This work was supported by Institut Carnot France Futur Elevage (&#34;OrganoPig&#34; project) and by INRAE HOLOFLUX (&#34;Holopig&#34; project). The authors are grateful to the Genotoul core facilities (TRI). We acknowledge Christelle Knudsen (GenPhySE, INRAE, Toulouse) for&#160; careful proofreading.

<ol>
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The authors have nothing to disclose.

This protocol describes a method used to cryopreserve epithelial crypts from the piglet intestine for the long-term storage and subsequent culture of 3D organoids. This protocol uses a freezing solution containing DMSO, FBS, the Y27632 ROCK inhibitor, DMEM, and antibiotics. Another study in pigs obtained organoids from crypts cryopreserved in a similar freezing solution but without the ROCK inhibitor15. The Y27632 ROCK inhibitor was included to prevent apoptosis and maintain the stem cell pool since, after thawing, epithelial crypt cells are dissociated, which may lead to cell death (&#39;anoikis&#39;)27,28. Interestingly, equine enteroids have been obtained from epithelial crypts frozen in a culture medium containing only DMEM and DMSO16; this simple method has not been tested for pig epithelial crypts yet. Other methods have been published to grow human and pig organoids from frozen tissues or biopsies instead of epithelial crypts4,17. The advantage of this method is the ability to directly cryopreserve the intestinal tissues without performing the crypt isolation procedure, which requires time and laboratory equipment. This might be convenient when the tissues have to be collected far from the lab. However, when isolating the crypts immediately after slaughter, large segments of the intestine can be processed to obtain a very high number of crypts, which is not the case when starting from small frozen tissue fragments. After the thawing of epithelial crypts, organoids were observed from 3-4 days post-seeding and split&#160;after 10 days. This is a slower growth rate than when starting the culture from fresh epithelial crypts, for which the organoids were obtained from day 1 post-seeding, and can usually be split at around day 511. Khalil et al. also reported a delayed growth of pig enteroids when starting from frozen crypts15, suggesting that stem cells might require time to recover their proliferative capacity. We also obtained a lower number of organoids when starting from frozen crypts compared to fresh crypts, which might be due to the death of stem cells during the freezing process. In some attempts of crypts thawing (&#60;20%), we did not obtain organoids from frozen crypts, probably due to a suboptimal cryopreservation procedure (e.g., delayed freezing after crypt isolation of probably more than 1 h). Thus, we recommend keeping the crypts on ice until counting, and freezing them as quickly as possible.
For 3D organoids, we chose to use a commercial organoid culture medium formulated for humans. Indeed, previous reports have showed that pig intestinal organoids grow efficiently with this medium8,11,14,19,25,26,29,30. It is of interest for this culture medium to be ready-to-use and have a standardized concentration of growth factors within a batch. However, this culture medium is costly, its composition is undisclosed, and it is thus not possible to modulate its composition. In contrast, other studies have cultured pig intestinal organoids in customized media containing pharmacological inhibitors, recombinant growth factor, and/or conditioned media5,6,7,21. Although highly flexible and cheaper, this method is time-consuming for the production of conditioned media and might lack reproducibility due to potential variability in the concentration of growth factors in conditioned media. Thus, the quality of each batch of conditioned media should be validated by measuring organoid growth or marker gene expression31.
A study has showed that pig jejunal organoids cultured in the same commercial organoid culture medium used here grew faster and seemed less differentiated, compared to enteroids cultured with media containing recombinant growth factor and/or conditioned media23. A high proliferative state facilitates the culture of 3D organoids, but might require inducing differentiation to be more representative of intestinal physiological characteristics. In this protocol, for the passage of 3D organoids, the cells are fully dissociated for counting, allowing to control the number of cells seeded in ECM. This increases the reproducibility of the phenotype of organoids, which is highly influenced by their density. Moreover, counting the cells avoids obtaining too low or an overcrowded culture, that requires adapting the culture schedule. Most other studies prepared organoid fragments not fully dissociated to single cells, and used a dilution ratio for passaging. This method is more straightforward, but might induce variability according to the organoid density of the culture.
