Name: studying cryptosporidium infection in 3d tissue-derived human organoid culture systems by microinjection
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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 at 4 &#176;C and should never be frozen. We normally use oocysts within one month. Organoids can be infected with either intact oocysts, or sporozoites may be isolated from excysted oocysts and used to infect organoids if it is important not to have oocysts carryover from the original inoculum.
<ol>
	<li>Prepare Cryptosporidium oocysts for infecting cells (Figure 1A).
	<ol>
		<li>Keep oocysts on ice throughout all manipulations until they are added to the organoids.</li>
		<li>Calculate the number of oocysts needed for a full six-well plate of organoids (usually about 5 x 105&#8211;2.5 x 105 for the plate). Count the numbers of oocysts in a hemocytometer to verify the quantity and transfer to a centrifuge tube. 
		NOTE: To aid in visualization, oocysts may be mixed 1:1 with an oocyst-specific fluorescent antibody (see the Table of Materials) before being loaded onto the hemocytometer. The fluorophore-labeled oocysts can then be easily visualized and enumerated using a fluorescence microscope. We suggest injecting about 100&#8211;1,000 oocysts/organoid. In general, 1,000&#8211;2,000 organoids can be grown in a six-well plate.</li>
		<li>Bring the volume of the oocysts suspension up to 900 &#181;L with PBS. Add 100 &#181;L of sodium hypochlorite (e.g., Clorox) bleach (at 4 &#176;C). Incubate for 10 min on ice.</li>
		<li>Centrifuge for 3 min in a microcentrifuge at 8,000 x g at 4 &#176;C. Orient the tubes in the centrifuge with the cap opening facing inward. The pellet can be hard to see so knowing where the parasites have pelleted in the tube is essential.</li>
		<li>Remove the supernatant with a pipette being careful to avoid the pellet. Add 1 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) and vortex to mix.</li>
		<li>Centrifuge for 3 min in a microcentrifuge at 8,000 x g at 4 &#176;C.</li>
		<li>Repeat washes with DMEM two more times.</li>
		<li>Prepare expansion medium (OME) or differentiation organoid medium (OMD) to which taurocholate has been added to a final concentration of 0.5% (w/v) (See Table of Materials). Taurocholate should always be prepared and added fresh. 
		NOTE: We have successfully used 0.5% taurocholate in our infection assays where the inoculum is intact oocysts, and saw improved rates of infection without deleterious effects on the host cells. However, taurocholate may have unanticipated effects on cells, and lower concentrations have been used successfully in infection assays16.</li>
		<li>Resuspend oocysts in 100 &#956;L of organoid culture medium supplemented with 0.5% (w/v) sodium taurocholate. Count oocysts again as described in step 1.1.2.</li>
		<li>Add Fast Green dye to the suspension in order to visualize injection.</li>
		<li>Fill micro-loader tips (see the Table of Materials) with the oocyst suspension and use it to fill pulled capillaries. 
		CAUTION: The whole procedure should be done in a tissue culture hood with level-2 safety protocols. Use of masks is recommended as Cryptosporidium oocysts can also be infectious when airborne.</li>
	</ol>
	</li>
</ol>
2. In Vitro Purification of Sporozoites from C. parvum Oocysts
<ol>
	<li>Purify sporozoites from C. parvum oocysts after bleaching and washing out the bleach as described above.
	<ol>
		<li>Transfer the oocysts to a 15 mL tube. Resuspend oocysts in room temperature excystation medium (0.75% w/v sodium taurocholate in DMEM) to obtain 1 x 107 oocysts/mL. The addition of taurocholate improves the excystation rate of the oocysts, improving sporozoite yield.</li>
		<li>Incubate oocyst suspension at 37 &#176;C for 1&#8211;1.5 h.</li>
		<li>Check the sample microscopically for extent of excystation; 60&#8211;80% excystation is reasonable for good recovery of sporozoites. If the level of excystation is low, incubate longer (another 30 min to 1 h).</li>
		<li>Determine the percent excystation relative to the number of starting oocysts. Excystation is calculated as: 
		% excystation = [1 &#8211; (number of intact oocysts/number of oocysts at start)] x 100</li>
