This work has been supported by the German Cancer Research Center (DKFZ), the EU-consortium &#8220;Tolerage&#8221;, the Deutsche Forschungsgemeinschaft (SFB 938) and the Landesstiftung Baden-W&#252;rttemberg.

This study has been approved by the ethics committee of the Regierungspr&#228;sidium 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.
<ol>
	<li>Decapitate the mouse pups and remove the thymus. Place the thymi on ice in a Petri plate containing RPMI 1640 medium (containing 5% FCS).</li>
	<li>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.</li>
	<li>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 &#176;C, followed by collagenase/dispase (0.2 mg/ml and 1.2 U/ml final concentration) for 25 min each at 37 &#176;C in a water bath with magnetic stirring until the thymi are completely digested. Use 1 ml enzyme per two to three thymi.</li>
	<li>Agitate the tissue once every 7-10 min with a Pasteur pipette. Pool the collagenase/dispase fractions and filter through a 70 &#181;m gauze.</li>
	<li>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.</li>
	<li>After MACS purification (purity of immature mTECs = 83.1 &#177; 6.3 % and mature mTECs = 79.23 &#177; 3.42%), seed the mTECs onto the organotypic cultures as described below (Section 2.3).</li>
	<li>Alternatively, sort mTECs by FACS (using 100 &#181;m 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.</li>
</ol>
2. 3D Organotypic Co-cultures (OTCs)
NOTE: The 3D-dermal constructs for organotypic cultivation of keratinocytes were prepared as described previously9,13. At all steps cells were incubated at 37 &#176;C and 5% CO2. The OTCs using mTECs were prepared with slight modifications as follows.
<ol>
	<li>Preparation of Human Fibroblasts 
	NOTE: The human dermal fibroblasts were obtained from explant cultures of de-epidermised dermis as described previously9.
	<ol>
		<li>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 &#176;C.</li>
		<li>Thereafter, separate the epidermis from the dermis using forceps.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Preparation of the Scaffold
	<ol>
		<li>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 &#181;m pore size) as a scaffold. Place the complete filter setup into a sterile 12-well plate.</li>
		<li>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.
		<ol>
			<li>Dilute 100 &#181;l fibrinogen (8 mg/ml) with 100 &#181;l phosphate buffered saline (PBS) without Ca2+ and Mg2+, pH 7.0. Dilute 100 &#181;l thrombin (10 units/ml) with 100 &#181;l FCS containing 270,000 fibroblasts.</li>
			<li>Dispense 200 &#181;l of the thrombin containing fibroblasts cell-mix onto the scaffold, to which add 200 &#181;l 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 &#176;C a clot enclosing the fibroblasts will have formed, filling completely the internal spaces of the scaffold and forming a smooth upper surface.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Co-culture with mTECs
	<ol>
		<li>For pre-culture, submerse the organ cultures in DMEM with 10 % FBS, 50 &#181;g/ml L-ascorbic acid and 1 ng/ml TGF-&#946;1 with a medium change every other day for 4-5 days.</li>
		<li>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 &#181;g/ml L-ascorbic acid, RANK ligand (0.1 &#956;g/ml) and 500 units/ml of Aprotinin, thus preventing precocious fibrinolysis by serine proteases secreted by fibroblasts.</li>
		<li>7-8 hr later, set-up the co-cultures by seeding 250,000 mTECs (either complete mTECs, or CD80lo and CD80hi subsets), in a volume of 100 &#181;l 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.</li>
		<li>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).</li>
		<li>In order to assess the proliferative activity of mTECs, add EdU (6.7 &#181;M/ml, i.e.,&#160;10 &#181;M/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).</li>
		<li>Following 4-7 days of co-culture, terminate the OTCs and process for RNA isolation, cryo-sectioning or FACS analysis (Day 8-11).
		<ol>
			<li>Terminate the cultures using a forceps, separating the scaffold/dermal equivalent from the filter of the well insert.</li>
			<li>For cryo-sectioning, embed the entire OTC in OCT compound and freeze in liquid nitrogen vapor before cryo-sectioning. Prepare 5-7 &#181;m thick OTC sections using a cryostat and store at -20 &#176;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.</li>
			<li>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 &#176;C after this point). If frozen at -80 &#176;C, thaw on ice. Centrifuge the tubes at 11,500-13,000 rpm for 10 min at 4 &#176;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.</li>
