This study was supported by ERC StG TroyCAN (851241). E.E. is a Cancerfonden Postdoctoral Associate. M.G. is a Ragnar S&#246;derberg Fellow and Cancerfonden Junior Investigator. We are grateful for the technical assistance from Karolinska Institutet core facilities, including the Biomedicum Flow Cytometry Core Facility, the Biomedicum Imaging Core (BIC), and the Comparative Medicine Biomedicum (KMB) animal facility. We thank members of the Genander lab for carefully reading and commenting on the protocol.

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The authors declare no conflict of interest.

The protocol presented here establishes an in vitro model for investigating functional esophageal epithelial-fibroblast interactions.
The epithelial layer is separated from the stroma, allowing for an optimized dissociation protocol for both the epithelial and stromal compartment. Despite optimization of the epithelial dissociation protocol, tissue clumps remain apparent. Pipetting up and down vigorously every 15 min decreases the number and size of clumps substantially. Other protocols use trypsin to further dissociate the epithelial layer5,6. Here, the use of trypsin, or increasing the dissociation time further, is not recommend, as this tends to result in decreased epithelial cell viability and organoid forming efficiency. In contrast to the epithelium, the stroma is easily dissociated, and 30 min in dissociation solution results in a single-cell suspension with ~90% fibroblast viability (Figure 1E). Excluding the epithelial-stomal separation step in the protocol increases the dissociation time substantially, resulting in a decreased fibroblast viability and a lower yield of epithelial cells. Additionally, separating the epithelium from the stroma provides an opportunity to determine cell numbers of each population and mix epithelial cells and fibroblasts from different mouse lines when setting up the co-cultures.
Studying fibroblast function on organoid growth is a commonly used method in stem cell biology 9,10,11,15,16. Established co-culture media are either DMEM supplemented with 10% fetal calf serum (FCS)9,15 or growth factor reduced medium10,16. In this protocol, the growth factor reduced medium is used to mimic the conditions in the in vivo stem cell niche, where fibroblasts are largely quiescent. FCS is a growth factor rich serum which results in activation and proliferation of fibroblasts in the co-cultures, likely corresponding to a fibroblast cell state distinct from the in vivo state. By excluding FCS and reducing growth factors, so that the medium alone (ERlow) does not support organoid growth and does not stimulate fibroblast proliferation, it is possible to isolate the effect of the fibroblasts on the organoid growth. In this medium, NOGGIN is removed and RSPO reduced to a minimum (10% RPSO). Both NOGGIN and RSPO have been demonstrated to be essential for esophageal organoid growth6. EGF was retained in the co-culture medium, as it does not support organoid growth by itself. However, fibroblasts are also capable of supporting organoid growth in an EGF-reduced medium (ElowRlow; Figure 2B,D).
Organoid co-cultures cannot be sustained through passaging as fibroblasts are lost during trypsinization. However, organoid passaging was included in the protocol since esophageal organoids can be maintained, expanded, and used for further experiment as mono-cultures. Passaged organoids from mono-cultures can be used to set up co-cultures with freshly isolated fibroblasts. A disadvantage of using primary cells is the number of mice needed to set up multiple organoid co-cultures. When focusing on small subpopulations of fibroblasts, the number of co-cultures obtained is limited. In other protocols, fibroblasts are first expanded in culture before using them to set up organoid co-cultures10. However, fibroblasts change morphology and identity during passaging, shown by using primary skin and cardiac fibroblasts17,18. Conventional 2D passaging of esophageal fibroblasts results in both morphology and phenotype changes, demonstrating that in vitro enrichment of fibroblasts is not suitable for co-cultures aiming to phenocopy the endogenous stem cell niche.
Whole mount staining provides a tool to maintain and visualize fibroblast-organoid interaction. It should be noted that, while not all organoids will have fibroblasts directly attached to them, most organoids are in contact with fibroblasts (see Figure 2C). To maintain epithelial-fibroblast interactions, it is important to handle the organoids with care and avoid vigorous pipetting, vortexing, and high-speed spinning. Optimal fixation is important to maintain 3D tissue architecture, as well as keep endogenous fluorescence. A 30 min fixation suffices to retain the H2BeGFP signal and is optimal for the antibodies used in this protocol, however this might vary between the fluorophores and antibodies used. Clearing of the organoids reduces light scattering and improves visualization of the whole 3D structure substantially. As the organoids are small, clearing is easy and fast; however, imaging whole organoids using laser-scanning confocal microscopy can be time-consuming, as multiple Z-stacks need to be made. Confocal microscopes, like the spinning disc, can be used to reduce imaging time.
Overall, esophageal organoids grown in the presence of fibroblasts provide a valuable tool to understand aspects of the esophageal stem cell niche. In addition, whole mount clearing provides an accessible method to visualize the interaction between fibroblasts and organoids.