		<li>Wash cells to remove excystation reagents by adding 14 mL of PBS or medium, mixing, and recovering cells (intact oocysts, oocyst shells, and sporozoites) by centrifugation at 3,400 x g for 20 min to recover sporozoites. Aspirate carefully to avoid losing cells.</li>
		<li>Resuspend the sporozoite pellet in 1&#8211;2 mL of DMEM to obtain 3 x 107 oocysts/mL (based on the number of starting oocysts).</li>
		<li>To remove remaining oocysts and shells, filter the suspension through a 3 &#181;m filter (Figure 1B). Use a 47 mm filter holder apparatus fitted with polycarbonate filter (3 &#181;m pore size) attached to a 10 mL syringe barrel. Place the filter holder apparatus on top of a 15 mL tube. Place the assembly in an ice bucket or in cold room.</li>
		<li>Add 7.5 mL of the sporozoite suspension to the filter assembly and allow to filter through by gravity. Wash through with another 7.5 mL of DMEM. 
		NOTE: To ensure success in sporozoite isolation fresh oocysts and good excystation are critical. If there are too many unexcysted oocysts, the suspension will not flow through by gravity. Applying pressure on the syringe can force unexcysted oocysts through. Microinjection of sporozoites is more challenging than that of oocysts because sporozoites may clump and block the capillary. To avoid this, we recommend making a wider capillary tip when injecting organoids with sporozoites. To achieve sufficient levels of infection, 2&#8211;4 times the number of sporozoites need to be injected into each organoid as compared to organoids infected with oocysts.</li>
		<li>Centrifuge the filtered sporozoite suspension at 3,400 x g using a swinging bucket rotor for 20 min to pellet sporozoites.</li>
		<li>Resuspend in 50&#8211;100 &#956;L of OME or OMD organoid culture medium (see the Table of Materials) supplemented with 0.05% (w/v) Fast Green dye and L-glutathione, betaine, L-cysteine, linoleic acid and taurine-containing reducing buffer5 (see the Table of Materials). 
		NOTE: Incubating oocysts for too long may result in the lysis of sporozoites and poor recovery and therefore should be avoided.</li>
	</ol>
	</li>
</ol>
3. In Vitro Culture of Human Intestinal and Lung Organoids for Microinjection
<ol>
	<li>Culture intestinal organoids under expansion and differentiation media conditions. 
	NOTE: The details of intestinal and lung organoid propagation have been previously described in other articles8,13 (see Table of Materials for media recipes). Here, we briefly describe the organoid culture method with specific reference to optimization for Cryptosporidium injection and growth. We have found that for imaging of parasites in organoids, organoids grown in expansion medium are preferable to those in grown differentiation media as there is less debris accumulation than that seen in organoids grown in differentiation medium. However, if the goal is to isolate oocysts, organoids grown in differentiation media produce far higher numbers of oocysts.
	<ol>
		<li>Maintain organoids in 3D cultures in extracellular matrix (see the Table of Materials) at 37 &#176;C. Add OME (expansion media) on top and refresh every day. 
		NOTE: For lung organoids, we do not have separate expansion and differentiation media.</li>
		<li>To split and plate organoids for microinjection, remove media from the 6-well plate containing human organoids and add F12+++ (See the Table of Materials) to the well and break up the matrix by pipetting with a 1 mL pipette tip several times. Collect cells into a 15 mL tube (2 mL of F12+++ per tube is enough for further procedures).</li>
		<li>Add 10&#8211;12 mL of F12+++ into another 15 mL tube and place a fire-polished glass pipet into the medium, pipette up and down 3 times to break up the human intestinal and lung organoids. 
		NOTE: Use a long glass pipet (20&#8211;30 cm) and fire-polish it briefly. Do not make the opening (1 mm diameter) very small because organoids can be damaged. Make the tip of the pipet smooth by briefly fire-polishing it. Break organoids into smaller pieces of ~50 &#181;m. Lung organoids have a thicker outer membrane and therefore require stronger shearing with the glass pipette as compared to intestinal organoids. Moreover, they have a slower growth rate than intestinal organoids (up to 14 days between each passaging).</li>