			<li>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 &#176;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 &#181;m filter; stain the single cell suspension using the antibodies as described in 1.7 and analyze by flow cytometry.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Derbinski, J., Schulte, A., Kyewski, B., Klein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promiscuous+gene+expression+in+medullary+thymic+epithelial+cells+mirrors+the+peripheral+self.">Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self.</a> Nat Immunol. 2, 1032-1039 (2001).</li><li>Kyewski, B., Klein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+central+role+for+central+tolerance.">A central role for central tolerance.</a> Annual review of immunology. 24, 571-606 (2006).</li><li>Bonfanti, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+reprogramming+of+thymic+epithelial+cells+to+skin+multipotent+stem+cells.">Microenvironmental reprogramming of thymic epithelial cells to skin multipotent stem cells.</a> Nature. 466, 978-982 (2010).</li><li>Kont, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Aire+regulates+the+expression+of+tissue-restricted+antigens.">Modulation of Aire regulates the expression of tissue-restricted antigens.</a> Molecular Immunology. 45, 25-33 (2008).</li><li>Mohtashami, M., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+architecture+of+the+thymus+is+required+to+maintain+delta-like+expression+necessary+for+inducing+T+cell+development.">Three-dimensional architecture of the thymus is required to maintain delta-like expression necessary for inducing T cell development.</a> J Immunol. 176, 730-734 (2006).</li><li>Palumbo, M. O., Levi, D., Chentoufi, A. A., Polychronakos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+proinsulin-producing+medullary+thymic+epithelial+cell+clones.">Isolation and characterization of proinsulin-producing medullary thymic epithelial cell clones.</a> Diabetes. 55, 2595-2601 (2006).</li><li>White, A., Jenkinson, E., Anderson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaggregate+thymus+cultures.">Reaggregate thymus cultures.</a> Journal of visualized experiments : JoVE. , (2008).</li><li>Stark, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+homeostasis+in+long-term+scaffold-enforced+skin+equivalents.">Epidermal homeostasis in long-term scaffold-enforced skin equivalents.</a> J Investig Dermatol Symp Proc. 11, 93-105 (2006).</li><li>Boehnke, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+fibroblasts+and+microenvironment+on+epidermal+regeneration+and+tissue+function+in+long-term+skin+equivalents.">Effects of fibroblasts and microenvironment on epidermal regeneration and tissue function in long-term skin equivalents.</a> Eur J Cell Biol. 86, 731-746 (2007).</li><li>Pinto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+organotypic+coculture+model+supporting+proliferation+and+differentiation+of+medullary+thymic+epithelial+cells+and+promiscuous+gene+expression.">An organotypic coculture model supporting proliferation and differentiation of medullary thymic epithelial cells and promiscuous gene expression.</a> J Immunol. 190, 1085-1093 (2013).</li><li>Gabler, J., Arnold, J., Kyewski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promiscuous+gene+expression+and+the+developmental+dynamics+of+medullary+thymic+epithelial+cells.">Promiscuous gene expression and the developmental dynamics of medullary thymic epithelial cells.</a> Eur J Immunol. 37, 3363-3372 (2007).</li><li>Farr, A., Nelson, A., Truex, J., Hosier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+heterogeneity+in+the+murine+thymus:+a+cell+surface+glycoprotein+expressed+by+subcapsular+and+medullary+epithelium.">Epithelial heterogeneity in the murine thymus: a cell surface glycoprotein expressed by subcapsular and medullary epithelium.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 39, 645-653 (1991).</li><li>Stark, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Authentic+fibroblast+matrix+in+dermal+equivalents+normalises+epidermal+histogenesis+and+dermoepidermal+junction+in+organotypic+co-culture.">Authentic fibroblast matrix in dermal equivalents normalises epidermal histogenesis and dermoepidermal junction in organotypic co-culture.</a> Eur J Cell Biol. 83, 631-645 (2004).</li><li>Schoop, V. M., Mirancea, N., Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+organization+and+differentiation+of+HaCaT+keratinocytes+in+organotypic+coculture+with+human+dermal+fibroblasts.">Epidermal organization and differentiation of HaCaT keratinocytes in organotypic coculture with human dermal fibroblasts.</a> J Invest Dermatol. 112, 343-353 (1999).</li><li>Rouse, R. V., Bolin, L. M., Bender, J. R., Kyewski, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+reactive+with+subsets+of+mouse+and+human+thymic+epithelial+cells.">Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 36, 1511-1517 (1988).</li></ol>

