Name: studying cryptosporidium infection in 3d tissue-derived human organoid culture systems by microinjection
Uploaded: 2019-09-14T13:00:00
Description: Watch this Scientific Journal Video about Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection at JoVE.com

We are grateful to Deborah A. Schaefer from the School of Animal and Comparative Biomedical Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, AZ, USA for helping us with oocyst production and analysis. We also thank Franceschi Microscopy and Imaging Center and D.L. Mullendore at Washington State University for TEM preparation and imaging of isolated organoid oocysts.
D.D. is the recipient of a VENI grant from the Netherlands Organization for Scientific Research (NWO-ALW, 016.Veni.171.015). I.H. is the recipient of a VENI grant from the Netherlands Organization for Scientific Research (NWO-ALW, 863.14.002) and was supported by Marie Curie fellowships from the European Commission (Proposal 330571 FP7-PEOPLE-2012-IIF). The research leading to these results has received funding from the European Research Council under ERC Advanced Grant Agreement no. 67013 and from NIH NIAIH under R21 AT009174 to RMO. This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society and was funded by a grant from the Dutch Cancer Society.

All tissue handling and resection was performed under Institutional Review Board (IRB) approved protocols with patient consent.
1. Preparation of C. parvum Oocysts for Injection
NOTE: Cryptosporidium oocysts were purchased from a commercial source (see the Table of Materials). These oocysts are produced in calves and are stored in phosphate-buffered saline (PBS) with antibiotics. They can be stored for about 3 months...

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Culture of Cryptosporidium parasites in intestinal and airway organoids provides an accurate model to study host-parasite interactions10 but also has many other applications. For example, current methods of selecting and propagating genetically modified Cryptosporidium parasites require passage in mice19 which does not allow isolation of parasites that have modifications essential for in vivo infection. Organoid culture of Cryptosporidium provides...

Development of drugs or vaccines for treatment and prevention of Cryptosporidium infection has been hindered by the lack of in vitro systems that precisely mimic the in vivo situation in humans1,2. Many of the currently available systems either only allow short term infection (&#60;5 days) or do not support the complete life cycle of the parasite3,4. Other systems which enable the complete development of the parasite are based on immortalized cell lines or cancer cell lines which do not faithfully recapitulate the physiological situation in humans5,6,7. Organoids or &#8216;mini-organs&#8217; are 3D tissue derived structures which are grown in an extracellular matrix supplemented with various tissue specific growth factors. Organoids have been developed from various organs and tissues. They are genetically stable and recapitulate most functions of the organs of their origin, and can be maintained in culture for extended periods of time. We have developed a method for infecting human intestinal and lung organoids with Cryptosporidium that provides an accurate in vitro model for the study of host-parasite interactions relevant to intestinal and respiratory cryptosporidiosis8,9,10,11,12,13. In contrast to other published culture models, the organoid system is representative of real-life host parasite interactions, allows for completion of the life cycle so that all stages of the parasite life cycle can be studied, and maintains parasite propagation for up to 28 days10.
Cryptosporidium parvum is an apicomplexan parasite that infects the epithelium of the respiratory and intestinal tracts, causing prolonged diarrheal disease. The resistant environmental stage is the oocyst, found in contaminated food and water14. Once ingested or inhaled, the oocyst excysts and releases four sporozoites that attach to epithelial cells. Sporozoites glide on hosts cells and engage host cell receptors, but the parasite does not fully invade the cell, and appears to induce the host cell to engulf it15. The parasite, which is internalized within an intracellular but extracytoplasmic compartment, remains at the apical surface of the cell, replicating within a parasitophorous vacuole. It undergoes two rounds of asexual reproduction&#8212;a process called merogony. During merogony, type I meronts develop which contain eight merozoites that are released to invade new cells. These merozoites invade new cells to develop into type II meronts containing four merozoites. These merozoites, when released, infect cells and develop into macrogamonts and microgamonts. Microgametes are released and fertilize the macrogametes producing zygotes that mature into oocysts. Mature oocysts are subsequently released into the lumen. Oocysts are either thin-walled which immediately excyst to reinfect the epithelium, or thick walled which are released into the environment to infect the next host14. All stages of the Cryptosporidium life cycle have been identified in the organoid culture system previously developed by our group10.
Since human organoids faithfully replicate human tissues9,11,13, and support all replicative stages of Cryptosporidium10, they are the ideal tissue culture system to study Cryptosporidium biology and host-parasite interactions. Here we describe the procedures for infecting organoids with both Cryptosporidium oocysts and excysted sporozoites, and isolating the new oocysts produced in this tissue culture system.