Organoids are used as 3D in vitro assays to characterize stem and progenitor cells, as well as to understand the signaling cues derived from the cellular components of the stem cell niche1,2,3,4. Mouse esophageal organoids were first described in 2014 and several papers have identified growth factors, like R-Spondin (RSPO), NOGGIN, and epidermal growth factor (EGF), needed to maintain and passage esophageal organoids5,6,7, arguing that similar signaling cues are required for in vivo progenitor cell renewal. However, growth factors are commonly added in non-physiological concentrations, resulting in organoid growth conditions which do not necessarily reflect the in vivo signaling environment.
Fibroblasts are heterogeneous stromal cell populations that support progenitor cell properties in many stem cell niches8. Combining epithelial progenitor cells and fibroblasts in the same organoid culture enables organoid formation in reduced concentrations of exogenously supplemented growth factors. Organoid co-culture systems from intestinal and hepatic epithelia are&#160;described, but a protocol to establish esophageal organoid-fibroblast co-cultures is still outstanding9,10,11.
In this protocol, two fluorescence-activated cell sorting (FACS) strategies for fibroblasts from the esophagus, using either transgenic Pdgfr&#945;H2BeGFP mice12 or wild-type mice with classical antibody staining are outlined. Different subpopulations of fibroblasts can be isolated using cell surface markers of choice, thereby providing flexibility to the protocol. In addition, a 3D fluorescence imaging technique preserving organoid morphology is used to characterize fibroblast-organoid interactions. Organoid clearing provides a quick method to increase the light penetration depth in the organoids, improving the visualization of organoid-fibroblast connections and enabling the recapitulation of the organoid structure in its entirety. This protocol combines esophageal organoid co-culture with a whole mount imaging strategy, enabling functional characterization of the fibroblast-organoid interaction.