The authors declare no financial or commercial conflict of interest.

Alongside RTOCs, the 3D OTCs have been by far superior in terms of TEC differentiation and pGE maintenance/induction (Table 1) compared to other (i) &#8216;simplified 3D cultures&#8217; using - fibroblasts alone without the scaffold; (ii) 2D systems using - fibroblasts/feeder cells co-cultured with TECs10, (iii) 3T3-J2 cells wherein TEC clones develop, but pGE is lost, (iv) matrigel or (v) ECM components (unpublished data). PGE was maintained for up to 7 days in the 3D OTCs, 4 days being the o...

Developing thymocytes make up about 98 % of the thymus, while the remaining 2 % consists of a variety of cells that collectively compose the thymic stroma (i.e., epithelial cells, dendritic cells, macrophages, B cells, fibroblasts, endothelial cells). The outer cortical epithelial cells (cTECs) procure immigration of pro-T cells from the bone marrow, T cell lineage induction in multipotent pre-T cells and positive selection of self-MHC restricted immature thymocytes. The inner medullary thymic epithelial cells (mTECs) are involved in tolerance induction of those thymocytes with a high-affinity TCR for self-peptide/MHC complexes by either inducing negative selection or their deviation into the T regulatory cell lineage. In the context of central tolerance induction, mTECs are unique in that they express a wide spectrum of tissue-restricted self-antigens (TRAs) thus mirroring the peripheral self. This phenomenon is called promiscuous gene expression (pGE)1,2.
Most current studies on this fascinating cell type rely on ex vivo isolated cells, as various short-term 2D culture systems invariably resulted in the loss of pGE and key regulator molecules like MHC class II, FoxN1 and Aire within the first 2 days3-6. It remained however unclear, which particular components and features of the intact 3D meshwork of the thymus were missing in 2D models. The re-aggregation thymic organ culture (RTOC) has been so far the only 3D system that allows the study of T cell development, on the one hand, and stromal cell biology, on the other hand, in an intact thymic microenvironment7. Yet, RTOCs have certain limitations, i.e., they already contain a complex mixture of cells, require the input of fetal stromal cells and endure a maximal culture period of 5 to 10 days.
The lack of reductionist in vitro culture systems has hampered the study of several aspects of T cell development and thymic organogenesis not least the molecular regulation of pGE and its relationship to the developmental biology of mTECs.
Owing to the close-relatedness of the structured organization of the epithelial cells of skin and thymus, we opted for a 3D organotypic culture (OTC) system that had been developed originally to emulate the differentiation of keratinocytes in vitro and thus create a dermal equivalent. The OTC system consists of an inert scaffold matrix overlaid with dermal fibroblasts that are trapped in a fibrin gel, onto which keratinocytes are seeded8,9. Here, we replaced keratinocytes with purified mTECs. While keeping the basic features of this model, we optimized certain parameters.
In the adopted OTC model mTECs proliferated, underwent terminal differentiation and maintained mTEC identity and pGE, thus closely mimicking in vivo mTECs development10. This technical note provides a detailed protocol allowing the stepwise set-up of thymus OTCs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pregnant C57BL/6 mice&#160;</td>
			<td>Charles River WIGA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Microbeads, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr>
			<td>EpCAM (G8.8 -Alexa 647 and -biotin)</td>
			<td>Ref. 12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD80-PE antibody</td>
			<td>BD Pharmingen</td>
			<td>553769</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-PerCP antibody</td>
			<td>BD Pharmingen</td>
			<td>557235</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly51-FITC antibody</td>
			<td>BD Pharmingen</td>
			<td>553160</td>
			<td></td>
		</tr>
		<tr>
			<td>CDR1-Pacific Blue</td>
			<td>Ref. 15</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Keratin 14 antibody</td>