		<li>Add F12+++ up to 5&#8211;7 mL and centrifuge at 350 x g for 5 min. 
		NOTE: The centrifugation speed in this step is higher than normal in order to make a good cell pellet that is well separated from the extracellular matrix (see the Table of Materials). We have observed that compared to mouse small intestine organoids, human small intestine organoids are harder to disrupt.</li>
		<li>Remove as much medium as possible without disturbing the cells, then resuspend the pellet with matrix maintained at 4 &#176;C; 200&#8211;300 &#181;L of matrix per well of a six-well plate is required. Organoids should be split one in three to maintain a fairly high cell density.</li>
		<li>Plate the organoids in matrix droplets of about 5&#8211;10 &#181;L each in the well of a six-well plate. Incubate for 20&#8211;30 min at 37 &#176;C and then add expansion medium (OME) on top.</li>
		<li>Change the medium every 2&#8211;3 days. 
		NOTE: In about 5&#8211;7 days, organoids growing in EM reach a size of 100&#8211;200 &#956;m and are ready for injection.</li>
		<li>To differentiate the organoids, after 5&#8211;6 days in EM, change the media to differentiation media (DM) conditions and keep for 5&#8211;6 additional days before injecting the parasites. 
		NOTE: For expansion of organoids, it is recommended to plate the organoids densely. For microinjection, use of a six-well plate is recommended with organoids plated at a lesser density. For example, plate organoids from three wells of a six-well plate into a full six-well plate for microinjections. Matrix should be maintained at -20 &#176;C for long term storage and thawed at 4 &#176;C or on ice before use. Expansion of lung organoids is done is a similar manner but using lung organoid specific media components (Table 1)8 .</li>
	</ol>
	</li>
</ol>
4. Microinjection of Oocysts/Sporozoites into the Organoid Lumen
<ol>
	<li>Microinject parasites into the apical side of the 3D organoid (Figure 2).
	<ol>
		<li>Prepare glass capillaries of 1 mm diameter using a micropipette puller. 
		NOTE: Settings used on the micropipette puller (See Table of Materials) are: Heat = 663, Pull = 100, Velocity = 200, Time = 40 ms. Settings will need to be adjusted according to the user instructions for a particular machine.</li>
		<li>Cut the tip of the capillary with forceps. The size/diameter of the capillary end measures about 9&#8211;12 &#956;m; this enables easy flow of oocysts (4&#8211;5 &#956;m size).</li>
		<li>Fill capillaries with oocyst or sporozoite suspension using micro-loader tips.</li>
		<li>Load the oocyst-filled capillary onto a microinjector.</li>
		<li>Microinject 100&#8211;200 nL suspension into each organoid under an inverted microscope at 5x magnification, keeping the pressure constant. After microinjection, refresh media with OME or OMD every day and maintain the plate at 37 &#176;C. 
		NOTE: We do not use a micromanipulator for microinjection. Use of the same capillary is recommended for the entire experiment to ensure equal injection in every sample.</li>
	</ol>
	</li>
</ol>
5. Immunofluorescence Staining of Organoids
<ol>
	<li>Collect organoids (1&#8211;2 x 24 wells) with a P1000 pipette into a 15 mL tube containing cold F12+++.</li>
	<li>Pellet organoids at 300 x g for 2 min, remove the supernatant without disrupting the pellet, and gently pipet the pellet loose into the remaining volume.</li>
	<li>Add 5 mL of 2% paraformaldehyde in PBS. To prevent the organoids from sticking to the wall do not invert the tube. Allow the organoids to settle to the bottom of the tube and fix at 4 &#176;C overnight or 1 h at room temperature.</li>
	<li>Remove the fixative and add 10 mL of permeabilization buffer (0.2% Triton in PBS).</li>
	<li>Rotate the tube at room temperature for 20 min (this ensures that all the organoids remain in suspension).</li>
	<li>Pellet the organoids at 300 x g for 2 min, and then discard the supernatant.</li>
	<li>Resuspend the organoids gently in 500 &#181;L of blocking solution (See the Table of Materials) and transfer to a 2 mL microcentrifuge tube.</li>