<table><tbody><tr><td>B-27 Supplement (50X), serum free</td><td>Thermo Fisher (Gibco)</td><td>17504001</td><td></td></tr><tr><td>Corning&amp;nbsp;Matrigel&amp;nbsp;Growth Factor Reduced (GFR) Basement Membrane Matrix</td><td>fisher scientific</td><td>356231</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>276855-100ML</td><td></td></tr><tr><td>DMEM/F-12</td><td>Thermo Fisher (Gibco)</td><td>11320074</td><td></td></tr><tr><td>DPBS</td><td>Thermo Fisher (Gibco)</td><td>14190250</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Thermo Fisher (Gibco)</td><td>35050061</td><td></td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red</td><td>Thermo Fisher (Gibco)</td><td>14175-129</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson Immuno</td><td>017-000-121</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher (Gibco)</td><td>15140122</td><td></td></tr><tr><td>Triton X-100 solution</td><td>Merck</td><td>93443-100ML</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Thermo Fisher (Gibco)</td><td>25200-056</td><td></td></tr><tr><td>&lt;strong&gt;Chemicals, Peptides, and recombinant proteins&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DAPI Solution</td><td>Thermo Fisher</td><td>62248</td><td></td></tr><tr><td>Dissociation solution: 0.25 mg/ml Liberase TM, 0.25 mg/ml Dnase in HBSS</td><td></td><td></td><td></td></tr><tr><td>Dnase I</td><td>Sigma-Aldrich</td><td>11284932001</td><td></td></tr><tr><td>Formaldehyde, 37%, with 10-15% methanol</td><td>Sigma-Aldrich</td><td>252549-1L</td><td></td></tr><tr><td>Liberase</td><td>Sigma-Aldrich</td><td>5401127001</td><td></td></tr><tr><td>N-Acetyl-cysteine</td><td>Sigma-Aldrich</td><td>A9165-25G</td><td></td></tr><tr><td>Noggin murine</td><td>Peprotech</td><td>250-38</td><td></td></tr><tr><td>RapiClear 1.47</td><td>SunJin Lab</td><td>#RC147001</td><td></td></tr><tr><td>Recombinant Mouse EGF Protein, CF</td><td>R&amp;amp;D systems</td><td>2028-EG-200</td><td></td></tr><tr><td>R-spondin-1 murine</td><td>Peprotech</td><td>315-32</td><td></td></tr><tr><td>SYTOX Blue Dead Cell Stain</td><td>Thermo Fisher</td><td>S34857</td><td></td></tr><tr><td>Thermolysin</td><td>Sigma-Aldrich</td><td>T7902-25MG</td><td></td></tr><tr><td>Y-27632 dihydrochloride</td><td>Sigma-Aldrich</td><td>Y0503-5MG</td><td></td></tr><tr><td>&lt;strong&gt;Plastic &amp;amp; Glassware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Corning Sterile Cell Strainers 40um</td><td>VWR</td><td>15360801</td><td></td></tr><tr><td>Corning Sterile Cell Strainers 70um</td><td>VWR</td><td>431751</td><td></td></tr><tr><td>Menzel Deckgl&amp;auml;ser/ cover slips</td><td>Thermo Fisher</td><td>Q10143263NR15</td><td></td></tr><tr><td>SafeSeal reaction tube, 1.5 mL, PP</td><td>Sarstedt</td><td>72.706</td><td></td></tr><tr><td>Snap Cap Low Retention Microcentrifuge Tubes 0.6 mL</td><td>Thermo Fisher</td><td>3446</td><td></td></tr><tr><td>SuperFrost Slides</td><td>VWR</td><td>631-9483</td><td></td></tr><tr><td>&lt;strong&gt;Tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.05 mm 4 circular well iSpacer</td><td>SunJin Lab</td><td>#IS204</td><td></td></tr><tr><td>Dumont #5 forceps, biology tip</td><td>F.S.T</td><td>11252-20</td><td></td></tr><tr><td>ImmEdge Pen</td><td>VectorLaboratories</td><td>H-4000</td><td></td></tr><tr><td>Spring Scissors Angled to Side Ball Tip 8mm Cutting Edge</td><td>F.S.T</td><td>15033-09</td><td></td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Confocal microscope Stellaris 5&amp;nbsp;</td><td>Leica</td><td></td><td></td></tr><tr><td>Dissection microscope ZEISS Stemi 305</td><td>Zeiss</td><td></td><td></td></tr><tr><td>FACS ARIA III</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Conjugated Antibodies for FACS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 647 anti-mouse CD104 Antibody&lt;br/&gt; Clone: 346-11A</td><td>123608</td><td>123608</td><td></td></tr><tr><td>APC anti-mouse CD26 (DPP-4) Antibody</td><td>H194-112</td><td>H194-112</td><td></td></tr><tr><td>PE/Cy7 anti-mouse/human CD324 (E-Cadherin) Antibody</td><td>147310</td><td>147310</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies for Immunofluorescence&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD104 (&amp;szlig;-integrin 4)&lt;br/&gt; Clone: 346-11A</td><td>BioLegend</td><td>553745</td><td></td></tr><tr><td>Cytokeratin 14</td><td>Acris Antibodies (AbD Serotec)</td><td>BP5009</td><td></td></tr><tr><td>Cytokeratin13&lt;br/&gt; Clone: EPR3671</td><td>abcam</td><td>ab92551</td><td></td></tr><tr><td>E-cadherin (CD324)&lt;br/&gt; Clone: 2.40E+11</td><td>Cell Signaling Technology</td><td>3195</td><td></td></tr><tr><td>Keratin 5 Polyclonal Chicken Antibody, Purified&lt;br/&gt; Clone: Poly9059</td><td>BioLegend</td><td>905901</td><td></td></tr><tr><td>p63&lt;br/&gt; Clone: 4a4</td><td>abcam</td><td>ab735</td><td></td></tr><tr><td>Recombinant Anti-KLF4 antibod&lt;br/&gt; Clone: EPR20753-25</td><td>abcam</td><td>ab214666</td><td></td></tr><tr><td>Vimentin</td><td>Sigma-Aldrich</td><td>AB5733</td><td></td></tr><tr><td>&lt;strong&gt;Secondary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Donkey anti-species* antibodies with fluorophore of choice</td><td>Jackson Immuno</td><td></td><td></td></tr></tbody></table>

functional characterization and visualization of esophageal fibroblasts using organoid co-cultures

