Name: Generation and Manipulation of Rat Intestinal Organoids (Video) | JoVE
Uploaded: 2023-06-23T12:55:00
Duration: 1 min 58 s
Description: Watch this Scientific Journal Video about Generation and Manipulation of Rat Intestinal Organoids at JoVE.com

We thank members of the Sumigray and Ameen laboratories for their thoughtful discussions. This work was supported by a Charles H. Hood Foundation Child Health Grant and a Cystic Fibrosis Foundation grant (004741P222) to KS and by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health to NA under award number 2R01DK077065-12.

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

The development of a rat intestinal organoid model preserves important functional characteristics found in the organ in vivo and is a promising tool for preclinical testing, drug screening, and functional assays. This in vitro model can be used in parallel to in vivo preclinical gastroenterology studies, for which rats are often a preferred model due to their larger intestinal size, shared physiological aspects with humans, and in some cases being better disease models38. Here, a robust step-by-step protocol for the isolation of rat intestinal crypts, generation and long-term culture of rat intestinal organoids, as well as downstream applications including functional forskolin swelling assays, whole mount immunofluorescence, 2D monolayer culture, and lentiviral genetic manipulation is outlined. Rat intestinal organoids are likely to be relevant in many disease contexts where the pathophysiology of mouse models is inappropriate and may provide a better model for human intestinal physiology compared to mouse intestinal organoids.
To establish long-lived organoid cultures that can be passaged and expanded, it is essential to identify the key growth factors required for maintaining intestinal epithelial proliferation. Mouse organoids are most often grown in a simple cocktail of EGF, R-spondin, and Noggin, though it has been reported that Noggin is not necessary for intestinal organoid culture39. Conditioned media can replace recombinant growth factors and the most commonly used cell lines are L-WRN, which secretes Wnt3a, Rspondin-3, and Noggin39, L-Wnt3a, and HA-Rspondin1-Fc 293T cells40. L-WRN conditioned media is sufficient to support not only mouse intestinal39 organoid growth, but the growth of intestinal organoids from several farm animals and companion animals, including dogs, cats, chickens, horses, cows, sheep, and pigs12. However, human intestinal organoids are very different in their growth factor requirements, as they require distinct media formulations for their expansion growth phase (i.e., the progression from small to large spheroids) versus their differentiation phase (i.e., the generation and maturation of differentiated cell types)10. The media requirements of rat intestinal organoids closely mirror those of the expansion growth media for human intestinal organoids, yet, notably, rat organoids are capable of both growth and differentiation in this media environment, considerably simplifying their culture requirements. While our initial attempts focused on establishing and growing rat intestinal organoids in L-WRN-conditioned media, long-term culture was tenuous, and rat intestinal organoid lines suffered from a lack of robustness (data not shown). This may be because L-WRN cell lines are engineered to secrete R-spondin 3, while the 293T-Rspo1 cell line recommended here is engineered to secrete R-spondin 1. It is possible that rat and human organoids prefer R-spondin 1, potentially accounting for the failure of rat organoid lines in L-WRN conditioned media.
To most closely recapitulate the in vivo setting, it is important to develop organoid culture conditions that allow for stem cell survival, maintenance, and proliferation, and that can maintain cellular turnover and simultaneous differentiation events into discrete cell types. Therefore, the concentrations of recombinant proteins and/or proteins in conditioned media need to be tightly titrated and controlled to strike this perfect balance. In particular, optimal Wnt levels are essential to avoid the loss of intestinal organoid cultures. Too little Wnt in conditioned media will be incapable of supporting growth, leading to a loss of stem cells and subsequent organoid death; overactivation of Wnt will cause organoids to be cystic and undifferentiated10. While not detailed here, it is strongly recommended to test each batch of L-Wnt3a and 293T-Rspo1 conditioned media using a Wnt reporter luciferase assay, such as a Topflash cell line41. Previous studies have described that an optimal batch of L-Wnt3a media should result in a 15-fold signal increase at 12.5% and a 300-fold signal increase at 50%, compared to 1% L-Wnt3a10. As rat organoids are more sensitive than mouse organoids to culture requirements, particularly Wnt activation levels, these additional quality control steps greatly assist in facilitating the robustness and reliability of rat organoid cultures. Because a similar reporter line is not available for testing Bmp activity and relative Noggin concentrations in Noggin conditioned media, it is advisable to use recombinant Noggin when possible to precisely control Noggin levels. While mouse intestinal organoids can be grown and maintained in the absence of Noggin39, this has not been attempted for rat intestinal organoid cultures.
