Name: the ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids
Uploaded: 2016-05-18T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids at JoVE.com

The authors would like to thank Dr. Sheryl Coutermarsh-Ott, Dylan McDaniel and Bettina Heid for technical discussions. The authors thank Dr. Nanda Nanthakumar for providing the Caco-2 cells. The authors also thank The Multicultural Academic Opportunities Program (MAOP) at Virginia Tech. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Award K01DK092355 (to I.C.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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The authors declare that they have no competing financial interests.

The culture and maintenance of intestinal organoids is a procedure that can be mastered by any individual with adequate tissue culture technique. There are subtleties in passaging when compared to growing cells in a more conventional monolayer, but these subtleties are not difficult to overcome. The critical steps of this method involve being able to grow the organoids to a high enough density for optimal seeding. Experiments must be scaled down with organoids as large seeding densities that can commonly be achieved with cell lines are not practical. This becomes especially apparent when there are multiple treatment groups.
This protocol is intended to provide a step-by-step method to study host-pathogen interactions of the intestinal epithelium with a variety of different bacterial, viral, and fungal pathogens, as well as address difficulties using this system with respect to normalization. Reports are available that describe the interactions of intestinal organoids with the bacterial pathogen Salmonella, yet do not address normalization methods when measuring secreted cytokines13.
There are several difficulties that are encountered when normalizing organoid cultures by different methods. Normalizing secreted protein via bicinchoninic acid assay (BCA) is an option; however, the growth components required for organoid culture (N2 and/or Vitamin B27) interfere with the BCA assay (data not shown). Normalizing via cellular viability, such as a modified MTT assay has been described14; however, a treatment that will alter the mitochondrial metabolic activity of the organoids will introduce an inaccurate method for normalization via this technique as MTT is based on reduction by the action of mitochondrial dehydrogenases15. It is also necessary to remove N-acetyl-cysteine (NAC) from the media, not only if normalization via the MTT technique is desired, but if treating with a bacterial pathogen as NAC can inhibit bacterial growth16.
Advantages of this technique are that secreted cytokines and protein products from organoids can now be normalized against a standard curve of Caco-2 cells. Limitations of this technique are that the normalization is correlative because nuclear staining of non-transformed primary epithelial organoids are normalized against the nuclear staining of a colon cancer cell line. The authors find that fixing with methanol and staining nuclei with nuclear staining dye is an effective normalization technique against a standard curve of Caco-2 cells. Although the growth characteristics of this colon cancer cell line do not exactly mimic the growth characteristics of intestinal organoids, using a nuclear stain and measuring mean fluorescence intensity is a good strategy to normalize against total cell number.
Taken together, the technique described here provides a good starting point to mimic host-pathogen interactions with adequate normalization that is essential for making accurate interpretations using this model system. The significance of this technique with respect to alternative methods is that proper normalization must be performed when conducting any evaluation of secreted protein, such as ELISA based assays.