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell populations are tucked away in anatomically distinct locations, enabling exclusive interactions with niche signals that maintain stemness. While the development of epithelial organoid cultures provides a powerful tool for understanding the role of stem and progenitor cells in homeostasis and disease, the interaction within the niche environment is largely absent, thereby hindering the identification of factors influencing stem cell behavior. Fibroblasts play a key role in directing epithelial stem and progenitor fate. Here, a comprehensive organoid-fibroblast co-culture protocol enabling the delineation of fibroblast subpopulations in esophageal progenitor cell renewal and differentiation is presented. In this protocol, a method to isolate both epithelial cells and fibroblasts in parallel from the esophagus is described. Distinct fluorescence-activated cell sorting strategies to isolate both the esophageal progenitor cells as well as the fibroblast subpopulations from either transgenic reporter or wild-type mice are outlined. This protocol provides a versatile approach that can be adapted to accommodate the isolation of specific fibroblast subpopulations. Establishing and passaging esophageal epithelial organoid mono-cultures is included in this protocol, enabling a direct comparison with the co-culture system. In addition, a 3D clearing approach allowing for detailed image analysis of epithelial-fibroblast interactions is described. Collectively, this protocol describes a comparative and relatively high-throughput method for identifying and understanding esophageal stem cell niche components in vitro.