Beyond cell culture requirements, the successful initial establishment of a rat organoid line depends critically on the efficient depletion of differentiated villi during crypt isolation. High levels of villar contamination cause crypt death, presumably due to either signals from the dying cells or sequestration of essential factors. To deplete these differentiated villi from epithelial preparations precisely and consistently, it is recommended to perform epithelial isolations with the aid of a stereoscope. Visual examination of the epithelium being released provides a clear cue when to discard the PBS and replace it (Figure 1). Crypts should not be collected until there is sufficient depletion of villi. Villar cells are terminally differentiated and cannot generate organoids in culture. Additionally, subsequent passaging of rat intestinal organoids and their use for any downstream application requires delicate care. Incubation in dissociation reagents for longer periods of time (10 min) result in significant cell death and loss of the organoid line.
Here, a simple and fast protocol for generating intestinal monolayers from rat organoids is described. EME and collagen I substrates have different effects on the epithelium, that can be leveraged depending on the purpose of the study. EME allows for the rapid and efficient adhesion of single cells and the formation of cell projections. In contrast, coating the surface with collagen I delays these processes. Once monolayers reach approximately 80% confluency, cells grown on EME begin to generate 3D organoid structures again. However, they lack sufficient physical and chemical support for continued growth. This reversion back to the organoid state can be prevented by maintaining monolayers in EME at a confluency of 50%-80%. The addition of diluted EME to the apical surface of monolayers promotes the rapid recovery and formation of de novo organoids, generating regions of convergence more quickly and readily. On a collagen I surface, cells can form a uniform monolayer and generate small clusters. However, the addition of collagen I on top of monolayers is not sufficient to induce organoid formation. EME must be diluted when adding to the monolayer surface, as there will be a stronger mechanical resistance for the nascent organoid to overcome. However, this diluted EME does not allow for the robust formation of large organoids. Any de novo generated rat organoids that naturally detach from the surface must be immediately removed and transferred to undiluted EME so that structural support and growth can be restored. Due to the small size of the organoids in this step, the passage of organoids is not recommended until robust growth has been established. The underlying biological significance of why EME can support the reformation of organoids, but whether collagen I can or cannot do this, is not clear. However, there have been reports that cells grown in 3D collagen cannot form budded organoids42,43 or support long-term maintenance. Commercially available EME products are heterogeneous mixtures of extracellular proteins, primarily laminin and collagen IV44. Therefore, the distinct composition of proteins and the ability of an epithelial cell to engage with the extracellular matrix using different cellular complexes could allow for remodeling in EME but not collagen I. Whether collagen I-derived monolayers can be put into EME to support organoid formation and growth has not been tested.
Genetic manipulation of the rat intestinal organoid model is described here, and protocols for lentiviral transduction of 3D organoids and transient transfection of 2D monolayers are outlined. To overcome the low efficiency of lentiviral organoid transduction, a protocol was developed for the transient transfection of 2D monolayers. The flat morphology and exposed apical domains of monolayers provide easier access to viruses and DNA-containing complexes. The expression of an EGFP reporter using the pLJM1-EGFP vector was used for the validation of this technique. GFP reporter expression was observed after 24 h, and was maintained for 5-6 days in monolayers. Future studies focusing on lentiviral transduction of monolayers are likely to have higher efficiency than 3D organoid transduction. Using the above protocols, 3D organoids can be reformed from infected 2D monolayers to facilitate the creation of stable lines. With care, rat intestinal organoids lines can be successfully maintained for over a year, remaining stable over many passages, cryopreserved, successfully thawed, and genetically modified using lentiviral transduction, thereby addressing the need for an accessible and tractable in vitro intestinal organoid model that retains physiological relevance to humans.