For the culture in monolayers, organoid cells are seeded in culture inserts precoated with a thin layer of ECM, that allows the attachment of cells but avoids the growth of organoids in 3D. This protocol used collagen type IV as an ECM protein, as described previously in pigs23. Other studies with pig organoid monolayers used the same tumor-derived ECM used here to culture 3D organoids6,8,9,21,25,30. The advantage of using collagen is the ability to standardize the protein concentration with a fully defined composition, which is not the case in the tumor-derived ECM. A critical step for the success of the culture of cell monolayers is to pay attention to the visual appearance of the precursor 3D organoids, which should have well-defined edges and an empty lumen without black debris. Indeed, organoids with a high maturation level and a low proliferative rate are not an appropriate source of cells for 2D culture. Thus, the timing of the dissociation of 3D organoids into single cells is crucial for the success of this step.
The culture of 2D monolayers from single cells allows standardizing the number of cells seeded, which is more difficult when starting from organoid fragments, as performed in some other methods. We seeded 7.6 x 105 cells per cm2, which is high compared to most other studies21,22,23 in pigs that used a lower cell density, ranging from 0.25 x 105 cells per cm2 to 1.78 x 105 cells per cm2. The requirement of a high number of organoid cells constitutes a limitation of this protocol, but it allowed us to quickly obtain a confluent monolayer, fully covering the culture insert after 1 day. In contrast, Vermeire et al.23 obtained confluency after 4-7 days with a lower density of cells seeded (from 0.25 x 105 cells/cm2 to 0.4 x 105 cells/cm2). Some studies have also used pig organoid cell monolayers that did not fully cover the culture surface for infections with viruses8,30. In these conditions, the apical side of epithelial cells is accessible for treatments, but fully confluent monolayers are required if the objective is to study nutrient absorption or epithelial permeability.
For organoid cell monolayers, a commercial organoid culture medium supplemented with 20% FBS was used, based on a recent study on bovine enteroid-derived monolayers32. In our tests, the supplementation with 20% FBS was necessary to obtain fully confluent monolayers, probably due to a high growth factor requirement. On the contrary, other studies using the same commercial medium have established monolayers without additional FBS8,25,30, but without reaching full confluency. Other studies have also used supplementation with 20% FBS in a customized medium for the culture of pig organoid cell monolayers21,22. In our experiments, TEER is high 1 day after seeding (around 700 &#8486;&#183;cm2), and remains high until day 3 (around 1,500 &#8486;&#183;cm2; this is consistent with the formation of tight junctions, as indicated by the expression of occludin. Van der Hee et al. obtained similar TEER values over 72 h for jejunum organoid cell monolayers21. They also demonstrated that monolayers can be maintained until day 12-15 with daily media changes. In contrast, other studies have reported much lower TEER values (around 200 &#8486;&#183;cm2) for pig organoid cell monolayers6,22. These differences between studies might be related to the gut segment studied or to the media used that influence epithelial differentiation.
In conclusion, the above protocol to grow pig intestinal 3D organoids from frozen epithelial crypts facilitates the organization of the culture work. It reduces the need for fresh tissues to be obtained from living animals. We also explain how to establish fully confluent cell monolayers derived from pig organoids in less than 3 days. Thus, our protocols could be useful resources for scientists studying the pig intestinal epithelium for veterinary or biomedical research.