	<li>Incubate for 20 min at room temperature on a shaker. Allow organoids to settle to the bottom of the tube by gravity. Replace the blocking solution with primary antibody solution (See Table of Materials) and incubate for 1&#8211;2 h at room temperature or overnight at 4 &#176;C.</li>
	<li>Wash 3x with PBS containing 0.1% Tween. Let the organoids settle every time and remove the supernatant.</li>
	<li>Add secondary antibody solution (See the Table of Materials) and incubate for 2 h at room temperature.</li>
	<li>Wash 3x with PBS containing 0.1% Tween. Leave 50 &#956;L of PBS after the third wash.</li>
	<li>Mount on the slide by pipetting the organoids suspended in 50 &#956;L of PBS on the slide. Remove excess PBS, add a drop of mounting agent (See the Table of Materials) and add the coverslip on top. Seal the sides with nail polish and let it dry.</li>
</ol>
6. Isolation of Oocysts from Organoids
<ol>
	<li>Isolate newly formed oocysts from the organoid lumen. 
	NOTE: Oocysts are isolated from the organoids by immunomagnetic separation using an oocyst isolation kit (see the Table of Materials) with the modifications described below. The isolated oocysts can then be analyzed by immunofluorescence and electron microscopy.
	<ol>
		<li>Start with differentiated organoids that have been maintained in OMD for 5&#8211;7 days and that are uninfected, infected for 1 day and infected for 5 days. Use the first two as negative controls. 
		NOTE: We have found that differentiated organoids produce more oocysts than lung or expanding intestinal organoids10.</li>
		<li>Collect organoids into 15 mL centrifuge tubes. Centrifuge the organoids for 20 min at 3,000 x g and 10 &#176;C. 
		NOTE: This high speed is needed to make sure no oocysts are lost out of any organoids that may be broken.</li>
		<li>Remove the organoid media and replace it with 5 mL of water.</li>
		<li>Disrupt the organoids by repeated vigorous pipetting with a fire-polished glass Pasteur pipette.</li>
		<li>If clumps are visible, transfer the organoid suspension to a glass dounce homogenizer, and homogenize until organoids are well disrupted. The dounce homogenizer will not affect the oocysts.</li>
		<li>Once there are no visible clumps, add 5 mL of buffer A from the oocyst isolation kit. Mix and then add 120 &#181;L of the magnetic beads coated with anti-oocyst IgM.</li>
		<li>Incubate the cell suspension and magnetic beads for 2 h at room temperature with continuous mixing on a rocker platform.</li>
		<li>At the end of the incubation, place the tubes containing cells and beads on a magnetic separation rack designed for 15 mL tubes.</li>
		<li>Rotate the tubes in magnetic separation rack manually for 3 min. The beads will adhere to the side of the tube next to the magnet.</li>
		<li>Carefully, with a 10 mL pipette, remove the supernatant from the beads. Resuspend the beads in 450 &#181;L of Buffer B and transfer to a 1.5 mL microcentrifuge tube. 
		NOTE: Keep the supernatant until the isolation of the oocysts is confirmed.</li>
		<li>To collect any remaining beads and oocysts, wash the 15 mL tube with 450 &#181;L of Buffer B and add this wash to the magnetic beads in the microcentrifuge tube.</li>
		<li>Repeat step 6.1.11 one more time. All beads and captured oocysts should now be transferred to the microcentrifuge tube.</li>
		<li>Place the microcentrifuge tube on a magnetic separation rack designed for holding microcentrifuge tubes.</li>
		<li>Rotate the tube in the magnetic separation rack by hand for 3 min.</li>
		<li>Carefully remove the supernatant with a pipette into a new tube. 
		NOTE: Keep the supernatant until the isolation of the oocysts is confirmed.</li>
		<li>Remove the microcentrifuge tube containing magnetic beads and oocysts from the magnetic separation rack.</li>
		<li>Add 100 &#181;L of 0.1 N HCl to magnetic beads to elute the oocysts off the beads. Vortex for 30 s. 
		NOTE: Vortexer should be set to slightly less than maximum speed.</li>
		<li>Incubate the beads in 0.1 N HCl for 10 min at room temperature.</li>