<table>
<tbody>
<tr>
<td>Basement membrane extract (extracellular matrix)</td>
<td>amsbio</td>
<td>3533-010-02</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Crypt-a-Glo antibody (Oocyst specific antibody)</td>
<td>Waterborne, Inc</td>
<td>A400FLR-1X</td>
<td>Final Concentration = Use 2-3 drops/slide</td>
</tr>
<tr>
<td>Crypto-Grab IgM coated Magnetic beads</td>
<td>Waterborne, Inc</td>
<td>IMS400-20</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dynamag 15 rack</td>
<td>Thermofisher Scientific</td>
<td>12301D</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dynamag 2 rack</td>
<td>Thermofisher Scientific</td>
<td>12321D</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>EMD Millipore&nbsp;Isopore Polycarbonate Membrane Filters- 3&micro;m</td>
<td>EMD-Millipore</td>
<td>TSTP02500</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fast green dye</td>
<td>SIGMA</td>
<td>F7252-5G&nbsp;&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Femtojet 4i Microinjector</td>
<td>Eppendorf</td>
<td>5252000013</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Glass capillaries of 1 mm diameter</td>
<td>WPI</td>
<td>TW100F-4</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Matrigel (extracellular matrix)</td>
<td>Corning</td>
<td>356237</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Microfuge tube 1.5ml</td>
<td>Eppendorf</td>
<td>T9661-1000EA</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Micro-loader tips</td>
<td>Eppendorf</td>
<td>612-7933</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Micropipette puller P-97</td>
<td>Shutter instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Normal donkey Serum</td>
<td>Bio-Rad</td>
<td>C06SB</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Penstrep</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium hypoclorite (use 5%)</td>
<td>Clorox</td>
<td>50371478</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Super stick slides</td>
<td>Waterborne, Inc</td>
<td>S100-3</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Swinnex-25 47mm Polycarbonate filter holder</td>
<td>EMD-Millipore</td>
<td>SX0002500</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Taurocholic acid sodium salt hydrate</td>
<td>SIGMA</td>
<td>T4009-5G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Tween-20</td>
<td>Merck</td>
<td>8221840500</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Vectashield mounting agent</td>
<td>Vector Labs</td>
<td>H-1000</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Vortex Genie 2</td>
<td>Scientific industries, Inc</td>
<td>SI0236</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Adv+++ (DMEM+Penstrep+Glutamax+Hepes)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Final amount</td>
</tr>
<tr>
<td>DMEM</td>
<td>Invitrogen</td>
<td>12634-010</td>
<td>500ml</td>
</tr>
<tr>
<td>Penstrep</td>
<td>Gibco</td>
<td>15140-122</td>
<td>5ml of stock in 500ml DMEM</td>
</tr>
<tr>
<td>Glutamax</td>
<td>Gibco</td>
<td>35050038</td>
<td>5ml of stock in 500ml DMEM</td>
</tr>
<tr>
<td>Hepes</td>
<td>Gibco</td>
<td>15630056</td>
<td>5ml of stock in 500ml DMEM</td>
</tr>
<tr>
<td> INTESTINAL ORGANOID MEDIA-OME (Expansion media)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Final concentration</td>
</tr>
<tr>
<td>A83-01</td>
<td>Tocris</td>
<td>2939-50mg</td>
<td>0.5&micro;M</td>
</tr>
<tr>
<td>Adv+++</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>make upto 100 ml</td>
</tr>
<tr>
<td>B27</td>
<td>Invitrogen</td>
<td>17504044</td>
<td>1X</td>
</tr>
<tr>
<td>EGF</td>
<td>Peprotech</td>
<td>AF-100-15</td>
<td>50ng/mL</td>
</tr>
<tr>
<td>Gastrin</td>
<td>Tocris</td>
<td>3006-1mg</td>
<td>10 nM</td>
</tr>
<tr>
<td>NAC</td>
<td>Sigma</td>
<td>A9125-25G</td>
<td>1.25mM</td>
</tr>
<tr>
<td>NIC</td>
<td>Sigma</td>
<td>N0636-100G</td>
<td>10mM</td>
</tr>
<tr>
<td>Noggin CM</td>
<td>In house*</td>
<td>&nbsp;</td>
<td>10%</td>
</tr>
<tr>
<td>P38 inhibitor (SB202190)</td>
<td>Sigma</td>
<td>S7076-25 mg</td>
<td>10&micro;M</td>
</tr>
<tr>
<td>PGE2</td>
<td>Tocris</td>
<td>2296/10</td>
<td>10 nM</td>
</tr>
<tr>
<td>Primocin</td>
<td>InvivoGen</td>