The human small intestinal epithelial architecture and cellular composition are complex, reflecting their physiological functions. The primary role of the small intestine is to absorb nutrients from food passing through its lumen1. To maximize this function, the intestinal surface is organized into finger-like protrusions called villi, which increase the absorptive surface area, and cup-like invaginations called crypts, which house and insulate the stem cells. Within the epithelium, various specialized absorptive and secretory cell types are generated to perform distinct functions1. Because of this complexity, it has been difficult to model tissues like the intestine in high passage-transformed immortalized cell lines. However, the study of stem cells, especially adult stem cells and their differentiation mechanisms, has allowed for the development of 3D intestinal organoid cultures. The use of organoid models has transformed the field, in part due to their recapitulation of some architectural components and cell type heterogeneity found in the intact intestine. Intestinal organoids can be cultured long-term in vitro due to the maintenance of the active stem cell population2.
Intestinal organoids have rapidly become an adaptable model to study stem cell biology, cell physiology, genetic disease, and nutrition3,4, as well as a tool to develop novel drug delivery methods5. In addition, patient-derived organoids are being utilized for disease modeling, drug discovery, and personalized treatment screening, among others6,7,8,9. However, human intestinal organoids still present challenges. Tissue availability, requirements for Institutional Review Board approval, and ethical issues limit the widespread use of human samples. Additionally, human intestinal organoids generated from intestinal crypts require two distinct culture conditions for the maintenance of undifferentiated stem cells or to induce the differentiation of mature cell types10. This contrasts with in vivo, where stem cells and mature differentiated cell types are simultaneously present and continuously generated/maintained1. On the other hand, mouse intestinal organoids, which are grown in a less complex cocktail of growth factors, do not require this switch in media composition and can maintain stem cells and differentiated cells in the same media context2,11. However, key differences in mouse intestine when compared to humans can make mouse organoids a suboptimal model in many instances. Overall, many intestinal organoids from larger mammals, including horses, pigs, sheep, cows, dogs, and cats, have been successfully generated in culture conditions more closely aligned with mouse intestinal organoids than the culture conditions of human intestinal organoids12. The differences in growth factor conditions between mouse and human organoids likely reflect differences in stem cell niche composition and differing requirements for stem cell survival, proliferation, and maintenance. Therefore, there is a need for an easily accessible model organoid system that 1) closely resembles human intestinal cell composition, 2) contains stem cells with growth factor requirements like those of human intestinal organoids, and 3) is capable of continuously maintaining undifferentiated and differentiated compartments. Ideally, the system would be from a commonly used preclinical animal model, such that in vivo and in vitro experiments can be correlated and used in tandem.