The intestinal epithelium is formed by a monolayer of cells covering the digestive mucosa at the interface with the luminal environment. This position is associated with diverse functions, such as nutrient absorption and barrier function, that are supported by the presence of stem cells and multiple differentiated epithelial cell types (absorptive, enteroendocrine, Paneth, and goblet cells)1. Immortalized cell lines traditionally used to study epithelial cells have major limitations, since they do not reflect the cellular complexity of the intestinal epithelium and present genomic abnormalities2. The development of three-dimensional (3D) organoids by Sato et al.3 provided a new model to study the intestinal epithelium with an improved physiological relevance. Indeed, intestinal organoids are derived from non-transformed stem cells, are composed of multiple cell types, and recapitulate the functionality of the intestinal epithelium. Intestinal organoids are increasingly being used to understand the development and functions of the intestinal epithelium and its interactions with pathogens, nutrients, toxins, drugs, the microbiota, and its metabolites2.
Initially developed for humans and mice, the methods used to culture intestinal organoids have recently been adapted to other species, including pigs4. Gonzales et al.5 were the first to culture pig organoids from the jejunum ; since then, porcine organoids have been described for other gut segments (duodenum, ileum, and colon)6,7,8, and have been shown to retain a location-specific phenotype9,10,11. Pig intestinal 3D organoids are now commonly used to study the effect of nutrients12,13 or enteric infections6,8,14.
Most of the studies have described the culture of intestinal organoids starting from freshly isolated epithelial crypts. However, this is not always feasible for logistical reasons, notably when working with large animals such as pigs. Indeed, animal facilities for pigs can be located far from the lab where organoids are cultured, which complicates the work organization. Moreover, organoid culture is time-consuming; thus, it is not practical to simultaneously grow multiple organoid lines, for instance, from different gut segments or several animals. To circumvent these issues, a few studies in humans, horses, and pigs have described methods to culture organoids from frozen intestinal tissues (or biopsies) or from isolated epithelial crypts4,15,16,17. These methods allow the cryopreservation of intestinal epithelial stem cells from multiple gut segments of a single animal, which can then be used to grow organoids when needed. Moreover, this allows a strong reduction in the number of live animals used as donors of stem cells, since large stocks of cryopreserved crypts can be created (principles of 3R). Another advantage of this method is the growth of intestinal organoids only from animals of interest after obtaining phenotypic or genotypic results, which is highly cost-effective.
In vivo, intestinal epithelial cells are polarized, with the apical side directed toward the lumen. In vitro, in 3D organoids, the apical side of epithelial cells is also facing the lumen (i.e., inside the organoids)4. This organization prevents access to the apical side, which is an issue when studying the effects of luminal components (e.g., nutrients, microbes, metabolites) on epithelial cells. To circumvent this disadvantage, several methods have been developed, such as the culture of organoid cells as 2D monolayers, microinjection, and polarity reversal (&#34;apical-out organoids&#34;)18,19. The culture of organoid cell monolayers is emerging as the most efficient and tractable system. The principle is to dissociate 3D organoids into single cells and seed them on a cell culture vessel previously coated with a thin layer of extracellular matrix (ECM)20. In these culture conditions, the apical side of the epithelial cells is facing upward, and is thus accessible to experimental treatments20. The culture of organoid cell monolayers was recently adapted for the pig intestine21,22; cell monolayers derived from pig 3D organoids have been used for multiple applications, including the study of enteric infections6,23,24,25, the transport of nutrients9, and digestive disease modeling26.
Here, this study first presents a detailed protocol for the culture and maintenance of pig intestinal 3D organoids derived from cryopreserved epithelial crypts (Figure 1). Then, a protocol is described to establish cell monolayers from pig intestinal 3D organoids. The methods described here provide experimental tools that can be used to study the pig intestinal epithelium for nutrient transport, barrier function, and host-microorganism interactions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA, pH 8.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9260G</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>Sarstedt</td>
			<td>62.554.502</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well cell culture plate </td>
			<td>Corning</td>
			<td>003526</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well cell culture plate </td>
			<td>Corning</td>
			<td>003548</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL polypropylene conical tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumine (BSA)</td>
			<td>Euromedex</td>
			<td>A6003</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Centrifuge Universal 320 R</td>
			<td>Hettich</td>
			<td>1406</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagene type IV from human placenta</td>
			<td>Sigma</td>
			<td>C5533</td>
			<td>Prepare the stock solution at 1 mg/mL in acetic acid according to the manufacturer&#39;s recommendation. Aliquot (500 &#181;L) and store at -20 &#176;C. </td>
		</tr>
		<tr>
			<td>CoolCell LX Cell Freezing Container</td>
			<td>Corning</td>
			<td>432003</td>
			<td>Used to cryopreserve crypts and organoids. </td>
		</tr>
		<tr>
			<td>Countess 3 Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>16842556</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 22 mm x 50 mm</td>
			<td>VWR</td>
			<td>630-1845</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotube ClearLine 1 mL</td>
			<td>Clear line </td>
			<td>390706</td>
			<td>Used to cryopreserve crypts and organoids. </td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Prepare the stock solution at 5 mg/mL in water according to the manufacturer's recommendation. Aliquot (20 &#181;L) and store at -20 &#176;C </td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT) </td>
			<td>Merck</td>
			<td>10197777001</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>31966047</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>DMSO (Dimethyl Sulfoxide)</td>
			<td>Corning</td>
			<td>25-950-CQC</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr>
			<td>Epredia Superfrost Plus Adhesion microscopic slide</td>
			<td>VWR</td>
			<td>631-9483 </td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270-106</td>
			<td>Store 5 mL aliquots at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile Cell Strainers</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363549</td>
			<td>Used for crypt isolation. </td>
		</tr>
		<tr>
			<td>Fixed Tilt 3D Platform Rotator </td>
			<td>VWR</td>
			<td> 97025-564 </td>
			<td>Used for incubations in the immunostaining protocol. </td>
		</tr>
		