<td>ant-pm-1</td>
<td>1ml/500ml media</td>
</tr>
<tr>
<td>RSpoI CM</td>
<td>In house*</td>
<td>&nbsp;</td>
<td>20%</td>
</tr>
<tr>
<td>Wnt3a CM</td>
<td>In house*</td>
<td>&nbsp;</td>
<td>50%</td>
</tr>
<tr>
<td>In house* - cell lines will be provided upon request</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>INTESTINAL ORGANOID MEDIA-OMD (Differentiation media)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To differentiate organoids, expanding small intestinal organoids were grown in a Wnt-rich medium for six to seven days after splitting, and then grown in a differentiation medium (withdrawal of Wnt, nicotinamide, SB202190, in a differentiation medium (withdrawal of Wnt, nicotinamide, SB202190, prostaglandin E2 from a Wnt-rich medium or OME)</td>
</tr>
<tr>
<td>LUNG ORGANOID MEDIA- LOM (Differentiation media)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Final concentration</td>
</tr>
<tr>
<td>Adv+++</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>make upto 100 ml</td>
</tr>
<tr>
<td>ALK-I A83-01</td>
<td>Tocris</td>
<td>2939-50mg</td>
<td>500nM</td>
</tr>
<tr>
<td>B27</td>
<td>Invitrogen</td>
<td>17504044</td>
<td>0.0763888889</td>
</tr>
<tr>
<td>FGF-10</td>
<td>Peprotech</td>
<td>100-26</td>
<td>100ng/ml</td>
</tr>
<tr>
<td>FGF-7</td>
<td>Peprotech</td>
<td>100-19</td>
<td>25ng/ml</td>
</tr>
<tr>
<td>N-Acetylcysteine</td>
<td>Sigma</td>
<td>A9125-25G</td>
<td>1.25mM</td>
</tr>
<tr>
<td>Nicotinamide</td>
<td>Sigma</td>
<td>N0636-100G</td>
<td>5mM</td>
</tr>
<tr>
<td>Noggin UPE</td>
<td>U-Protein Express</td>
<td>Contact company directly</td>
<td>10%</td>
</tr>
<tr>
<td>p38 MAPK-I</td>
<td>Sigma</td>
<td>S7076-25 mg</td>
<td>1&micro;M</td>
</tr>
<tr>
<td>Primocin</td>
<td>InvivoGen</td>
<td>ant-pm-1</td>
<td>1:500</td>
</tr>
<tr>
<td>RhoKI Y-27632</td>
<td>Abmole Bioscience</td>
<td>M1817_100 mg</td>
<td>2.5&micro;m</td>
</tr>
<tr>
<td>Rspo UPE</td>
<td>U-Protein Express</td>
<td>Contact company directly</td>
<td>10%</td>
</tr>
<tr>
<td>Reducing buffer (for resuspension of oocysts and sporozoites for injection)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Final concentration</td>
</tr>
<tr>
<td>L-Glutathione reduced</td>
<td>Sigma</td>
<td>G4251-10MG</td>
<td>0.5 &mu;g/&mu;l of OME/OMD/LOM</td>
</tr>
<tr>
<td>Betaine</td>
<td>Sigma</td>
<td>61962</td>
<td>0.5 &mu;g/&mu;l of OME/OMD/LOM</td>
</tr>
<tr>
<td>L-Cysteine</td>
<td>Sigma</td>
<td>168149-2.5G</td>
<td>0.5 &mu;g/&mu;l of OME/OMD/LOM</td>
</tr>
<tr>
<td>Linoleic acid</td>
<td>Sigma</td>
<td>L1376-10MG</td>
<td>6.8 &mu;g/ml of OME/OMD /LOM</td>
</tr>
<tr>
<td>Taurine</td>
<td>Sigma</td>
<td>T0625-10MG</td>
<td>0.5 &mu;g/&mu;l of OME/OMD/LOM</td>
</tr>
<tr>
<td>Blocking buffer (for immunoflourescence staining)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Final concentration</td>
</tr>
<tr>
<td>Donkey/Goat serum</td>
<td>Bio-Rad</td>
<td>C06SB</td>
<td>2%</td>
</tr>
<tr>
<td>PBS</td>
<td>Thermo-Fisher</td>
<td>70011044</td>
<td>Make upto 100ml</td>
</tr>
<tr>
<td>Tween 20</td>
<td>Merck</td>
<td>P1379</td>
<td>0.1%</td>
</tr>
<tr>
<td>List of Antibodies used </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Alexa 568 goat anti-rabbit</td>
<td>Invitrogen</td>
<td>A-11011</td>
<td>Dilution-1:500; RRID: AB_143157</td>
</tr>
<tr>
<td>Crypt-a-Glo Comprehensive Kit- Fluorescein-labeled antibody Crypto-Glo</td>
<td>Waterborne, Inc</td>
<td>A400FLK</td>
<td>Dilution- 1:200</td>
</tr>
<tr>
<td>Crypta-Grab IMS Beads- Magnetic beads coated in monoclonal antibody reactive</td>
<td>Waterborne, Inc</td>
<td>IMS400-20</td>
<td>Dilution-1:500</td>
</tr>
<tr>
<td>DAPI</td>
<td>Thermo Fisher Scientific</td>
<td>D1306</td>
<td>Dilution-1:1000; RRID : AB_2629482</td>
</tr>
<tr>
<td>Phalloidin-Alexa 674</td>
<td>Invitrogen</td>
<td>A22287</td>
<td>Dilution-1:1000; RRID: AB_2620155</td>
</tr>
<tr>
<td>Rabbit anti-gp15 antibody generated by R. M. O'Connor (co-author).</td>
<td>Upon request</td>
<td>Upon request</td>
<td>Dilution-1:500</td>
</tr>
<tr>
<td>Sporo-Glo</td>
<td>Waterborne, Inc</td>
<td>A600FLR-1X</td>
<td>Dilution- 1:200</td>
</tr>
</tbody>
</table>

