, to which add 200 µl fibrinogen (1:1 mixture), resulting in a final concentration of fibrinogen of 2 mg/ml and of thrombin of 2.5 units/ml. Mix well and distribute evenly over the whole area of the scaffold by gentle pipetting (Day 1).
         NOTE: After 30 min at 37 °C a clot enclosing the fibroblasts will have formed, filling completely the internal spaces of the scaffold and forming a smooth upper surface.
3. Co-culture with mTECs
   1. For pre-culture, submerse the organ cultures in DMEM with 10 % FBS, 50 µg/ml L-ascorbic acid and 1 ng/ml TGF-β1 with a medium change every other day for 4-5 days.
   2. On the day of mTEC seeding, replace the medium by rFAD medium (1:1 DMEM + DMEM/F12) with 10% FBS, 10^-10 M cholera toxin, 0.4 mg/ml hydrocortisone, 50 µg/ml L-ascorbic acid, RANK ligand (0.1 μg/ml) and 500 units/ml of Aprotinin, thus preventing precocious fibrinolysis by serine proteases secreted by fibroblasts.
   3. 7-8 hr later, set-up the co-cultures by seeding 250,000 mTECs (either complete mTECs, or CD80^lo and CD80^hi subsets), in a volume of 100 µl per well on top of the fibroblast scaffold. Count the cells using a Neubauer chamber (Day 4).
      NOTE: At all times the thymus 3D organotypic cultures are submersed in media unlike skin OTCs which are air-lifted.
   4. After 24 hr incubation, supply the cultures with medium (total medium exchange) as mentioned above (step 2.3.2) now containing reduced amounts of Aprotinin, 250 units/ml (Day 5).
   5. In order to assess the proliferative activity of mTECs, add EdU (6.7 µM/ml, i.e., 10 µM/well) to OTCs for 4 hr before termination of the cultures. Perform the staining of OTC cryo-sections as described in the EdU Imaging Kit combined with co-staining of keratin 14. Determine the proliferative indices of mTECs, by either counting the K14⁺ EdU⁺ cells in two sections of each culture specimen or by flow cytometry (EdU flow cytometry, Section 1.7 but instead of Ly51-FITC use CDR1-PB¹⁵ at a 1:100 dilution and 2.3.6.4).
   6. Following 4-7 days of co-culture, terminate the OTCs and process for RNA isolation, cryo-sectioning or FACS analysis (Day 8-11).
      1. Terminate the cultures using a forceps, separating the scaffold/dermal equivalent from the filter of the well insert.
      2. For cryo-sectioning, embed the entire OTC in OCT compound and freeze in liquid nitrogen vapor before cryo-sectioning. Prepare 5-7 µm thick OTC sections using a cryostat and store at -20 °C until use. For immuno-histochemistry of cryo-sectioned OTCs use anti-keratin 14 (AF64, use at 1:1,000 dilution), and anti-vimentin (GP53, use at 1:100 dilution) antibodies. Perform the indirect immuno-histochemistry staining using respective secondary antibodies.
      3. For RNA isolation, add 1 ml Denaturing solution (containing phenol and guanidinium thiocyanate) in a screw cap of a 2 ml RNase free tube. Cut the entire OTC into pieces with a scalpel and add into the tube containing denaturing solution. Mechanically shred the OTCs with FastPrep instrument twice for 30 sec at a speed of 6.0, place in between on ice for 2 min (the sample can be stored at -80 °C after this point). If frozen at -80 °C, thaw on ice. Centrifuge the tubes at 11,500-13,000 rpm for 10 min at 4 °C. Transfer the supernatant to a fresh tube, incubate for 5 min at RT and follow RNA isolation protocols using Acid guanidinium thiocyanate-phenol-chloroform extraction as described by the manufacturer.
      4. For FACS analysis, remove the scaffold and separate the membrane from the fibrin/fibroblast/mTEC gel. Finely cut the gel with a scalpel and digest it in a FACS tube for ~20 min or until completely digested with 2 ml of collagenase/dispase at 37 °C in a water bath with magnetic stirring. Agitate the enzyme solution with a Pasteur pipette once every 5 min. After complete digestion, filter the cell suspension through a 70 µm filter; stain the single cell suspension using the antibodies as described in 1.7 and analyze by flow cytometry.