		<tr>
			<td>Gibco PBS, pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010015</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Gibco TrypLE Express Enzyme (1x), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605-010</td>
			<td>Enzyme dissociation reagent. Store at room temperature.</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, Alexa fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11008</td>
			<td>Secondary antibody. Store at 4 &#176;C. Working dilution 1:1000. </td>
		</tr>
		<tr>
			<td>IntestiCult Organoid Growth Medium (Human)</td>
			<td>Stem Cell Technology</td>
			<td>6010</td>
			<td>Organoid culture medium. Store at -20 &#176;C. Thaw the basal medium and organoid supplement at room temperature and mix (1:1). Store the mix at 4 &#176;C for up to 1 week. </td>
		</tr>
	
	<tr>
			<td>Insert with PET membrane transparent Falcon for plate 24 wells</td>
			<td>Corning</td>
			<td>353095</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td> Eclipse TS2</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Tumor-derived extracellular matrix used for the 3D culture of organoids. Matrigel polymerizes at room temperature. Use cooled tips to pipet the Matrigel. Prepare 500 &#181;L aliquots and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Mounting medium for fluorescence with DAPI</td>
			<td>Vectashield</td>
			<td>H1250</td>
			<td>
Store at 4 &#176;C.
</td>
		</tr>
		<tr>
			<td>Occludin polyclonal antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>71-1500</td>
			<td>Primary antibody. Store at -20 &#176;C. Working dilution 1:200. </td>
		</tr>
		<tr>
			<td>Paraformaldehyde 32%</td>
			<td>Electron microscopy science</td>
			<td>15714</td>
			<td>Prepare 4% paraformaldehyde (PFA) solution under chemical hood by adding 5 mL of 32% PFA to 35 mL of PBS. Aliquot by 10 mL and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td>Antibacterial. Store 5 mL aliquots at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Phalloidin TRITC</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>Probe for actin staining. 1 mg/mL stock solution. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen</td>
			<td>ant-pm-05</td>
			<td>Antimicrobial agent for primary cells acting on bacteria, mycoplasma and fungi. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>ROCK Inhibitor (Y27632)</td>
			<td>ATCC</td>
			<td>ACS-3030</td>
			<td>Used to maintain the stem cells. Prepare the stock solution at 10 mM in sterile water according to the manufacturer&#39;s recommendation and store aliquots (50 &#181;L) at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Rotating shaker mix XL</td>
			<td>Clear line </td>
			<td>062646CL</td>
			<td>Used for crypt isolation. </td>
		</tr>
		<tr>
			<td>Stripette Serological Pipets 10 mL</td>
			<td>Corning</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish </td>
			<td>TPP</td>
			<td>93100</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>Sigma</td>
			<td>8787</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr>
			<td>Trypan Blue stain 0.4% </td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr>
			<td>Vacuum system Vacusip</td>
			<td>Integra</td>
			<td>159000</td>
			<td>Used to remove the medium of organoid wells. </td>
		</tr>
	</tbody>
</table>