studying cryptosporidium infection in 3d tissue-derived human organoid culture systems by microinjection

Cryptosporidium parvum is one of the major causes of human diarrheal disease. To understand the pathology of the parasite and develop efficient drugs, an in vitro culture system that recapitulates the conditions in the host is needed. Organoids, which closely resemble the tissues of their origin, are ideal for studying host-parasite interactions. Organoids are three-dimensional (3D) tissue-derived structures which are derived from adult stem cells and grow in culture for extended periods of time without undergoing any genetic aberration or transformation. They have well defined polarity with both apical and basolateral surfaces. Organoids have various applications in drug testing, bio banking, and disease modeling and host-microbe interaction studies. Here we present a step-by-step protocol of how to prepare the oocysts and sporozoites of Cryptosporidium for infecting human intestinal and airway organoids. We then demonstrate how microinjection can be used to inject the microbes into the organoid lumen. There are three major methods by which organoids can be used for host-microbe interaction studies&#8212;microinjection, mechanical shearing and plating, and by making monolayers. Microinjection enables maintenance of the 3D structure and allows for precise control of parasite volumes and direct apical side contact for the microbes. We provide details for optimal growth of organoids for either imaging or oocyst production. Finally, we also demonstrate how the newly generated oocysts can be isolated from the organoid for further downstream processing and analysis.