Rats are a commonly used preclinical model for intestinal physiology and pharmacology studies due to their very similar intestinal physiology and biochemistry to humans13, particularly with regards to intestinal permeability14. Their relatively larger size compared to mice makes them more amenable to surgical procedures. While large animal models, including pigs, are sometimes used, rats are a more affordable model, require less space for husbandry, and have readily commercially available standard strains15. A drawback of using rat models is that the genetic toolkit for in vivo studies is not well developed compared to mice, and the generation of novel rat lines, including knockouts, knock-ins, and transgenics, is often cost-prohibitive. The development and optimization of a robust rat intestinal organoid model would allow for genetic manipulation, pharmacological treatments, and higher throughput studies in an accessible model that retains key physiological relevance to humans. However, the advantages of one rodent organoid model versus another is highly dependent on the particular process or gene being studied; certain genes found in humans may be pseudogenes in mice but not rats16,17. Additionally, species-specific cell subtypes are increasingly being unveiled by single-cell RNAseq18,19,20. Finally, rat and mouse intestinal disease models often display considerable variations in phenotypes21,22 such that the model that more closely recapitulates the symptoms and disease process seen in humans must be selected for downstream work. The generation of a rat intestinal organoid model provides additional flexibility and choice for researchers in selecting a model system most appropriate for their circumstances. Here, existing protocols23,24 are expanded upon for the generation of rat intestinal organoids and a protocol is outlined for the generation and maintenance of rat intestinal organoids from the duodenum or jejunum. In addition, several downstream applications, including lentiviral infection, whole mount staining, and forskolin swelling assays, are described.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-D Culture Matrix Rat Collagen I</td>
			<td>Cultrex/R&#38;D Systems</td>
			<td>3447-020-01</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>Corning/Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco/Thermo Fisher</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma Aldrich</td>
			<td>A2942-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Cayman Chemical</td>
			<td>13122</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor</td>
			<td>Stem Cell Technologies</td>
			<td>100-1061</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex HA-Rspondin1-Fc 293T cells</td>
			<td>R &#38; D Systems</td>
			<td>3710-001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Molecular Probes/Thermo Fisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco/Thermo Fisher</td>
			<td>16-000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gastrin I (human)</td>
			<td>Sigma Aldrich</td>
			<td>G9145</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>Stem Cell Technologies</td>
			<td>100-0485</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Thermo Fisher</td>
			<td>35-050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth factor-reduced Matrigel, phenol red-free</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AmericanBio</td>
			<td>AB06021</td>
			<td></td>
		</tr>
		<tr>
			<td>Lanolin</td>
			<td>Beantown Chemical</td>
			<td>144255-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco/Thermo Fisher</td>
			<td>A2916801</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Wnt3a cells</td>
			<td>ATCC</td>
			<td>CRL-2647</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Thermo Fisher</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Sigma Aldrich</td>
			<td>A9165-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide&#160;</td>
			<td>Sigma Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Gibco/Thermo Fisher</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Fisher Scientific</td>
			<td>P31-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma Aldrich</td>
			<td>P7793</td>
			<td>transparent film</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo Fisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco/Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>pLJM1-EGFP</td>
			<td>Addgene</td>
			<td>19319</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Millipore</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine hydrochloride (PEI)</td>
			<td>Sigma Aldrich</td>
			<td>764965</td>
			<td></td>
		</tr>
		<tr>
			<td>p-phenylenediamine&#160;</td>
			<td>Acros Organics/Thermo Fisher</td>
			<td>417481000</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>VWR</td>
			<td>J593-25mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human FGF2 protein</td>
			<td>Peprotech</td>
			<td>100-18B-250ug</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IGF-1 protein</td>
			<td>Biolegend</td>
			<td>B356441</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human Noggin protein</td>
			<td>R &#38; D Systems</td>
			<td>6057-NG-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse EGF protein</td>
			<td>Thermo Fisher</td>
			<td>PMG8041</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley rat</td>
			<td>Charles River Laboratories</td>
			<td>Strain 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>American Bioanalytical</td>
			<td>AB02025-00500</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Gibco/Thermo Fisher</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632 dihydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>Y0503</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and manipulation of rat intestinal organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Generation and Manipulation of Rat Intestinal Organoids

Rat duodenal and jejunal organoids were generated using the protocol outlined in section 2. It is very important during the crypt isolation steps that villi are efficiently depleted from the PBS. If too many villi are plated in the EME with crypts, it can cause death of the entire culture and failure to establish an organoid line. Because of this, it is useful to isolate crypts under a dissecting scope, allowing for visual confirmation of villar depletion. Figure 1 depicts representative villar fragments and crypts (Figure 1A). Note the significantly smaller size of crypts compared to villi (Figure 1B). After plating, the crypts will expand into spheroids over the next few days and will begin to bud and differentiate by day 4-7 (Figure 2). Once the organoids reach an extensively budded stage, they should be passaged. During passaging, it is important to disrupt the organoids enough to split the crypt buds apart, so that organoid numbers can be expanded (Figure 3B).