			<td>Covance</td>
			<td>PRB-155P</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin antibody</td>
			<td>Progen</td>
			<td>GP58</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated AffiniPure Goat anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>111-165-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated AffiniPure F(ab&#39;)2 Fragment Goat anti-Guinea Pig IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>106-546-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Molecular Probes (Invitrogen GmbH)</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10339">Click-iT EdU Alexa Fluor 594 Imaging Kit</a></td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10339">C10339</a></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10425">Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit</a></td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10425">C10425</a></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well filter inserts (thincerts)</td>
			<td>Greiner bio-one</td>
			<td>657631</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Greiner</td>
			<td>665180-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Jettex 2005/45</td>
			<td>ORSA, Giorla Minore, Italy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen TISSUECOL-Kit Immuno</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin TISSUECOL-Kit Immuno</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Serva</td>
			<td>47302.03</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Lonza</td>
			<td>BE12-719F</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-049&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Gold</td>
			<td>GE Healthcare</td>
			<td>A11-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin (Trasylol)</td>
			<td>Bayer</td>
			<td>4032037</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Biomol</td>
			<td>G117</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Seromed (Biochrom)</td>
			<td>K3520</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#223;1</td>
			<td>Invitrogen</td>
			<td>PHG9214</td>
			<td></td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>R&#38;D systems</td>
			<td>462-TR-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermolysin</td>
			<td>Sigma Aldrich</td>
			<td>&#160;T-7902</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>TissueTek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol (aka. Denaturing solution - Acid guanidinium thiocyanate-phenol-chloroform extraction)</td>
			<td>Invitrogen</td>
			<td>10296028</td>
			<td></td>
		</tr>
		<tr>
			<td>FastPrep FP120</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV&#160;</td>
			<td>CellSystems</td>
			<td>LS004189</td>
			<td>0.2 mg/ml and 57U/ml final conc.</td>
		</tr>
		<tr>
			<td>Neutrale Protease (Dispase)</td>
			<td>CellSystems</td>
			<td>LS002104</td>
			<td>0.2 mg/ml and 1.2U/ml final conc.</td>
		</tr>
		<tr>
			<td>DNase I&#160;</td>
			<td>Roche</td>
			<td>11 284 932 001</td>
			<td>25 &#181;g/ml final conc.</td>
		</tr>
	</tbody>
</table>

3d organotypic co-culture model supporting medullary thymic epithelial cell proliferation, differentiation and promiscuous gene expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

We adopted a 3D organotypic co-culture model (3D OTC) which had been originally developed for in vitro long term culture of keratinocytes9. MACS-enriched mTECs (see MACS enrichment scheme Figure 1) were seeded onto a scaffold comprising of a fibrin gel and entrapped fibroblasts. The fibroblasts provide the essential extracellular matrix (ECM) supporting mTECs in vitro. MTECs were cultivated in OTCs for 4-14 days in the presence of RANKL in submerged cultures unlike keratinocy...

Watch this Scientific Journal Video about 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression at JoVE.com

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

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

3d-organotypic-co-culture-model-supporting-medullary-thymic

Research

JoVE Journal

Developmental Biology

10.6K Views.  German Cancer Research Center (DKFZ). Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression. 