The esophagus is divided into different layers: epithelium, lamina propria, submucosa, and muscularis externa (Figure 1A). Fibroblasts reside within the submucosa and lamina propria, referred to as the stroma. In this protocol, the muscularis externa is mechanically removed (Figure 1B), which does not lead to a loss of fibroblasts (Pdgfr&#945;H2BeGFP+) residing in the stroma (Figure 1C). Before dissociation, the epithelium is separated from the stroma resulting in two tissue segments (Figure 1B). Separating the two layers provides the opportunity to increase dissociation time for the more robust epithelial layer compared to the fragile stromal layer. In this way, an efficient isolation protocol yielding both viable epithelial progenitor cells as well as stromal fibroblasts is established (Figure 1B). Esophageal progenitor cells are sorted based on their high INTEGRIN-&#946;4 and E-CADHERIN expression (Figure 1C,D).
Subpopulations of fibroblasts can be isolated by using distinct markers. In this protocol, a strategy for fibroblast isolation based on commonly used fibroblast markers PDGFR&#945;&#160;and DPP4 (CD26) is provided. Isolation by either the Pdgfr&#945;H2BeGFP reporter expression or DPP4 antibody shows that around 50% of the isolated cells are fibroblasts (Figure 1E,F). Additionally, 70% of the PDGFR&#945;+ fibroblasts are DPP4+, indicating that a largely overlapping, but not identical, fibroblast population is obtained. After isolating both epithelial and stromal cell populations, esophageal progenitor cells are either cultured alone or together with fibroblasts in a matrix dome. To study the contribution of fibroblasts to organoid formation, the co-culture is maintained in a growth factor reduced medium (Figure 1G).
Esophageal progenitor cells form organoids in the presence of EGF, NOGGIN and RSPO (ENR). Removing NOGGIN and reducing the amount of RSPO (25 ng/&#181;L; ERlow) is sufficient to prevent organoid formation (Figure 2A). Interestingly, adding either DPP4+ or PDGFR&#945;+ fibroblasts to the esophageal progenitor cells in the ERlow medium restores the organoid forming ability, demonstrating a supportive function for both fibroblast populations (Figure 2A-D). Visualization of the Pdgfr&#945;H2BeGFP transgene shows that fibroblasts are in close contact with the epithelial progenitor cells during organoid formation (Figure 2A). At day 6, Pdgfr&#945;H2BeGFP+&#160;fibroblasts are still abundantly present in the co-culture. Fibroblasts are present throughout the dome, near and touching the organoids (full arrow), or attached to the organoids (arrowhead; Figure 2D).
Whole mount staining shows a 3D representation of the interaction of the fibroblasts with the organoids (Figure 3). While it is possible to perform the whole mount protocol without the use of a clearing solution, it decreases transparency and laser penetration of the organoid (Figure 3B, z-view). When mounting organoids, the spacer helps to maintain organoid morphology. In contrast, plating the coverslip directly on the organoids (without a spacer) flattens the organoids and results in loss of organoid structure (Figure 3A,B).
Both the DPP4+ and PDGFR&#945;+ fibroblasts are found to be wrapped around the organoids (Figure 3C, Video1,&#160;and Video 2). Differentiation of esophageal organoids can be assessed using different markers. Figure 4 shows that the staining protocol provided is suitable for easier-to-stain keratins (KRT14/13) as well as more-difficult-to-stain transcription factors (TRP63/KLF4). The co-culture protocol generates organoids with a similar differentiation pattern, as demonstrated in vivo13,14 and as seen in organoids grown in ENR medium; KRT14+ or TRP63+ progenitor cells form the outer layer and KRT13+ or KLF4+ differentiated cells oriente inwards.
This protocol provides a tool to study the esophageal stem cell niche in vitro and visualizes the interaction between organoids and fibroblasts. By implementing a protocol for the isolation of fibroblasts using antibodies, the method is adaptable and can be used to study fibroblast subpopulations without the need of transgenic mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64905/64905fig01.jpg" /> 
Figure 1: Isolation of progenitor cells and fibroblast subpopulations from the esophagus. (A) Schematic overview of the different layers in the esophagus. The stroma contains the lamina propria and submucosa. (B) Schematic overview of the isolation protocol. The muscle (muscularis externa) is mechanically removed using forceps; the remaining esophagus is cut open and incubated in thermolysin to separate the epithelial layer from the stroma. The epithelium and stroma are separated, mechanically minced, and enzymatically digested to single cell suspensions. Dissociated cells are then stained and prepared for FACS. (C) Cross section of the esophagus stripped from the muscularis externa showing Pdgfr&#945;H2BeGFP+ fibroblasts in the stroma. INTEGRIN-&#946;4 (ITG&#946;4) and E-CADHERIN (ECAD) double positive cells are the epithelial progenitor cells of the esophagus. Scale bar = 100 &#181;m. (D) Representative flow cytometry plot of epithelial cell isolation showing the percentage of live cells (upper panel) from all single cells. The lower panel shows the percentage of isolated ITG&#946;4+ ECAD+ progenitor (Prog.) cells from all live cells. (E) Representative flow cytometry plot of stromal cell isolation showing the percentage of live cells (upper left panel). Representative flow cytometry plots showing the percentage of isolated DPP4+ fibroblasts (Fibr.; upper right panel) and Pdgfr&#945;+ fibroblasts (lower left panel) of all live cells. 