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. 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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 optimum time-point thereafter, pGE starts to decline. Other morphological TEC features were maintained for up to 14 days. Intriguingly, the OTCs supported the end-stage of mTEC differentiation seen by the occasionally formed Hassall-like structures10.
The thymi used for OTCs were derived from young post-natal mice as they have a higher propensity to survive in culture compared to adult thymi. TECs derived from embryonic thymi have not yet been tested in OTCs. More so, after the long digestion period (not using trypsin due to cleavage of certain epitopes) the cells appeared more viable using MACS rather than FACS sorting cells. Using a two-step positive enrichment protocol on MACS columns (anti-PE beads) of CD80+ mTECs additionally improved TEC purity.
For the OTC setup it is essential to make sure that the viscose, nonwoven fibrous material is cut into sharp, well demarcated circles without loose fragments or serrated ends as stated above that exactly fit into 12 well-culture inserts. Alternatively, scaffolds such as BEMCOT- viscose wiper M-3 (http://www.bemliese.com) can also be used. The well size for the OTC is also critical and should be tested, if plate formats other than 6-well and 12-well formats should be used.
The fibrin gel should be prepared such that the fibrinogen and thrombin components do not come into contact with each other before being set onto the OTC, make sure to exchange the pipette tip. Mix the two components thoroughly in the plate over the scaffold to form a smooth upper surface. Always prepare a test gel alongside in a well without scaffold to check for proper clot formation and to have a good estimate of the time required for clotting. Make sure that the fibrin gel in the OTC has completely clotted before supplying the cultures with media.
Upon termination of the cultures the seeded mTECs can be easily retrieved by enzymatic digestion (collagenase/dispase) of the fibrin gel, which can be used for characterization of cultured mTECs e.g., by flow cytometry or PCR.
The 3D OTCs described herein has the potential to study or manipulate several TEC parameters namely: 1) to study and/or manipulate (via siRNA, morpholino oligos) developmental pathways and functions of mTECs; 2) to study the role of mTECs in T cell development; 3) to culture cTECs; 4) to test the progenitor potential of thymic derived stem cells; 5) to culture human TECs and stem cells; 6) to perform single TEC clonal assays. The 3D OTCs represent an elaborate culture technique emulating part of the in vivo thymus microenvironment. It should be emphasized that any additional assays applied to TECs in the current OTC setup will need to be thoroughly tested and optimized separately.