		<li>Vortex again. Then place the tube back on magnetic separation rack. Wait for beads to adhere to the side of the tube and then transfer the supernatant to a new microcentrifuge tube.</li>
		<li>Repeat steps 6.1.17 through 6.1.19 and combine the second eluate with the first elution.</li>
		<li>Neutralize the eluate with 20 &#181;L of 1 N NaOH, or another neutralizing buffer such as 1 M Tris, pH 8.</li>
		<li>To count oocysts, take 10 &#181;L of the eluate, combine it with 10 &#181;L of oocyst-specific antibody (See Table of Materials) and count fluorescent oocysts on a hemocytometer. 
		NOTE: Isolated oocysts can be stored at 4 &#176;C or used immediately for immunofluorescence or electron microscopy imaging.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 an alternative to this procedure. However, we have noted that electroporated sporozoites clump together and block the micropipette. For the purpose of selecting genetically modified parasites, organoids can be grown on collagen coated transwells in a two-dimensional format under differentiation conditions to allow infection with transfected sporozoites and consequently the selection of the genetically modified oocysts. The transwells allow access to both the apical and basolateral surfaces and are stable for extended periods of time.
Currently, we culture organoids in a two-dimensional format for high throughput screening of drugs for cancer tissue-derived organoids (unpublished data). This method of organoid culture can also be adapted for testing of anti-Cryptosporidium drugs using the genetically-modified luciferase tagged Cryptosporidium strains19. Moreover, even though the infection is not tightly synchronized, infection of organoids with sporozoites provides sufficient synchronization of the life cycle that drugs can be tested for their efficacy against specific life cycle stages.
Organoid co-culture systems are now being developed taking into account some other aspects of the host system such as microbiota and immune cells20. Thus, the ability to dissect interactions between the parasite and host cells, immune cells and microbiota will soon be possible in vitro. Genetic manipulation of Cryptosporidium is also now possible19, and the combination of fluorescent reporter strains of Cryptosporidium and organoid culture will provide the tools for single cell sequencing of infected cells, and even more specifically single cell sequencing of cells infected with specific stages of the parasite.
The success of the experiments described here is highly dependent on the viability and infectivity of the oocysts. Different batches of Cryptosporidium oocysts can vary widely in excystation rates and ability to infect host cells. Sufficient yields of sporozoites is dependent on good excystation rates and the excystation rate is not always correlated to infectivity. If low levels of infection or poor excystation are observed with a particular batch of oocysts, time and effort may be saved by obtaining a new lot of oocysts rather than attempting to increase oocyst numbers, or lengthening incubation times.
Organoid culture media should be refreshed every alternate day. Use of earlier passages of organoid cultures is advisable. It is important to thaw a new vial of organoids if organoids start to differentiate in later passages as health of organoid cultures vastly determine viability of the parasite. After infection, organoid media should be refreshed every day to avoid accumulation of toxic substances in the media.
Organoid culture of Cryptosporidium is limited in that the parasite cannot be propagated indefinitely, and the infection peters out after three passages over 28 days10. Microinjection of sufficient organoids for mouse experiments such as we have described can be time-consuming and physically taxing. Nevertheless, to date, no other method enables the complete life cycle in an in vitro system completely representative of human infection, nor has any culture system been described that allows exploration of the host-pathogen interactions important for respiratory infection. Organoid culture of Cryptosporidium provides a powerful new tool that opens up avenues of exploration into host-parasite interactions not previously possible for Cryptosporidium.

