Name: Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures (Video) | JoVE Journal
Uploaded: 2023-01-06T15:00:00.000+00:00
Duration: 11 min 58 s
Description: Learn esophageal organoid-fibroblast co-culture methods to study stem cell niche interactions and cancer-associated fibroblast function using whole mount imaging techniques.

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5 minutes

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.

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

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 (PdgfrαH2BeGFP+) 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-β4 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 PDGFRα and DPP4 (CD26) is provided. Isolation by either the PdgfrαH2BeGFP reporter expression or DPP4 antibody shows that around 50% of the isolated cells are fibroblasts (Figure 1E,F). Additionally, 70% of the PDGFRα+ 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/µL; ERlow) is sufficient to prevent organoid formation (Figure 2A). Interestingly, adding either DPP4+ or PDGFRα+ 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 PdgfrαH2BeGFP transgene shows that fibroblasts are in close contact with the epithelial progenitor cells during organoid formation (Figure 2A). At day 6, PdgfrαH2BeGFP+ 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 PDGFRα+ fibroblasts are found to be wrapped around the organoids (Figure 3C, Video1, 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="Esophageal cross-section diagram with epithelial and stromal cell analysis using flow cytometry." class="xfigimg" src="/files/ftp_upload/64905/64905fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
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 PdgfrαH2BeGFP+ fibroblasts in the stroma. INTEGRIN-β4 (ITGβ4) and E-CADHERIN (ECAD) double positive cells are the epithelial progenitor cells of the esophagus. Scale bar = 100 µm. (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 ITGβ4+ 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 Pdgfrα+ fibroblasts (lower left panel) of all live cells. 70% of the Pdgfrα+ fibroblasts are also DPP4+ (lower right panel). (F) Representative flow cytometry plot of the stroma showing DPP4+ only cells (2.5%), DPP4+ PDGFRα+ cells (37.5%), and PDGFRα+ 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/µL EGF, 100 ng/µL NOGGIN, and 250 ng/µL RSPO (ENR), or together with fibroblasts in the presence of EGF and a low concentration of RSPO (25 ng/µL). <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="Organoid formation, eGFP expression, fibroblast influence; microscopy, chart, process analysis." class="xfigimg" src="/files/ftp_upload/64905/64905fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
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 PdgfrαH2BeGFP+ fibroblasts also show the nuclear eGFP signal. Scale bar = 25 µm. (B) Brightfield images of the whole matrix dome at day 6. The left column shows organoid co-cultures grown in the presence of Pdgfrα+ 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/µL), NOGGIN (100 ng/µL), and RSPO (250 ng/µL). ERlow = EGF and 25 ng/µL RSPO. ElowRlow = 5 ng/µL EGF and 25 ng/µL RSPO. Scale bar = 500 µm. (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 PdgfrαH2BeGFP+ fibroblasts. PdgfrαH2BeGFP+ fibroblasts are present throughout the dome, attached to the organoids (arrowhead), and unattached but in contact with the organoids (full arrow). Scale bar = 250 µm. <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="Cellular staining method diagram; 3D tumor spheroid clearing results; microscopy imaging." class="xfigimg" src="/files/ftp_upload/64905/64905fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
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 µm. <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="3D cell culture diagram showing mono-culture and co-culture; protein expression analysis in 3D and 2D." class="xfigimg" src="/files/ftp_upload/64905/64905fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Whole mount images reveal distinct basal and suprabasal cell populations. (A) Whole mount staining of mono- and co-cultured organoids with PdgfrαH2BeGFP+ fibroblasts showing KRT14+ basal cells in the outer layer and KRT13+ differentiated suprabasal cells. Scale bar = 50 μm. (B) Whole mount staining of mono- and co-cultured organoids with PdgfrαH2BeGFP+ fibroblasts showing TRP63+ basal cells in the outer layer and KLF4+ differentiated suprabasal cells. Scale bar = 50 µm. <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: PdgfrαH2BeGFP+ fibroblast wrapped around and in close contact with the organoid. The video accompanies the upper panel of Figure 3C. The scale bar in Figure 3C is 50 µm, and the organoid is ~120 µm in diameter. VIMENTIN is shown in white, E-CADHERIN in red, PdgfrαH2BeGFP in green, and DAPI in blue. <a href="https://www.jove.com/files/ftp_upload/64905/Video%201_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 in Figure 3C is 50 µm, and the organoid is ~120 µm 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%202_RE.avi" target="_blank">Please click here to download this Video.</a>

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

Research

JoVE Journal

Biology

This video describes an in vitro culture technique to generate human organotypic esophageal constructs by co-culturing primary human esophageal fibroblast and squamous epithelial cells within a decellularized porcine scaffold. These 3D culture models mimic the function and physiology of their in vivo tissue counterparts.

Esophageal 3D Raft Culture Model: An In Vitro Culture Technique to Generate Human Esophageal Mucosa Model

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 α-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.

Isolation of Myofibroblasts from Mouse and Human Esophagus

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.

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

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

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.

Culture and Imaging of Human Nasal Epithelial Organoids

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.

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

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.

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

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.

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

The present protocol describes the establishment and histological analysis of esophageal organoid models that represent different stages of tumor progression. This method enables researchers to study changes in cellular morphology, spatial organization, and molecular marker expression patterns during the transition from normal to cancerous tissues.

Establishment and Histological Analysis of Esophageal Organoids Modeling the Progression from Normal to Cancerous Tissues

This protocol describes a method for simultaneously isolating cancer organoids, carcinoma-associated fibroblasts (CAFs), and non-tumor-associated fibroblasts (CFs) from the same patient with esophageal squamous cell carcinoma (ESCC). Employment of these patient-matched materials could avoid issues from inter-patient variability and provide an appropriate experimental platform for ESCC research.

Establishment of Tumor Organoids, Carcinoma-Associated Fibroblasts, and Counterpart Fibroblasts from the Same Esophageal Cancer Patient

Learn esophageal organoid-fibroblast co-culture methods to study stem cell niche interactions and cancer-associated fibroblast function using whole mount imaging techniques.

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

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