Name: co-culture of murine small intestine epithelial organoids with innate lymphoid cells
Uploaded: 2022-03-23T13:20:00
Description: Watch this Scientific Journal Video about Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells at JoVE.com

E.R. acknowledges a Ph.D. fellowship from the Wellcome Trust (215027/Z/18/Z). G.M.J. acknowledges a Ph.D. fellowship from the Wellcome Trust (203757/Z/16/A). D.C. acknowledges a Ph.D. studentship from the NIHR GSTT BRC. J.F.N. acknowledges a Marie Sk&#322;odowska-Curie Fellowship, a King&#39;s Prize fellowship, an RCUK/UKRI Rutherford Fund fellowship (MR/R024812/1), and a Seed Award in Science from the Wellcome Trust (204394/Z/16/Z). We also thank the BRC flow cytometry core team based at Guy&#39;s Hospital. Rorc(&#947;t)-GfpTG C57BL/6 reporter mice were a generous gift from G. Eberl (Institut Pasteur, Paris, France). CD45.1 C57BL/6 mice were kindly given by T. Lawrence (King&#39;s College London, London) and P. Barral (King&#39;s College London, London).

All experiments must be completed in accordance and compliance with all relevant regulatory and institutional guidelines for animal use. Ethical approval for the study described in the following article and video was acquired in accordance and compliance with all relevant regulatory and institutional guidelines for animal use.
All mice were culled by cervical dislocation according to the standard ethical procedure, conducted by trained individuals. Before organ and tissue harvesting, slicing o...

<ol>
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This protocol describes the methods for establishing murine small intestine organoids, isolating rare ILC1 by minimizing the loss of lymphocytes during the intestinal dissociation protocol, and establishing co-cultures between these two compartments. There are many steps to this protocol, and while some are specific to ILC1s, this approach can be applied to other intestinal immune cell types, and co-culture setups can be modularly adapted to suit individual research questions. Several critical steps (that are recommended...