70% of the Pdgfr&#945;+ fibroblasts are also DPP4+ (lower right panel). (F) Representative flow cytometry plot of the stroma showing DPP4+ only cells (2.5%), DPP4+ PDGFR&#945;+ cells (37.5%), and PDGFR&#945;+ only cells (17.7%). The percentages are of all live cells. (G) Epithelial only cells are plated in a matrix dome in the presence of 50 ng/&#181;L EGF, 100 ng/&#181;L NOGGIN, and 250 ng/&#181;L RSPO (ENR), or together with fibroblasts in the presence of EGF and a low concentration of RSPO (25 ng/&#181;L). <a href="https://www.jove.com/files/ftp_upload/64905/64905fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64905/64905fig02.jpg" /> 
Figure 2: Representative results of organoid co-cultures. (A) Brightfield images showing growth of the organoids from day 1 to day 6. The brightfield images with the organoids co-cultured with Pdgfr&#945;H2BeGFP+ fibroblasts also show the nuclear eGFP signal. Scale bar = 25 &#181;m. (B) Brightfield images of the whole matrix dome at day 6. The left column shows organoid co-cultures grown in the presence of Pdgfr&#945;+ fibroblasts in ERlow or ElowRlow medium. The middle column shows organoid co-cultures grown in the presence of DPP4+ fibroblasts in ERlow or ElowRlow medium. The right column shows organoid mono-cultures grown in ENR medium. ENR medium = EGF (50 ng/&#181;L), NOGGIN (100 ng/&#181;L), and RSPO (250 ng/&#181;L). ERlow = EGF and 25 ng/&#181;L RSPO. ElowRlow = 5 ng/&#181;L EGF and 25 ng/&#181;L RSPO. Scale bar = 500 &#181;m. (C) Graph showing the organoid forming efficiency (%) (i.e., the percentage of cells forming organoids in different culture conditions). Each dot represents a matrix dome and the bar represents the mean of all dots per condition. (D) Brightfield and fluorescent image of day 6 organoids co-cultured with Pdgfr&#945;H2BeGFP+ fibroblasts. Pdgfr&#945;H2BeGFP+ fibroblasts are present throughout the dome, attached to the organoids (arrowhead), and unattached but in contact with the organoids (full arrow). Scale bar = 250 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64905/64905fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64905/64905fig03.jpg" /> 
Figure 3: Whole mount staining protocol for the study of fibroblast-organoid interactions. (A) Schematic overview of the whole mount immunofluorescence protocol. AB = antibody. (B) Immunofluorescence picture of uncleared whole mount staining showing a decreased transparency and penetration of the laser light compared to the cleared organoids. The absence of a spacer results in flattening of the organoid and loss in organoid morphology. (C) Whole mount images of the co-cultured organoids show 3D surfaces of the organoids with VIMENTIN+ fibroblasts (Fibr.) wrapped around and in close contact with the organoid. 3D cross sections and 2D plane images show the lumen of the organoid. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64905/64905fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64905/64905fig04.jpg" /> 
Figure 4: Whole mount images reveal distinct basal and suprabasal cell populations. (A) Whole mount staining of mono- and co-cultured organoids with Pdgfr&#945;H2BeGFP+ fibroblasts showing KRT14+ basal cells in the outer layer and KRT13+ differentiated suprabasal cells. Scale bar = 50 &#956;m. (B) Whole mount staining of mono- and co-cultured organoids with Pdgfr&#945;H2BeGFP+ fibroblasts showing TRP63+ basal cells in the outer layer and KLF4+ differentiated suprabasal cells. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64905/64905fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Table describing the organoid culture media components. <a href="https://www.jove.com/files/ftp_upload/64905/Table1_revised_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Video 1: Pdgfr&#945;H2BeGFP+ fibroblast wrapped around and in close contact with the organoid. The video accompanies the upper panel of Figure 3C. The scale bar&#160;in Figure 3C is 50 &#181;m, and the organoid is ~120 &#181;m in diameter. VIMENTIN is shown in white, E-CADHERIN in red, Pdgfr&#945;H2BeGFP&#160;in green, and DAPI in blue. <a href="https://www.jove.com/files/ftp_upload/64905/Video 1_Re.avi" target="_blank">Please click here to download this Video.</a>
Video 2: DPP4+ fibroblast wrapped around and in close contact with the organoid. The video accompanies the lower panel of Figure 3C. The scale bar&#160;in Figure 3C is 50 &#181;m, and the organoid is ~120 &#181;m in diameter. VIMENTIN is shown in white, E-CADHERIN in red, and DAPI in blue. <a href="https://www.jove.com/files/ftp_upload/64905/Video 2_RE.avi" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures at JoVE.com

Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures

Organoid-fibroblast co-cultures provide a model to study the in vivo stem cell niche. Here, a protocol for esophageal organoid-fibroblast co-cultures is described. Additionally, whole mount imaging is used to visualize the fibroblast-organoid interaction.

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell ...

functional-characterization-visualization-esophageal-fibroblasts

Nanyang Technological University

Research

JoVE Journal

Biology

2.4K Views.  Karolinska Institutet. Organoid-fibroblast co-cultures provide a model to study the in vivo stem cell niche. Here, a protocol for esophageal organoid-fibroblast co-cultures is described. Additionally, whole mount imaging is used to visualize the fibroblast-organoid interaction.

Video: Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Bioengineering

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

Cancer Research

Advancing 3D Bioprinting with &#954;-Carrageenan Sub-Microgel Medium

Embedded Bioprinting of Tissue-like Structures Using &#954;-Carrageenan Sub-Microgel Medium

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic scaffolds. However, engineering a suitable gel suspension medium that balances precise bioink deposition with cell viability and function presents multiple challenges, particularly in achieving the desired viscoelastic properties. Here, a novel &#954;-carrageenan gel supporting bath is fabricated through an easy-to-operate mechanical grinding process, producing homogeneous sub-microscale particles. These sub-microgels exhibit typical Bingham flow behavior with small yield stress and rapid shear-thinning properties, which facilitate the smooth deposition of bioinks. Moreover, the reversible gel-sol transition and self-healing capabilities of the &#954;-carrageenan microgel network ensure the structural integrity of printed constructs, enabling the creation of complex, multi-layered tissue structures with defined architectural features. Post-printing, the &#954;-carrageenan sub-microgels can be easily removed by a simple phosphate-buffered saline wash. Further bioprinting with cell-laden bioinks demonstrates that cells within the biomimetic constructs have a high viability of 92% and quickly extend pseudopodia, as well as maintain robust proliferation, indicating the potential of this bioprinting strategy for tissue and organ fabrication. In summary, this novel &#954;-carrageenan sub-microgel medium emerges as a promising avenue for embedded bioprinting of exceptional quality, bearing profound implications for the in vitro development of engineered tissues and organs.

Author Spotlight: Development of Homogeneous &#954;-Carrageenan Sub-Microgel Baths for High-Resolution 3D Bioprinting

This study introduces a novel &#954;-carrageenan sub-microgel suspension bath, displaying remarkable reversible jamming-unjamming transition properties. These attributes contribute to the construction of biomimetic tissues and organs in embedded 3D bioprinting. The successful printing of heart/esophageal-like tissues with high resolution and cell growth demonstrates high-quality bioprinting and tissue engineering applications.

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic ...

Engineering

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Human Esophageal Organoids and Air-Liquid Interface Culture Protocols

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

Author Spotlight: Investigating the Pathophysiology of Eosinophilic Esophagitis

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and ...

Immunology and Infection

Advanced Two-Layered 3D Co-Culture System for Epithelial and Fibroblast Cells

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(&#949;-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

Author Spotlight: Advancing 3D Coculture Systems with PVA-PCL Nanofibrous Membranes 

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Medicine

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of the human intestinal epithelium, in which they exhibit crypt/villus-like structures. Here, we describe the generation of hiPSC-derived intestinal organoids by the stepwise differentiation of hiPSCs into definitive endoderm, which is then posteriorized to form hindgut epithelium before being transferred into 3D culture conditions. The 3D culture environment consists of extracellular matrix (ECM) (e.g., Matrigel or other compatible ECM) supplemented with SB202190, A83-01, Gastrin, Noggin, EGF, R-spondin-1 and CHIR99021. Organoids undergo passaging every 7 days, where they are mechanically disrupted before transfer to fresh extracellular matrix and allowed to expand. QPCR and immunocytochemistry confirm that hiPSC-derived intestinal organoids contain mature intestinal epithelial cell types including goblet cells, Paneth cells and enterocytes. Additionally, organoids show evidence of polarization by expression of villin localized on the apical surface of epithelial cells.
The resulting organoids can be used to model human intestinal development as well as numerous human intestinal diseases including inflammatory bowel disease. To model intestinal inflammation, organoids can be exposed to inflammatory mediators such as TNF-&#945;, TGF-&#946;, and bacterial LPS. Organoids exposed to proinflammatory cytokines display an inflammatory and fibrotic phenotype in response. Pairing of healthy versus hiPSCs derived from patients with IBD may be useful in understanding mechanisms driving IBD. This may reveal novel therapeutic targets and novel biomarkers to assist in early disease diagnosis.

This protocol allows for the differentiation of human pluripotent cells into intestinal organoids. The protocol mimics normal human development by differentiating cells into a population of definitive endoderm, hindgut endoderm and then intestinal epithelium. This makes the protocol suitable for studying both intestinal development as well as disease modelling applications.

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of ...