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...

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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

9.3K vistas.  UMC Utrecht. Usando este procedimiento de purificación y microinyección de esporozoíto, esencialmente cualquier microbio huésped puede ser estudiado en cualquier sistema de órganos derivados del tejido. Usando esta técnica, podemos controlar la cantidad de esporozoítos puros inyectados en el lumen organoide o en el lado apical del organoide. Para purificar los esporozoitos de C.parvum, transfiera el ócicle a un tubo de 15 mililitros y resuspendir las células a una multiplicación por 10 al sépti...