culture of piglet intestinal 3d organoids from cryopreserved epithelial crypts and establishment of cell monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Following the protocol described above, epithelial crypts are obtained from the pig intestine and cryopreserved for long-term storage in liquid nitrogen (Figure 1 and Figure 2A). After thawing, the crypt stem cells are seeded in the ECM (Figure 2B). The crypt structure is usually lost after this step, due to the disintegration of the crypt structure in the ECM. The organoids can be observed within 3-4 days, and then rapidly grow and develop budding structures (Figure 2B). We successfully obtained organoids after thawing frozen crypts in &#62;80% of attempts. Approximately 10 days after thawing (according to the growth rate of organoids), a passage of organoids is performed to expand the culture (Figure 3). The organoids grow faster after splitting, and they present diverse morphologies, with some cystic and a majority of budding organoids. For optimal maintenance of the culture, the organoids used for passaging must present a clear and empty lumen and well-defined edges (examples indicated by green arrows) with no black debris in the lumen (indicated by red arrows), as observed in mature organoids (Figure 3). We found that black cell debris starts accumulating around day 6 post-passaging. Thus, splitting or freezing the organoids at day 4-5 post-passaging is recommended.
The maturity stage of the organoids is also an important point in obtaining cell monolayers. Organoids with advanced maturity (indicated by the presence of black cell debris in the lumen) are not optimal to seed monolayers. We usually dissociate 3D organoids 4 days after passage to collect cells for 2D culture. Approximately one to three wells of 3D organoids cultured at 3,000 cells per 50 &#181;L dome of the ECM are needed for seeding one culture insert with 2.5 x 105 cells. Cells attach and form a fully confluent monolayer within 1 day (Figure 4A), which is confirmed by the high TEER of around 700 &#8486;&#183;cm2 (Figure 4B). However, the cell edges are difficult to visualize by brightfield microscopy at this early time point, probably due to a low differentiation level. The TEER remains high for 3 days (Figure 4B).
Actin staining indicates that the apical side of the epithelial cells is oriented toward the lumen in 3D organoids (Figure 5A). Organoid cells seeded in culture insert form a confluent single layer of epithelial cells, with the apical side oriented toward the upper compartment (Figure 4B,C). Occludin staining reveals the presence of tight junctions at the apical side of the epithelial cells in 3D organoids and in cell monolayers (Figure 5A-C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64917/64917fig01.jpg" /> 
Figure 1: Schematic representation of the methods used to culture 3D organoids and cell monolayers derived from organoids. Epithelial crypts are isolated from the piglet intestine. These crypts can i) be used immediately to culture 3D organoids or ii) be frozen and stored in a biobank in liquid nitrogen. Cryopreserved crypts can be thawed and used to culture 3D organoids. The 3D organoid culture can be maintained with successive splitting, or frozen and stored in the biobank. Cell monolayers can be obtained from the 3D organoid culture to allow access to the apical side of the cells and study the epithelial barrier function. <a href="https://www.jove.com/files/ftp_upload/64917/64917fig01large.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/64917/64917fig02v2.jpg" /> 
Figure 2: Culture of pig intestinal 3D organoids from cryopreserved epithelial crypts. (A) Representative microscopic image of freshly isolated jejunal crypts. (B)&#160;Representative microscopic images of 3D organoids obtained after thawing of the jejunal crypts. The organoids were cultured in a 25 &#181;L dome of ECM in a 48-well plate. The figure shows images of the 3D organoids at 4, 7, 8, 9, and 10 days post-seeding. The scale bar represents 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64917/64917fig02large.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/64917/64917fig03v2.jpg" /> 
Figure 3: Morphology of pig intestinal 3D organoids derived from cryopreserved epithelial crypts after the first passage. Representative microscopic images showing the development of organoids derived from cryopreserved jejunum crypts after the first passage from day 4 to day 7. Green arrows indicate clear organoids appropriate for the passage or seeding of monolayers. Red arrows indicate mature organoids not suitable for splitting or seeding monolayers; thus, wells should be used before the apparition of this morphology. The scale bar represents 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64917/64917fig03large.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/64917/64917fig04v2.jpg" /> 
Figure 4: Characteristics of cell monolayers derived from pig intestinal 3D organoids. (A) Representative microscopic images of the monolayer morphology over 3 days. Jejunum organoid cells were seeded at 2.5 x 105 cells into 0.33 cm2 cell culture inserts coated with collagen IV at 50 ng/mL. The scale bar represents 500 &#181;m. (B) Transepithelial electrical resistance (TEER) of organoid cell monolayers over 3 days. Dots connected by a line correspond to the same well at different times. <a href="https://www.jove.com/files/ftp_upload/64917/64917fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64917/64917fig05v2.jpg" /> 
Figure 5: Imaging of pig jejunum organoids cultured in 3D or 2D. Confocal microscopy imaging of (A) 3D organoids 5 days after splitting and (B,C) organoid cell monolayers 3 days after seeding (B: XZ section; C:&#160;XY section). DNA (blue) was stained with DAPI. Actin (red) was stained with phalloidin. Occludin (green) was stained with a polyclonal antibody. White arrows indicate occludin localized at the tight junction. The scale bar represents 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64917/64917fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers at JoVE.com