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>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</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>&amp;nbsp;</td></tr><tr><td>Dynamag 15 rack</td><td>Thermofisher Scientific</td><td>12301D</td><td>&amp;nbsp;</td></tr><tr><td>Dynamag 2 rack</td><td>Thermofisher Scientific</td><td>12321D</td><td>&amp;nbsp;</td></tr><tr><td>EMD Millipore&amp;nbsp;Isopore Polycarbonate Membrane Filters- 3&amp;micro;m</td><td>EMD-Millipore</td><td>TSTP02500</td><td>&amp;nbsp;</td></tr><tr><td>Fast green dye</td><td>SIGMA</td><td>F7252-5G&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>Femtojet 4i Microinjector</td><td>Eppendorf</td><td>5252000013</td><td>&amp;nbsp;</td></tr><tr><td>Glass capillaries of 1 mm diameter</td><td>WPI</td><td>TW100F-4</td><td>&amp;nbsp;</td></tr><tr><td>Matrigel (extracellular matrix)</td><td>Corning</td><td>356237</td><td>&amp;nbsp;</td></tr><tr><td>Microfuge tube 1.5 mL</td><td>Eppendorf</td><td>T9661-1000EA</td><td>&amp;nbsp;</td></tr><tr><td>Micro-loader tips</td><td>Eppendorf</td><td>612-7933</td><td>&amp;nbsp;</td></tr><tr><td>Micropipette puller P-97</td><td>Shutter instrument</td><td>P-97</td><td>&amp;nbsp;</td></tr><tr><td>Normal donkey Serum</td><td>Bio-Rad</td><td>C06SB</td><td>&amp;nbsp;</td></tr><tr><td>Penstrep</td><td>Gibco</td><td>15140-122</td><td>&amp;nbsp;</td></tr><tr><td>Sodium hypoclorite (use 5%)</td><td>Clorox</td><td>50371478</td><td>&amp;nbsp;</td></tr><tr><td>Super stick slides</td><td>Waterborne, Inc</td><td>S100-3</td><td>&amp;nbsp;</td></tr><tr><td>Swinnex-25 47 mm Polycarbonate filter holder</td><td>EMD-Millipore</td><td>SX0002500</td><td>&amp;nbsp;</td></tr><tr><td>Taurocholic acid sodium salt hydrate</td><td>SIGMA</td><td>T4009-5G</td><td>&amp;nbsp;</td></tr><tr><td>Tween-20</td><td>Merck</td><td>8221840500</td><td>&amp;nbsp;</td></tr><tr><td>Vectashield mounting agent</td><td>Vector Labs</td><td>H-1000</td><td>&amp;nbsp;</td></tr><tr><td>Vortex Genie 2</td><td>Scientific industries, Inc</td><td>SI0236</td><td>&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Adv+++ (DMEM+Penstrep+Glutamax+Hepes)&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Final amount&lt;/strong&gt;</td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>12634-010</td><td>500 mL</td></tr><tr><td>Penstrep</td><td>Gibco</td><td>15140-122</td><td>5 mL of stock in 500 mL DMEM</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050038</td><td>5 mL of stock in 500 mL DMEM</td></tr><tr><td>Hepes</td><td>Gibco</td><td>15630056</td><td>5 mL of stock in 500 mL DMEM</td></tr><tr><td>&lt;strong&gt; INTESTINAL ORGANOID MEDIA-OME (Expansion media)&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Final concentration&lt;/strong&gt;</td></tr><tr><td>A83-01</td><td>Tocris</td><td>2939-50mg</td><td>0.5 &amp;micro;M</td></tr><tr><td>Adv+++</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>make up to 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>50 ng/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>1.25 mM</td></tr><tr><td>NIC</td><td>Sigma</td><td>N0636-100G</td><td>10 mM</td></tr><tr><td>Noggin CM</td><td>In house*</td><td>&amp;nbsp;</td><td>10%</td></tr><tr><td>P38 inhibitor (SB202190)</td><td>Sigma</td><td>S7076-25 mg</td><td>10 &amp;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>1 mL/500 mL media</td></tr><tr><td>RSpoI CM</td><td>In house*</td><td>&amp;nbsp;</td><td>20%</td></tr><tr><td>Wnt3a CM</td><td>In house*</td><td>&amp;nbsp;</td><td>50%</td></tr><tr><td>In house* - cell lines will be provided upon request</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;INTESTINAL ORGANOID MEDIA-OMD (Differentiation media)&lt;/strong&gt;</td><td>&amp;nbsp;</td><td>&amp;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>&lt;strong&gt;LUNG ORGANOID MEDIA- LOM (Differentiation media)&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Final concentration&lt;/strong&gt;</td></tr><tr><td>Adv+++</td><td>&amp;nbsp;</td><td>&amp;nbsp;</td><td>make up to 100 mL</td></tr><tr><td>ALK-I A83-01</td><td>Tocris</td><td>2939-50mg</td><td>500 nM</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>100 ng/mL</td></tr><tr><td>FGF-7</td><td>Peprotech</td><td>100-19</td><td>25 ng/mL</td></tr><tr><td>N-Acetylcysteine</td><td>Sigma</td><td>A9125-25G</td><td>1.25 mM</td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636-100G</td><td>5 mM</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 &amp;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 &amp;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>&lt;strong&gt;Reducing buffer (for resuspension of oocysts and sporozoites for injection)&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Final concentration&lt;/strong&gt;</td></tr><tr><td>L-Glutathione reduced</td><td>Sigma</td><td>G4251-10MG</td><td>0.5 &amp;mu;g/&amp;mu;L of OME/OMD/LOM</td></tr><tr><td>Betaine</td><td>Sigma</td><td>61962</td><td>0.5 &amp;mu;g/&amp;mu;L of OME/OMD/LOM</td></tr><tr><td>L-Cysteine</td><td>Sigma</td><td>168149-2.5G</td><td>0.5 &amp;mu;g/&amp;mu;L of OME/OMD/LOM</td></tr><tr><td>Linoleic acid</td><td>Sigma</td><td>L1376-10MG</td><td>6.8 &amp;mu;g/mL of OME/OMD /LOM</td></tr><tr><td>Taurine</td><td>Sigma</td><td>T0625-10MG</td><td>0.5 &amp;mu;g/&amp;mu;L of OME/OMD/LOM</td></tr><tr><td>&lt;strong&gt;Blocking buffer (for immunoflourescence staining)&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Final concentration&lt;/strong&gt;</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 up to 100 mL</td></tr><tr><td>Tween 20</td><td>Merck</td><td>P1379</td><td>0.1%</td></tr><tr><td>&lt;strong&gt;List of Antibodies used &lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</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-1:1,000; RRID : AB_2629482</td></tr><tr><td>Phalloidin-Alexa 674</td><td>Invitrogen</td><td>A22287</td><td>Dilution-1:1,000; RRID: AB_2620155</td></tr><tr><td>Rabbit anti-gp15 antibody generated by R. M. O&amp;rsquo;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 ensure injection of all organoids and allows visualization of injected organoids for at least for 24 h after injection (Figure 2B).