Communication between the intestinal epithelium and gut-resident immune system is central to the maintenance of intestinal homeostasis1. Disruptions to these interactions are associated with both local and systemic diseases, including Inflammatory Bowel Disease (IBD) and gastrointestinal cancers2. A notable example of one more recently described critical regulator of homeostasis comes from the study of innate lymphoid cells (ILCs), which have emerged as key players in the intestinal immune landscape3. ILCs are a group of heterogenous innate immune cells that regulate intestinal homeostasis and orchestrate inflammation largely through cytokine-mediated signalling4.
Murine ILCs are broadly divided into subtypes based on transcription factor, receptor, and cytokine expression profiles5. Type-1 ILCs, which include cytotoxic Natural Killer (NK) cells and helper-like type-1 ILCs (ILC1s), are defined by expression of the transcription factor (eomesodermin) Eomes and T-box protein expressed in T cells (T-bet)6, respectively, and secrete cytokines associated with T helper type-1 (TH1) immunity: interferon-&#947; (IFN&#947;) and tumor necrosis factor (TNF), in response to interleukin (IL)-12, IL-15 and IL-187. During homeostasis, tissue-resident ILC1s secrete Transforming Growth Factor &#946; (TGF-&#946;) to drive epithelial proliferation and matrix remodelling8. Type-2 ILCs (ILC2s) primarily respond to helminth infection via secretion of T helper type-2 (TH2) associated cytokines: IL-4, IL-5, and IL-13, and are characterized by the expression of retinoic acid-related orphan receptor (ROR) &#945; (ROR-&#945;)9 and GATA Binding Protein 3 (GATA-3)10,11,12. In mice, intestinal &#34;inflammatory&#34; ILC2s are further characterized by expression of Killer cell lectin-like receptor (subfamily G member 1, KLRG)13 where they respond to epithelial tuft-cell derived IL-2514,15. Finally, type-3 ILCs, which include lymphoid tissue inducer cells and helper-like type-3 ILCs (ILC3s), are dependent on the transcription factor ROR-&#947;t16, and cluster into groups that secrete either Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), IL-17, or IL-22 in response to local IL-1&#946; and IL-23 signals17. Lymphoid tissue inducer cells cluster in Peyer&#39;s patches and are crucial for the development of these secondary lymphoid organs during development18, whereas ILC3s are the most abundant ILC subtype in the adult murine small intestine lamina propria. One of the earliest murine intestinal organoid co-culture systems with ILC3s was harnessed to tease apart the impact of the cytokine IL-22 on Signal Transducer And Activator Of Transcription 3 (STAT-3) mediated Leucine-Rich Repeat Containing G Protein Coupled Receptor 5 (Lgr5)+ intestinal stem cell proliferation19, a powerful example of a regenerative ILC-epithelial interaction. ILCs exhibit imprint-heterogeneity between organs20,21 and exhibit plasticity between subsets in response to polarizing cytokines22. What drives these tissue-specific imprints and plasticity differences, and what role they play in chronic diseases such as IBD23, remain exciting topics that could be addressed using organoid co-cultures.
Intestinal organoids have emerged as a successful and reliable model to study the intestinal epithelium24,25. These are generated by culturing intestinal epithelial Lgr5+ stem cells, or entire isolated crypts, which include Paneth cells as an endogenous source of Wnt Family Member 3A (Wnt3a). These 3D structures are maintained either in synthetic hydrogels26 or in biomaterials that mimic the basal lamina propria, for instance, Thermal-crosslinking Basal Extracellular Matrix (TBEM), and are further supplemented with growth factors that mimic the surrounding niche, most notably Epithelial Growth Factor (EGF), the Bone Morphogenetic Protein (BMP)-inhibitor Noggin, and an Lgr5-ligand and Wnt-agonist R-Spondin127. Under these conditions, organoids maintain epithelial apico-basal polarity and recapitulate the crypt-villi structure of the intestinal epithelium with budding stem cell crypts that terminally differentiate into absorptive and secretory cells in the center of the organoid, which then shed into the internal pseudolumen by anoikis28. Although intestinal organoids alone have been hugely advantageous as reductionist models of epithelial development and dynamics in isolation29,30, they hold tremendous future potential for understanding how these behaviors are regulated, influenced, or even disrupted by the immune compartment.
In the following protocol, a method of co-culture between murine small intestinal organoids and lamina propria derived ILC1s is described, which was recently used to identify how this population unexpectedly decreases intestinal signatures of inflammation and instead contributes to increased epithelial proliferation via TGF-&#946; in this system8.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 (BV510)</td>
			<td>BioLegend</td>
			<td>103137</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse NK1.1 (PE)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-5941-83</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>CD127 Monoclonal Antibody (APC)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17-1271-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19 Monoclonal Antibody (eFluor 450)</td>
			<td>Thermo Fisher Scientific</td>
			<td>48-0193-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3e Monoclonal Antibody (eFluor 450)</td>
			<td>Thermo Fisher Scientific</td>
			<td>48-0051-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD5 Monoclonal Antibody (eFluor 450)</td>
			<td>Thermo Fisher Scientific</td>
			<td>48-0031-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris</td>