The successful recovery of organoids after freezing is highly dependent on the state at which they are frozen. Organoids in a highly proliferative undifferentiated state recover with the highest efficiency. Therefore, we recommend inducing them to be spherical and cystic instead of budded and differentiated. To achieve this, Wnt can be hyperactivated by increasing the amount of the Wnt ligand R-spondin in the media and by including nicotinamide in the media, which has been shown to support organoid formation and cell survival in several culture systems30,31. Figure 3A demonstrates a healthy organoid culture just 2 days after thawing. Including BSA in the media during thawing has also helped in the survival of rat intestinal organoid cultures, which have proven to be more delicate than mouse intestinal organoids.
While 3D organoid culture is often preferred because it recapitulates some of the normal intestinal architecture, it makes other approaches, including live imaging, transfections, and lentiviral transductions, more technically challenging. The use of 2D monolayers generated from 3D organoids32 (Figure 4) allows for higher efficiency introduction of the plasmids. While 3D intestinal organoids are traditionally resistant to transient transfections, plasmids encoding for EGFP can be successfully introduced using lipid-based transfection methods. The most cost-effective approach using PEI is outlined in step 6.1 (Figure 5), but electroporation and commercially available transfection reagents have also yielded comparable results (data not shown). Future studies will be focused on whether these approaches can be used for introducing CRISPR constructs into monolayers.
It was important to be able to reform 3D organoids from 2D monolayers after transfection so that they could be maintained as a passageable line with 3D architectural components of crypts. Interestingly, 2D monolayers plated on the EME readily reformed into small spheroids when EME was added back to the top of the cells, whereas a collagen I substrate was not sufficient for the reformation of 3D structures (Figure 6).
While transient transfections are useful for many studies, the formation of stable lines are often more useful, requiring the introduction of lentivirus into the cells. Rat intestinal organoids were infected with lentivirus by modifying previously published protocols (Figure 7). A key step in the protocol is the disruption of organoids into small aggregates or cell clusters. If cultures are not efficiently disrupted and organoids remain intact, the lentiviral particles will not get into the cells. After infection, organoids must recover and regrow. The protocol outlined here allows for the uptake of viral particles by 10%-48% (mean: 19.4% &#177; 6.5%) of cells before selection.
Whole mount staining of organoids can prove difficult due to incomplete removal of EME residue or incomplete antibody penetrance. The protocol outlined here allows for the robust staining of organoids. The visualization of organoids on a confocal microscope can also prove difficult if they are too far away from the coverslip. By using VALAP, a well with some height is created such that organoids are not crushed by the coverslip, but are still allowed to settle close to the coverslip for ease of imaging. Representative staining against the apical anion channel cystic fibrosis transmembrane conductance regulator (CFTR) and phalloidin to label F-actin is shown in Figure 8.
Finally, organoids have utility in functional assays. Patient-derived organoids from cystic fibrosis patients have been used to screen CFTR function, as treatment with the cAMP agonist forskolin induces robust CFTR-mediated fluid secretion, causing organoid swelling29,33-37. One goal of this work was to identify and develop an organoid model that can be used in parallel to in vivo preclinical studies. Therefore, we aimed to determine whether rat intestinal organoids undergo forskolin-induced swelling. Indeed, within 30 min of forskolin treatment, rat organoids swelled, with a maximal effect observed by 120 min (Figure 9).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65343/65343fig01.jpg" /> 
Figure 1: Villar fragments and crypts during epithelial isolation. (A) Representative image of villar fragments in EDTA solution during the crypt isolation protocol. Yellow arrowheads mark villar fragments. Red arrows depict crypts attached to a villar fragment. Note the difference in relative sizes. (B) Higher magnification image of a single crypt (red arrow) so that morphology can be visualized. Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65343/65343fig01large.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/65343/65343fig02.jpg" /> 
Figure 2: Rat intestinal organoid progression. Rat jejunum crypts were plated in EME immediately after isolation (A,B). Within 2 days, the crypts became spheroids (C,D). By Day 5, they began to initiate crypt buds (E,F), which elaborated and grew by Day 7 (G). Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65343/65343fig02large.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/65343/65343fig03.jpg" /> 