The ability to harvest and culture primary organoids have been described for small intestine, colon, pancreas, liver and brain and are exciting advances germane to understanding a more physiologically representative phenomena for tissue biology1-5. The first methods describing the culture and maintenance of small intestinal organoids was reported by Sato et al. out of the lab of Hans Clevers1. Prior to this method, harvesting and culture of primary intestinal epithelial cells proved to be limited and ineffective in sustaining epithelial cell growth. Methods included dissociation of tissue via incubation with enzymes, such as collagenase and dispase, which would ultimately lead to the outgrowth of intermixed primary fibroblast cells6. These conditions would also be time restricted in sustaining the epithelial cell culture. Minimal to no epithelial cell niche would form, as the epithelial cells would enter apoptosis due to the lack of appropriate growth factors or loss of contact integrity, termed anokis7. The advent of the 3D-organoid culture system has provided a method to culture primary intestinal cells containing a spectrum of intestinal cell types in sustained culture1. These epithelial organoids have advantages over cell lines being that they are composed of several differentiated cells, and better mimic the organ they are derived from in vivo8. The process to ultimately &#34;grow a mini gut in a dish&#34; has proven to be a valuable tool for assessing the response of intestinal epithelium under different stimuli. Investigating the interaction of primary intestinal cells with microbial pathogen associated molecular patterns (PAMPs) is relevant to the field of immunology as these molecular patterns can regulate diverse responses from both host and microbe9. Not only can investigators now explore these interactions with mouse organoids, but they can be cultured from humans as well2. This technology has the potential to dramatically alter personalized medicine and it is tempting to speculate about advances that this technique will make possible in the near future.
The overall goal of this method is to provide a protocol for the culture, expansion, and treatment of intestinal organoids with a variety of stimuli. Such stimuli can ultimately range from vaccines, bacterial PAMPs, live pathogens, gastrointestinal (GI) and cancer therapeutics. The isolation and culture of mouse intestinal organoids has been adapted from Sato et al. Though there are slight deviations from the original method, the end product being organoid culture is still achieved when following this protocol. This method is focused on describing an adequate technique for proper normalization when working with non-homogenous cell structures, which must be taken into consideration when conducting an assay based on cell number.