These protocols for preparation of oocysts and sporozoites are straightforward and have been used for many years, so it is expected that the treated oocysts and purified sporozoites will be viable and infectious. However, in our studies, we used scanning electron microscopy to ensure that the excystation process did not damage the sporozoites or oocysts (Figure 2A)10. Injection of equal amounts of oocysts into the organoid lumen can be visually confirmed by simple microscopic imaging (Figure 2C). A portion of infected organoids should be set up to verify parasite propagation by quantitative PCR as we have described10.
Progress through the parasite life cycle can be visualized by collection of infected organoids at different time points post infection and analysis by transmission electron microscopy or by immunofluorescence combined with 4&#8242;,6-diamidino-2-phenylindole (DAPI) staining of parasite nuclei10. For example, antibodies to merozoite surface antigens, such as gp40 and gp1517 can be used to identify meront stages; type I meronts will have 8 nuclei and type II meronts, 4 nuclei10. Recently, a panel of monoclonal antibodies specific to trophozoites, merozoites, type I versus II meronts, and macrogamonts has become available18. These antibodies would also be very effective in marking progress of the parasite through its various life cycle stages in the organoids.
Immunofluorescence assays can also be used to explore which cell types are infected by Cryptosporidium. This was especially important to look at in the airway organoids as very little is known about respiratory cryptosporidiosis, and the exact host cell for the parasite was not known. We conducted immunofluorescence assays on Cryptosporidium-infected organoids, co-localizing CC10, a marker for club cells and found that Cryptosporidium infected both CC10- negative and positive cells (Figure 3). These results were corroborated by TEMs in which we observed Cryptosporidium infecting secretory and non-secretory cells in the airway organoids10.
After differentiated organoids have been infected for five days, there should be significant numbers of oocysts being produced. In our hands, infection of organoids from one six-well plate yielded about 4000 oocysts, which could be easily identified and counted on a hemocytometer by labeling with an oocyst-specific antibody. The presence of four sporozoites in the oocysts could be confirmed by drying down a portion of the oocysts onto an adhesive slide, fixing with methanol and combining DAPI staining with oocyst specific antibody (Figure 4). Verification of production of thick walled oocysts could be done by TEM analysis10.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59610/59610fig01.jpg" /> 
Figure 1: Preparation and purification of Cryptosporidium oocysts and sporozoites.&#160;(A) Schematic representation of the method used for oocyst and sporozoite preparation for infection. (B) Image showing in vitro excystation of oocysts. Filtration of unexcysted oocyts and shells gives a purified solution of sporozoites. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59610/59610fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59610/59610fig02.jpg" /> 
Figure 2: Microinjection of oocysts into the organoid lumen.&#160;This figure has been modified from Heo et al.10. (A) scanning electron microscopy (SEM) images of oocysts and sporozoites. (B) Image showing oocyst-injected organoids. The green dye helps visualize the injection of each organoid and persists over at least 24 h. (C) Image of an organoid injected with oocysts. <a href="https://www.jove.com/files/ftp_upload/59610/59610fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59610/59610fig03.jpg" /> 
Figure 3: Immunofluorescence image of Cryptosporidium-infected airway organoid.&#160;Mucin is labeled with anti-mucin 5 antibody (red) in the lumen of the organoid, club cells are labeled with anti-CC10 (yellow), Cryptosporidium is detected with oocyst-specific antibody (green), and cell nuclei are stained with DAPI (blue). Panel B is an enlargement of the area indicated in the square in panel A. <a href="https://www.jove.com/files/ftp_upload/59610/59610fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59610/59610fig04.jpg" /> 
Figure 4: Immunofluorescence image of oocyst isolated from differentiated intestinal organoids.&#160;Oocyst wall is labeled with oocyst-specific antibody in green and the four sporozoite nuclei are visualized with DAPI (blue) <a href="https://www.jove.com/files/ftp_upload/59610/59610fig04.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.5K Views.  UMC Utrecht. 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.