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

culture-piglet-intestinal-3d-organoids-from-cryopreserved-epithelial

Research

JoVE Journal

Biology

2.2K Views.  Université de Toulouse, INRAE, ENVT. Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells. 

Video: Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment.
Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues 1-6. The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties.
The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type1, 7-13. Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

A rotating cell culture system that allows epithelial cells to grow under physiological conditions resulting in 3-D cellular aggregate formation is described. The aggregates generated display in vivo-like characteristics not observed in conventional culture models and serve as a more accurate organotypic model system for a multitude of scientific investigations.

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Mucociliary Epithelial Organoids from Xenopus Embryonic Cells: Generation, Culture and High-Resolution Live Imaging

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated clearance. Many clinically relevant defects in the mucociliary epithelium are inferred as they occur deep within the body. Here, we introduce a tractable 3D model for mucociliary epithelium generated from multipotent progenitors that were microsurgically isolated from Xenopus laevis embryos. The mucociliary epithelial organoids are covered with newly generated epithelium from deep ectoderm cells and later decorated with distinct patterned multiciliated cells, secretory cells, and mucus-producing goblet cells that are indistinguishable from the native epidermis within 24 h. The full sequences of dynamic cell transitions from mesenchymal to epithelial that emerge on the apical surface of organoids can be tracked by high-resolution live imaging. These in vitro cultured, self-organizing mucociliary epithelial organoids offer distinct advantages in studying the biology of mucociliary epithelium with high-efficiency in generation, defined culture conditions, control over number and size, and direct access for live imaging during the regeneration of the differentiated epithelium.

We describe a simple protocol to develop mucociliary epithelial organoids from deep ectoderm cells isolated from Xenopus laevis embryos. The multipotent progenitors regenerate epithelial goblet cell precursors and allow live tracking of the initiation and progression of the cell transitions on the surface of organoids.

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.