			<td>4423/10</td>
			<td></td>
		</tr>
		<tr>
			<td>COLLAGENASE D, 500MG</td>
			<td>Merck</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex HA- RSpondin1-Fc HEK293T Cells</td>
			<td>Cell line was used to harvest conditioned RSpondin1 supernatant, the cell line and Materials Transfer Agreement was provided by the Board of Trustees of the Lelands Stanford Junior University (Calvin Kuo, MD,PhD, Stanford University)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DISPASE II (NEUTRAL PROTEASE, GRADE II)</td>
			<td>Merck</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1) (1X) Dulbecco&#39;s Modified Eagle Medium Nutrient Mixture F-12 (Advanced DMEM/F12)</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DNASE I, GRADE II</td>
			<td>Merck</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (1X)</td>
			<td>Gibco</td>
			<td>21969-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethilenediamine Tetraacetate Acid</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP2482-100</td>
			<td></td>
		</tr>
		<tr>
			<td>FC block</td>
			<td>2B Scientific</td>
			<td>BE0307</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, hear inactivated</td>
			<td>Gibco</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX (100X)</td>
			<td>Gibco</td>
			<td>3050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (10X)</td>
			<td>Gibco</td>
			<td>14065056</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (1X)</td>
			<td>Gibco</td>
			<td>12549069</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK-293T- mNoggin-Fc Cells</td>
			<td>Cell line was used to harvest conditioned Noggin supernatant, cell line acquired through Materials Transfer Agreement with the Hubrecth Institute, Uppsalalaan8, 3584 CT Utrecht, The Netherlands, and is based on the publication by Farin, Van Es, and Clevers Gastroenterology (2012).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution (1M)</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>KLRG1 Monoclonal Antibody (PerCP eFluor-710)</td>
			<td>Thermo Fisher Scientific</td>
			<td>46-5893-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Fixable Blue Dead Cell Stain Kit, for UV excitation</td>
			<td>Thermo Fisher Scientific</td>
			<td>L23105</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly-6G/Ly-6C Monoclonal Antibody (eFluor 450)</td>
			<td>Thermo Fisher Scientific</td>
			<td>48-5931-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced Basement Membrane Matrix, Phenol Red-free, LDEV-free</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine (500mM)</td>
			<td>Merck</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>NKp46 Monoclonal Antibody (PE Cyanine7)</td>
			<td>Thermo Fisher</td>
			<td>25-3351-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1 X) 7.2 pH</td>
			<td>Thermo Fisher Scientific</td>
			<td>12549079</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>Gibco</td>
			<td>70013032</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Cytiva</td>
			<td>17089101</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human EGF, Animal-Free Protein</td>
			<td>R&#38;D Systems</td>
			<td>AFL236</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-15 GMP Protein, CF</td>
			<td>R&#38;D Systems</td>
			<td>247-GMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-2 (carrier free)</td>
			<td>BioLegend</td>
			<td>589106</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-7 (carrier free)</td>
			<td>R&#38;D Systems</td>
			<td>407-ML-005/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraComp eBeads</td>
			<td>Thermo Fisher Scientific</td>
			<td>01-2222-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride (ROCK inhibitor)</td>
			<td>Bio-techne</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Falcon</td>
			<td>10788561</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tube</td>
			<td>Eppendorf</td>
			<td>30121023</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL pippette</td>
			<td>StarLab</td>
			<td>E4860-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Falcon</td>
			<td>11507411</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL pippette</td>
			<td>StarLab</td>
			<td>E4860-0025</td>
			<td></td>
		</tr>
		<tr>
			<td>p10 pippette tips</td>
			<td>StarLab</td>
			<td>S1121-3810-C</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 pippette tips</td>
			<td>StarLab</td>
			<td>I1026-7810</td>
			<td></td>
		</tr>
		<tr>
			<td>p200 pippette tips</td>
			<td>StarLab</td>
			<td>E1011-0921</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard tissue culture treated 24-well plate</td>
			<td>Falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 and temperature controled incubator</td>
			<td>Eppendorf</td>
			<td>Galaxy 170 R/S</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Assisted Cellular Sorter</td>
			<td>BD equipment</td>
			<td>FACS Aria II</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated shaker</td>
			<td>Stuart Equipment</td>
			<td>SI500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice box</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light microscope</td>
			<td>Thermo Fisher Scientific</td>
			<td>EVOS XL Core Imaging System (AMEX1000)</td>
			<td></td>
		</tr>
		<tr>
			<td>p10 pippette</td>
			<td>Eppendorf</td>
			<td>3124000016</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 pippette</td>
			<td>Eppendorf</td>
			<td>3124000063</td>
			<td></td>
		</tr>
		<tr>
			<td>p200 pippette</td>
			<td>Eppendorf</td>
			<td>3124000032</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippette gun</td>
			<td>Eppendorf</td>
			<td>4430000018</td>
			<td></td>
		</tr>
		<tr>
			<td>Wet ice</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