The protocols presented here result&#160;in the efficient purification of oocysts and sporozoites (Figure 1A) ready for microinjection. The excystation protocol results in the release of sporozoites from approximately 70&#8211;80% of the oocysts, therefore it is essential to filter out the remaining oocysts and shells through a 3 &#181;m filter. Filtration results in almost 100% sporozoite purification (Figure 1B). Furthermore, addition of a green dye helps ensu...

Watch this Scientific Journal Video about Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection at JoVE.com

Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection

We describe protocols to prepare oocysts and purify sporozoites for studying infection of human intestinal and airway organoids by Cryptosporidium parvum. We demonstrate the procedures for microinjection of parasites into the intestinal organoid lumen and immunostaining of organoids. Finally, we describe the isolation of generated oocysts from the organoids.

Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection

Cryptosporidium parvum is one of the major causes of human diarrheal disease. To understand the pathology of the parasite and develop efficient drugs, an ...

Using this sporozoite purification and microinjection procedure, essentially any host microbe can be studied in any tissue-derived organ system. Using this technique, we can control the amount of pure sporozoites injected into the organoid lumen or the apical side of the organoid. To purify C.parvum sporozoites, transfer the oocyst to a 15-milliliter tube and resuspend the cells at a one times 10 to the seventh oocyst per milliliter excystation medium concentration.After 60 to 90 minutes at 37 degrees Celsius, mix 14 milliliters of an appropriate wash solution with the oocysts, and sediment the cells by centrifugation. Aspirate the supernatant carefully to avoid losing oocysts and resuspend the sporozoic pellet at a three times 10 to the seventh oocyst per one to two milliliters of DMEM concentration. To remove any remaining oocysts and shells, place a 10-milliliter syringe barrel equipped with a 47-milliliter filter holder apparatus fitted with a three-micrometer pore size polycarbonate filter into a 15-milliliter tube at four degrees Celsius.Add 7.5 milliliters of the sporozoite suspension to the filter assembly. When the entire volume has passed through the strainer by gravity, wash any remaining cells through the strainer with another 7.5 milliliters of DMEM. When all of the wash has passed through the strainer, collect the filtered sporozoite suspension by centrifugation for 10 minutes and label the pellet in 50 to 100 microliters of an appropriate organoid culture medium, supplemented with 0.05%fast green dye, and L-glutathione, betaine, L-cysteine, linoleic acid, and taurine containing reducing buffer.To microinject the parasites into the apical side of a 3D organoid, first use a micropipette puller to prepare glass injection capillaries. Use forceps to cut the tip of the capillary to a nine to 12 micrometer diameter to enable an easy flow of sporozoites or oocysts, if you choose to inject oocysts directly, and use micro-loader tips to fill each capillary with a fast green dye labeled oocyst or sporozoite suspension. Then, load the first sporozoite-filled capillary into a microinjector and use an inverted microscope at a 5x magnification and a constant pressure to microinject 100 to 200 nanoliters of the suspension into each organoid.The excystation protocol results in the release of sporozoites from approximately 70 to 80%of the oocysts. Therefore, it is essential to filter out the remaining oocyst shells through a three-micrometer filtration. Remove almost 100%of the unexcysted oocysts.Moreover, the addition of a green dye helps ensure the injection of all of the organoids, and allows visualization of the injected organoids for at least 24 hours after the injection. Although this is a well-practiced method, scanning electron microscopy can be used to ensure that the excystation process did not damage the sporozoites or the oocysts. The injection of equal amounts of oocysts into the organoid lumen can be visually confirmed by simple microscopic imaging.Immunofluorescence assays can also be used to explore the types of cells that are infected by Cryptosporidium. After the differentiated organoids have been infected for five days, the presence of sporozoites within the oocysts can be confirmed by drying down a portion of the oocysts onto an adhesive slide, fixing the oocysts with methanol, and combining DAPI staining with a suitable oocyst-specific antibody. For maximum results, use fresh oocysts, use the same capillary for all of the injections, and change the media every day after injection.Using this technique, various labs are now starting to use organoids to study host-microbe interactions. We are studying commensals as well as other pathogens in similar organoid microculture settings. As Cryptosporidium is a human parasite, it is important to perform these experiments according to Level 2 safety regulations.

studying-cryptosporidium-infection-3d-tissue-derived-human-organoid

Research

JoVE Journal

Immunology and Infection

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 ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

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. ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...