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.

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Research

JoVE Journal

Immunology and Infection

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

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

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

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

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...