When successfully completed, freshly isolated crypts should form budding crypt structures within 2-4 days (Figure 1A). Healthy and robust organoid cultures should be actively growing and can be passaged and expanded as detailed in the protocol.
This protocol describes the isolation of small intestinal ILC1 from the ROR&#947;tGFP murine transgenic reporter line, which allows the isolation of live ILC1 by FACS (Figure 2). Usi...

Watch this Scientific Journal Video about Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells at JoVE.com

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

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 ...

This protocol provides a significant tool to study the interactions between intestinal immune cells and the epithelium and to dissect how these interactions may regulate in intestinal homeostasis and inflammation. The main advantage of this technique is the exquisite amount of experimental control facilitated by the reduction in vitro model, which can be used to address questions in epithelial and immune biology. This system has already been used to determine the role of ILC1 in the expansion of CD44-positive epithelial crypts and has the potential to further advance our understanding of inflammation-mediated gastrointestinal diseases.To begin, place thermal cross-linking basal extracellular matrix on ice to thaw and pre-warm a standard tissue culture-treated 24-well plate by placing it in a 37-degree-Celsius incubator. Afterward, remove the plate containing organoids from the incubator and discard the media from the well to be passaged. Then, add 500 microliters of ice-cold advanced DMEM F12 to the wells, and using a P-1000 tip, harvest the organoids from all the wells into a 15-milliliter tube.Rinse the bottom of the wells with 250 to 300 microliters of ice-cold advanced DMEM F12, ensuring no organoids remain in the well and pool into the 15-milliliter tube containing harvested organoids. Resuspend the one-to two-day-old harvested organoid pellet in one milliliter of ice cold advanced DMEM F12. Pre-coat one 1.5-milliliter tube with PBS2 per innate lymphoid cells replicate sample, and then using a pre-coated PBS2 tip, distribute approximately 100 to 200 organoids into the tube.Using a PBS2-coated P-1000 tip, add a volume of no less than 500 ILC1s as calculated from the sorting step to the PBS2-coated 1.5-milliliter tube containing the organoids. Spin down ILC1 and organoids at 300 G for five minutes at four degrees Celsius and ensure to avoid small-diameter tabletop centrifuges, as they will pull the cells along the edge of the tube interior instead of creating a pellet at the tip of the tube. Then, remove the supernatant slowly and gently without disturbing the pellet and place the sample on ice.While holding the tube on a cold surface, resuspend the organoids and ILC in 30 microliters of thermal-crosslinking basal extracellular matrix at least 10 to 15 times to ensure even distribution. Apply 30 microliters per well of ILC organoids in the thermal-crosslinking basal extracellular matrix to a pre-warmed 24-or 48-well plate to form a single dome. Afterward, place the plate directly in the incubator for 10 to 20 minutes at 37 degrees Celsius and 5%carbon dioxide.Then, add 550 microliters of complete ILC1 medium per well with any desired experimental cytokines or blocking antibodies and incubate at 37 degrees Celsius and 5%carbon dioxide for 24 hours. After 24 hours, gently remove the plate from the incubator and allow the plate to sit in a tissue culture hood for one minute to ensure that the lymphocytes are settled. Remove 200 to 250 microliters of media and place it into an empty well of a 24-well plate.Using an inverted microscope, check the supernatant to ensure that no lymphocytes were removed. If the supernatant is clear, add 300 microliters of fresh ILC1 medium to the co-culture in the original well. If lymphocytes are present in the supernatant, centrifuge at 300 to 400 G for three to five minutes at four degrees Celsius and resuspend the pellet in 300 microliters of fresh ILC1 medium.Then, add the cell suspension to the remaining 200 to 250 microliters of media in the original well. Perform downstream analysis using immunofluorescence, flow cytometry, or FACS purification of target populations into lysis buffer for gene expression analysis by a single-cell or bulk RNA seek or RT-qPCR as described in the text. After successful completion of passage, healthy and robust organoid cultures revealed that the freshly isolated crypts should form budding crypt structures within two to four days.The flow cytometric analysis was performed to isolate small intestinal ILC1 from the ROR gamma-T murine transgenic reporter line, and the expected ILC count range was found to be 350 to 3, 500 isolated cells. After being seeded with organoids, co-cultures can be visualized by immunochemistry cytochemistry. Confocal microscopy revealed the images of small intestinal organoids that are cultured alone and in the presence of ILC1s.Immuno-cytochemical staining reveals presence of CD45-positive ILC1 in close proximity to Zonula occludens protein-1-positive epithelial cells in organoid ILC1 co-cultures. Flow cytometry demonstrated that epithelial cellular adhesion molecule marks intestinal epithelial cells in organoids, while CD45 marks ILC1s. The flow cytometric plot and immunochemistry revealed increased expression of CD44 in intestinal epithelial cells when co-cultured with ILC1.The RT-qPCR studies show that ILC1 co-culture specifically upregulated CD44 variant 6, which was inhibited by TGF-beta 1, 2, 3 neutralizing antibody, and upregulated by recombinant TGF-beta 1 in small intestinal-organoids-only cultures. It is important to be gentle during the manipulation of the organoids to ensure the whole structures are maintained and to check that the structures have pelleted sufficiently after centrifugation. By increasing the system complexity by adding other immune and non-immune components, this in vitro system can be used to address further questions in intestinal biology.

Research

JoVE Journal

Immunology and Infection

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Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

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