<table><tbody><tr><td>Fetal Bovine Serum (FBS)</td><td>Atlanta Biologicals</td><td>S11050</td><td>(Section 1,3,6) Or equivalent brand</td></tr><tr><td>Sorvall Legend XTR Centrifuge</td><td>Thermo</td><td></td><td>(Section 1,3)</td></tr><tr><td>DMEM</td><td>GE Healthcare</td><td>Sh30243.01</td><td>(Section 1,6) For Caco-2 and HEK293 Rspondin1 cells</td></tr><tr><td>HEK293T-Rspondin1 secreting cell line</td><td></td><td></td><td>(Section 1) Described and modified from Kim, K.A. et al. Lentiviral particles contained RSPO1(NM_138683) ORF cDNA cloned into a pReceiver-Lv105 backbone custom ordered and purchased from GeneCopoeia.&amp;nbsp;</td></tr><tr><td>50 ml conical tube</td><td>Falcon</td><td>352070</td><td>(Section 1) Or equivalent brand</td></tr><tr><td>T-175 Flask</td><td>Corning</td><td>431079</td><td>(Section 1) Or equivalent brand&amp;nbsp;</td></tr><tr><td>Protein Matrix</td><td>Corning</td><td>356231</td><td>(Section 2,3,4,5,6) Matrigel Growth Factor Reduced&amp;nbsp;</td></tr><tr><td>HyClone Dulbecco&#039;s (DPBS)</td><td>GE Healthcare</td><td>SH30264.01</td><td>(Section 2,3)</td></tr><tr><td>DMEM/F12&amp;nbsp;</td><td>Life Technologies</td><td>12634-010</td><td>(Section 2,3) Advanced DMEM/F12</td></tr><tr><td>Corning 24 Well TC Plates</td><td>Corning</td><td>3524</td><td>(Section 2)</td></tr><tr><td>N2 Supplement 100x&amp;nbsp;</td><td>Life Technologies</td><td>17502-048</td><td>(Section 2)</td></tr><tr><td>B27 without vitamin A 50x&amp;nbsp;</td><td>Life Technologies</td><td>12587-010&amp;nbsp;</td><td>(Section 2)</td></tr><tr><td>Trizol</td><td>Life Technologies</td><td>15596-026</td><td>(Section 2)</td></tr><tr><td>Glutamine Supplement (Glutamax)</td><td>Life Technologies</td><td>35050-061</td><td>(Section 2) Can Combine with Advanced DMEM/ F12</td></tr><tr><td>HEPES (1 M)</td><td>Life Technologies</td><td>15630-080</td><td>(Section 2) Can Combine with Advanced DMEM/ F12&amp;nbsp;</td></tr><tr><td>10ml Serological Pipet</td><td>Falcon</td><td>357551</td><td>(Section 2) Or equivalent brand</td></tr><tr><td>Murine Noggin</td><td>Peprotech</td><td>250-38</td><td>(Section 2) Stock = 100 mg/ml</td></tr><tr><td>N-Acetyl-L-cysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td>(Section 2) Stock = 1M</td></tr><tr><td>Recombinant Mouse EGF</td><td>Biolegend</td><td>585608</td><td>(Section 2) Stock = 500 mg/ml</td></tr><tr><td>Rocker Variable</td><td>Bioexpres</td><td></td><td>(Section 3)</td></tr><tr><td>dissecting scissors</td><td></td><td></td><td>(Section 3)</td></tr><tr><td>forceps</td><td></td><td></td><td>(Section 3)</td></tr><tr><td>glass slides</td><td></td><td></td><td>(Section 3)</td></tr><tr><td>dissecting tweezers</td><td></td><td></td><td>(Section 3)</td></tr><tr><td>25 ml Serological Pipet</td><td>Falcon</td><td></td><td>(Section 3)</td></tr><tr><td>EDTA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SLBB9821</td><td>(Section 3) 0.5M or alternative TC grade EDTA</td></tr><tr><td>Sterile Petri Dish 100mm x 15mm</td><td>Fisher</td><td>FB0875712</td><td>(Section 3) Or equal sized TC dish</td></tr><tr><td>1ml Syringe</td><td>Becton Dickinson</td><td>309659</td><td>(Section 4)</td></tr><tr><td>Precision Glide Needle</td><td>Becton Dickinson</td><td>305120</td><td>(Section 4) 23G x 1 1/4 (0.6mm x 30mm)</td></tr><tr><td>Flagellin from Bacillus subtilis</td><td>Invivogen</td><td>tlrl-bsfla&amp;nbsp;</td><td>(Section 5,6)</td></tr><tr><td>Listeria monocytogenes</td><td>ATCC</td><td>19115</td><td>(Section 5,6)&amp;nbsp;(Murray&amp;nbsp;et&amp;nbsp;al.)&amp;nbsp;</td></tr><tr><td>Hemocytometer</td><td>Sigma-Aldrich</td><td>Z359629-1EA</td><td>(Section 5,6)Or equivalent brand</td></tr><tr><td>BBL Brain Heart Infusion Agar</td><td>Becton Dickinson</td><td>211065</td><td>(Section 5)</td></tr><tr><td>Bacto Brain Heart Infusion</td><td>Becton Dickinson</td><td>237500</td><td>(Section 5)</td></tr><tr><td>Caco-2</td><td>ATCC</td><td>HTB-37</td><td>(Section 6)</td></tr><tr><td>Trypsin&amp;nbsp;</td><td>gibco</td><td>25200056</td><td>(section 6)</td></tr><tr><td>Methanol</td><td>Fisher</td><td>A412-4</td><td>(Section 6)</td></tr><tr><td>SpectraMax M5</td><td>Molecuar Devices</td><td></td><td>(Section 6)</td></tr><tr><td>96 Well Assay Plate</td><td>Corning</td><td>3603</td><td>(Section 6) Black Plate, Clear Bottom TC treated</td></tr><tr><td>Nuclear Staining Dye</td><td>Life Technologies</td><td>H1399</td><td>(section 6) Hoechst 33342</td></tr><tr><td>T-75 Flask</td><td>Corning</td><td>430641</td><td>(Section 6) Or equivalent brand</td></tr><tr><td>15 ml conical tube</td><td>Falcon</td><td>352096</td><td>(Section1,3) Or equivalent brand</td></tr><tr><td>1.7 ml polypropylene tube</td><td>Bioexpress</td><td>C-3262-1</td><td>Or equivalent brand</td></tr><tr><td>Quick-RNA MiniPrep</td><td>Zymo Research</td><td>R1054</td><td>Or equivalent brand</td></tr><tr><td>TNF-alpha&amp;nbsp;</td><td>Applied Biosystems</td><td>Mm 00443260_g1</td><td>Taqman gene expression assay kit</td></tr><tr><td>IL-6&amp;nbsp;</td><td>Applied Biosystems</td><td>(Mm 00446190_m1</td><td>Taqman gene expression assay kit</td></tr><tr><td>IL-1beta</td><td>Applied Biosystems</td><td>Mm 00434228_m1</td><td>Taqman gene expression assay kit</td></tr><tr><td>IL-18</td><td>Applied Biosystems</td><td>Mm 00434225_m1</td><td>Taqman gene expression assay kit</td></tr><tr><td>18s</td><td>Applied Biosystems</td><td>Hs 99999901_s1</td><td>Taqman gene expression assay kit</td></tr><tr><td>7500 Fast Real Time PCR System</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>Nexus gradient Mastercycler</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>TaqMan Fast Universal PCR Master Mix</td><td>Life Technologies</td><td>4352042</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit</td><td>Life Technologies/Applied Biosystems</td><td>4368814</td><td></td></tr><tr><td>Fast Optical 96-Well Reaction Plate, 0.1 mL</td><td>Life Technologies/Applied Biosystems</td><td>4346907</td><td></td></tr><tr><td>Recombinant Mouse R-Spondin 1 Protein</td><td>R&amp;amp;D Systems</td><td>3474-RS-050</td><td>500 ng/ml</td></tr><tr><td>chloroform</td><td>Sigma-Aldrich</td><td>C7559</td><td></td></tr></tbody></table>

the ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

When following this protocol to cultivate intestinal organoids, characteristic sphere shaped organoids will be present after harvesting. The addition of R-spondin1 conditioned media daily will initiate the growth and budding of the organoids. The growth of organoids is shown in Figure 1A-F, and is representative of intestinal organoids on days 1, 2, 4, 5, 6 and day 14. Figure 1F represents the non-homogeneous growth characteristics of organoids on day 14.
Once the organoids are grown to an adequate number, they can be replated and challenged with various PAMPs and/or microbes. Expression analysis can be performed via standard techniques. This is represented in Figure 2A-C which shows the mRNA expression of inflammatory cytokines IL-18, IL-6, and TNF&#945; which were analyzed following a 24 hr challenge of organoids with heat killed L. monocytogenes and the PAMP flagellin. The rationale for evaluating the mRNA expression cytokines IL-18, IL-6, and TNF&#945; was that these were good candidates for being modulated by epithelial cells in response to pathogenic challenge, and modulation of these inflammatory cytokines would demonstrate the effectiveness of the technique.
Figure 3A-C demonstrates the relative nuclear staining of intestinal organoids with nuclear staining dye following fixation with minimal background staining of debris in the resident protein matrix. This method of fixation and staining can be applied to normalize assays, which will account for differing cell numbers in each well. This is apparent in Figure 4 when generating a standard curve of serial dilutions of Caco-2 cells plated in protein matrix, then fixed and stained with nuclear staining dye. The R2 value of 0.89 indicates a linear relationship between cell number and mean fluorescent intensity, and the linear equation can be used to normalize organoids to cell number. The standard curve depicted in Figure 4 begins to reach the saturation limit beyond 30,000 cells per well. A titration of cells above 30,000 cells per well has been shown in Figure 4 to include the saturation range of nuclear staining dye. Increased accuracy of normalization will be obtained with a cell number that reflects the linear range of the nuclear staining dye before the saturation limit is reached.
<img alt="Figure 1" src="/files/ftp_upload/54033/54033fig1.jpg" /> 
Figure 1: Small Intestinal Organoid Growth. Time course of growth for murine small intestine derived organoids following isolation. (A) Day 1. (B) Day 2. (C) Day 4. (D) Day 5. (E) Day 6. (F) Day 14. Images are representative of organoid growth for each given day. Scale bars equal 200 &#956;m in the left image for A, B, C, D, and E. Scale bars equal 100 &#956;m in the right image for A, B, C, D, and E. Scale bars equal 1,000 &#956;m and 200 &#956;m for left and right images for F, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54033/54033fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54033/54033fig2.jpg" /> 
Figure 2: mRNA Expression of Inflammatory Cytokines Following 24 hr PAMP Challenge. Relative mRNA expression of wild-type intestinal organoids challenged for 24 hr with PAMPs and heat killed Listeria monocytogenes. (A-C) represent fold change of the inflammatory cytokines IL-18, IL-6 and TNF&#945; respectively. Error bars represent Standard Deviation (S.D.) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54033/54033fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54033/54033fig3.jpg" /> 
Figure 3: Relative Fluorescent Staining of Organoids for Normalization. Nuclear staining of organoids with following fixation. (A) Bright field of organoids. (B) Fluorescent staining of organoids with nuclear staining dye. (C) Merged image of bright field and fluorescent staining. Scale bars equal 400 &#956;m in all images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54033/54033fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54033/54033fig4.jpg" /> 
Figure 4: Standard Curve generated from Caco-2 Cells. Standard curve generated from triplicate wells. Cell seeding had a range starting at 60,000 cells per well with dilutions down to 2,500 cells per well. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54033/54033fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54033/54033fig5.jpg" /> 
Figure 5: Overview of the Protocol Diagram. 1-Top Panel of the Figure illustrates harvesting the R-spondin1 conditioned media. 2-Middle Panel of the Figure illustrates aspects of protocol to harvest and culture mouse small intestinal organoids. 3-Bottom Panel of the Figure illustrates: (A) The plating of 14 day cultured organoids. (B) Challenge with PAMPs/ L. monocytogenes, and leaving unoccupied wells to generate a standard curve. (C) Following the PAMP challenge, the plating of Caco-2 cells in the previously unoccupied wells to generate a standard curve. (D) Following the fixation and addition of a nuclear staining dye, measuring the excitation/ emission of the wells and normalizing against a standard curve. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54033/54033fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids at JoVE.com