Figure 3: Organoids after thawing and passaging. (A)&#160;Rat jejunal organoids were thawed following the outlined protocols after cryopreservation. Note the presence of both spheroids and budded organoids just 2 days after thawing. (B) The same organoid line depicted in A immediately after passaging following the outlined protocol. Note the relative size difference between structures in A and B and the presence of single crypt-like domains in B. Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65343/65343fig03large.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/65343/65343fig04.jpg" /> 
Figure 4: 2D monolayer formation from 3D organoids. (A-C) 2D monolayer progression on EME. (D-F) 2D monolayer progression on collagen I. By day 5, each condition yielded ~80% confluency. Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65343/65343fig04large.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/65343/65343fig05.jpg" /> 
Figure 5: Transient transfection of a 2D monolayer. Representative image of a 2D monolayer grown on EME transiently transfected with pLJM1-EGFP plasmid using PEI. (A) Brightfield, (B) fluorescence (GFP), (C) overlay. The dotted red line marks the monolayer boundary. Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65343/65343fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65343/65343fig06.jpg" /> 
Figure 6: Reformation of 3D organoids from 2D monolayers on EME. (A) Formation of 3D organoids from 2D monolayers grown on EME. Organoids efficiently formed by 5 days after adding EME to the apical surface of the monolayer. Note the abundance of dead cells surrounding the small 3D spheroids. (B) Persistence of 2D monolayers 5 days after collagen I was added to the apical surface of 2D monolayers grown on collagen I. Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65343/65343fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65343/65343fig07.jpg" /> 
Figure 7: Lentiviral infection of 3D organoids. Rat jejunum organoids were infected with nuclear GFP lentiviral particles using the outlined protocol. After recovery and growth for 5 days, organoids were fixed and counterstained with DAPI. (A) DAPI: gray; nuclearGFP: green. (B) nuclearGFP:green. The dotted red line marks the organoid boundary. Scale bars: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65343/65343fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65343/65343fig08.jpg" /> 
Figure 8: Whole mount immunofluorescence of rat intestinal organoids. (A) CFTR, (B) phalloidin, and (C) merged whole mount immunofluorescence of rat jejunal organoids. Note the apical enrichment of CFTR staining in organoids (gray in A, magenta in C). Phalloidin marks F- actin and prominently labels the apical brush border (gray in B, cyan in C). Scale bars: 25 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65343/65343fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65343/65343fig09.jpg" /> 
Figure 9: Rat intestinal organoids swell upon forskolin stimulation. Representative time course of rat intestinal organoid swelling after addition of the cAMP agonist forskolin. The 0 min time represents the time point immediately prior to the addition of 10 &#181;M forskolin. Images show the same organoid at 30 min time intervals. Maximal swelling was observed at 120 min after forskolin addition. The dotted red line outlines the organoid boundary. The dark material in the middle of the organoid lumen is comprised of dead cells. Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65343/65343fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: AdDMEM+ recipe. Ingredients to make the standard AdDMEM+ media, which is the base media throughout the methods shown here. <a href="https://www.jove.com/files/ftp_upload/65343/Table1_Re.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Rat intestinal organoid media (rIOM) recipe. Detailed recipe of the standard rat intestinal organoid media, including solvent and storage conditions for recombinant proteins. <a href="https://www.jove.com/files/ftp_upload/65343/Table2_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Solutions. Recipes and instructions to make other solutions used throughout the protocol. <a href="https://www.jove.com/files/ftp_upload/65343/Table3_resub2_Re.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4. Rat intestinal organoid medium for 2D monolayer culture (rIOM2D). Modified recipe of organoid culture media optimized for the 2D growth of monolayers. <a href="https://www.jove.com/files/ftp_upload/65343/Table4_resub2_RE.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Generation and Manipulation of Rat Intestinal Organoids at JoVE.com

Generation and Manipulation of Rat Intestinal Organoids (Video) | JoVE 

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.