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><tbody><tr><td>Pregnant C57BL/6 mice&amp;nbsp;</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&amp;nbsp;</td><td>111-165-003</td><td></td></tr><tr><td>Alexa 488-conjugated AffiniPure F(ab&#039;)2 Fragment Goat anti-Guinea Pig IgG (H+L)</td><td>Jackson ImmunoResearch&amp;nbsp;</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>Click-iT EdU Alexa Fluor 594 Imaging Kit</td><td>Invitrogen</td><td>C10339</td><td></td></tr><tr><td>Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit</td><td>Invitrogen</td><td>C10425</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&amp;nbsp;</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-&amp;szlig;1</td><td>Invitrogen</td><td>PHG9214</td><td></td></tr><tr><td>RANKL</td><td>R&amp;amp;D systems</td><td>462-TR-010</td><td></td></tr><tr><td>Thermolysin</td><td>Sigma Aldrich</td><td>&amp;nbsp;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&amp;nbsp;</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&amp;nbsp;</td><td>Roche</td><td>11 284 932 001</td><td>25 &amp;micro;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 keratinocytes, which are air-exposed mimicking their in vivo environment (see OTC set-up scheme Figure 2).
Both mTEC subsets analyzed here (i.e., CD80lo and CD80hi mTECs) survived during the entire culture period of up to 14 days. MTECs were identified by keratin 14 expression and were easily distinguishable from vimentin-positive fibroblasts (Figure 3A, 3B). Interestingly, immature and mature mTECs grew in different patterns in culture. The CD80lo mTECs typically formed bi-layers (in close contact with fibroblasts), while CD80hi mTECs tended to form compact cell aggregates delimited by fibroblasts. These patterns were highly reproducible.
The mTEC subsets not only survived but also proliferated under 3D OTC conditions as assessed by EdU incorporation (Figure 3C, 3D). Interestingly, the CD80lo mTECs proliferated at a higher rate in the presence of RANKL, while the reverse was true for CD80hi mTECs10.
Additionally, CD80lo mTECs differentiated into CD80hi mTECs in the presence of RANKL within 4 days of culture, as indicated by the strong up-regulation of CD80. The differentiated mTECs also maintained the expression of Aire, FoxN1 (gene and protein) as well as promiscuously expressed Aire-independent and -dependent TRAs10.
<img alt="Figure 1" src="/files/ftp_upload/52614/52614fig1.jpg" /> 
Figure 1. MACS enrichment of TECs. After filtering, mTECs were enriched from the thymus single cell suspension using magnetic cell sorting (MACS). First, the hematopoietic cell lineages were depleted using anti-CD45 Microbeads. The CD45- cells were then incubated with anti-CD80 PE antibody, followed by anti-PE Microbeads. The eluate containing CD80+ -mature mTECs was directly cultured on the OTCs, while the flow-through contained CD80- immature mTECs and other stromal cells. MTECs were further enriched using anti-EpCAM-bio antibody and streptavidin Microbeads. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52614/52614fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52614/52614fig2.jpg" /> 
Figure 2.&#160;Scheme for the stepwise setup of the 3D OTCs. A scaffold matrix was placed into a 12-well filter insert. The dermal fibroblasts from explanted skin (270,000 cells/OTC well) were inoculated into a fibrin gel (consisting of 1:1 ratio of fibrinogen and thrombin). These dermal equivalents were sustained for 4-5 days with DMEM + nutrients until mTECs (250,000 cells/OTC well) were seeded on top and cultured with medium enriched with different nutrient (rFAD + nutrients).
<img alt="Figure 3" src="/files/ftp_upload/52614/52614fig3.jpg" /> 
Figure 3.&#160;Growth patterns and proliferation of mTECs within the OTCs. The enriched CD80lo mTECs tend to grow as a bi-layer in close contact with the fibroblasts (A), whereas the differentiated CD80hi mTECs grow as cell aggregates or tight clusters (B). OTCs were labeled on day 4 of culture with anti-keratin 14 (red) and anti-vimentin antibodies (green) along with nuclear staining (Hoechst, blue). To evaluate the proliferation of mTECs, the OTCs were pulsed with EdU (6.7 &#181;M/ml, i.e.,&#160;10 &#181;M/well) for 4 hr prior to termination of the cultures. The OTC cryo-sections were then stained with anti-keratin 14 (green), and EdU-Click-iT reaction mixture (magenta) along with nuclear staining (Hoechst, blue). Representative images depicting proliferating EdU+ CD80lo mTECs (C) and EdU+ CD80hi mTECs (D) are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52614/52614fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="always" border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reaggregation thymic organ cultures (RTOC)</td>
			<td>Organotypic co-cultures&#160; (OTC)</td>
		</tr>
		<tr>
			<td>Useful system to study cellular interactions that can be readily monitored and manipulated using a largely intact 3D thymic architecture; develops into a properly organized structure upon grafting.</td>
			<td>Useful system to study development and differentiation of enriched/purified thymic epithelial cells in an artificial 3D meshwork generated by human skin fibroblasts entrapped in a fibrin scaffold.</td>
		</tr>
		<tr>
			<td>Well established for the study of the development of thymic epithelial cells and thymocytes using inhibitors or modifiers, which penetrate the capsule.</td>
			<td>Amenable to manipulate thymus epithelial development or to study thymocyte development in co-culture with TECs.</td>
		</tr>
		<tr>
			<td>Since the system is air-lifted, the membrane pore size (0.8 &#181;m) is crucial when supplying factors to the RTOC: larger molecules may not penetrate through the membrane and/or capsule.</td>
			<td>Culture is submerged in media, which facilitates uptake of substances/factors (successfully tested using morpholino oligos).</td>
		</tr>
		<tr>
			<td>Adult and post-natal TECs or thymocytes can be spiked into fetal RTOCs.</td>
			<td>Post-natal TECs grow better than adult TECs; fetal TECs not yet tested.</td>
		</tr>
		<tr>
			<td>Require embryonic E14-14.5 thymi (optimal) to allow for reaggregation and integration of the added desired population; difficult to control the fetal thymic (acceptor) cell composition; RTOCs require cells from either fluorescently labelled or congenic mice as donor or acceptor cells.</td>
			<td>Possible to study single cells/clones; only &#8220;contaminants&#8221; are human skin fibroblasts required to provide ECM to support the TECs.</td>
		</tr>
		<tr>
			<td>Imaging limited to penetration depth of live two-photon microscopy; amenable to FACS analysis or sorting; gene expression studies require FACS sorting of the desired cell type after culture.</td>
			<td>Fully accessible to imaging; suitable for FACS analysis, immuno-histochemistry, and gene expression studies.</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Comparison between two 3D thymic culture systems.

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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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.7K 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. 

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

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Cancer Research

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 ...

Biology

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Bioengineering

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Preparation and Applications of Organotypic Thymic Slice Cultures

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.

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.

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Immunology and Infection

Recreating the Thymic Microenvironment with iPSC-Derived Organoids

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

Author Spotlight: Advancing Thymic Epithelial Cells and T-Cell Research with Human Thymic Organoids

Here, we describe a protocol for the formation of human iPSC-derived thymus organoids grown in 3D fibrin hydrogels aiming to support thymic epithelial cell (TEC) maturation and prolonged maintenance, as well as thymopoiesis in vitro.

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

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

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