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

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Nanyang Technological University

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Immunology and Infection

13.5K Views.  Virginia Maryland College of Veterinary Medicine, Virginia Tech.  Hier wordt een protocol te oogsten, te onderhouden en te behandelen muis dunne darm organoids met pathogeen geassocieerde moleculaire patronen (PAMPs) en Listeria monocytogenes beschreven, alsmede nadruk op genexpressie en correcte normalisatie technieken voor eiwit. 

Video: The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Medicine

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

Cancer Research

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberk&#252;hn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate1. Throughout the small intestine, Lgr5+ stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5+ stem cells through several key ligands as Wnt3, EGF, and Dll12.
When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine3. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5+ and Paneth cells.
Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5+ cells to associate and form organoids when co-cultured. This approach, here referred to as &#34;organoid reconstitution assay&#34; (ORA), allows the genetic and biochemical modification of Paneth or Lgr5+ stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5+) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis.

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

Bioengineering

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune cells, and stroma. All of these heterogeneous cells contribute to intestinal homeostasis and participate in antimicrobial host defense. Therefore, identifying a surrogate model for studying immune response and antimicrobial activity of the intestine in an in vitro setting is extremely challenging. In vitro studies using immortalized intestinal epithelial cell lines or even primary crypt organoid culture do not represent the exact physiology of normal intestine and its microenvironment. Here, we discuss a method of culturing mouse colon tissue in a culture dish and how this ex vivo organ culture system can be implemented in studies related to antimicrobial host defense responses. In representative experiments, we showed that colons in organ culture express antimicrobial peptides in response to exogenous IL-1&#946; and IL-18. Further, the antimicrobial effector molecules produced by the colon tissues in the organ culture efficiently kill Escherichia coli in vitro. This approach, therefore, can be utilized to dissect the role of pathogen- and danger-associated molecular patterns and their cellular receptors in regulating intestinal innate immune responses and antimicrobial host defense responses.

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

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

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

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

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

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Biology

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

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 ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

3D Culture of Organoids from Murine Intestinal Crypts and Single Stem Cell

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

Author Spotlight: The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

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