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

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Developmental Biology

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

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

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

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

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

Cancer Research

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. ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived from all sections of the GI tract (gastric, jejunum, duodenum, ileum, colon, rectum) and, upon differentiation, contain most of the cell types that are naturally found in each individual section. For example, intestinal organoids contain nutrient-absorbing enterocytes, secretory cells (Goblet, Paneth, and enteroendocrine), stem cells, as well as all lineage-specific differentiation intermediates (e.g.,&#160;early or immature cell types). The greatest advantage in using gastrointestinal tract-derived organoids to study infectious diseases is the possibility of precisely identifying which cell type is targeted by the enteric pathogen and to address whether the different sections of the gastrointestinal tract and their specific cell types similarly respond to pathogen challenges. Over the past years, gastrointestinal models, as well as organoids from other tissues, have been employed to study viral tropism and the mechanisms of pathogenesis. However, utilizing all the advantages of using organoids when employing highly pathogenic viruses represents a technical challenge and requires strict biosafety considerations. Additionally, as organoids are often grown in three dimensions, the basolateral side of the cells is facing the outside of the organoid while their apical side is facing the inside (lumen) of the organoids. This organization poses a challenge for enteric pathogens as many enteric infections initiate from the apical/luminal side of the cells following ingestion. The following manuscript will provide a comprehensive protocol to prepare human intestinal organoids for infection with enteric pathogens by considering the infection side (apical vs. basolateral) to perform single-cell RNA sequencing to characterize cell-type-specific host/pathogen interactions. This method details the preparation of the organoids as well as the considerations needed to perform this work under biosafety level 3 (BSL-3) containment conditions.

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 BSL-3 Conditions

This protocol describes how to infect human intestinal organoids from either their apical or basolateral side to characterize host/pathogen interactions at the single-cell level using single-cell RNA sequencing (scRNAseq) technology.

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived ...

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

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

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

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

Biology

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

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

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

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

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

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

Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria

Source: Bartfeld, S. et al. <a href="https://app.jove.com/v/53359">Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori.</a> J. Vis. Exp.(2015).
This video demonstrates a method to establish a gastric organoid infection model by microinjecting gastric pathogen Helicobacter pylori into the lumen of the organoids. This model helps in studying infection-induced changes in gastric cells.

1. Passage of Organoid Cultures

	Add 1 ml of cold basal medium to the basement matrix in the well. Using a 1 ml micropipette, vigorously pipette up and ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

CRC Organoid Culture: A Method to Obtain 3D Organoids from Colorectal Cancer Cells

Source: Okazawa, Y. et al. <a href="https://www.jove.com/v/57374"> High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor.</a> J. Vis. Exp., (2018).
This video describes the detailed protocol for generating 3D organoids from murine-model derived colorectal cancer cells by propagating them on artificial extracellular matrix coated plates. These CRC organoids can be used as a model to study the primary growth of human tumor cells and metastasis.

Source: Okazawa, Y. et al.  High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived ...

Colorectal Cancer

Assays & techniques

In Vitro Sporozoite Isolation from Cryptosporidium parvum Oocysts

Source: Dutta, D. et al., <a href="https://app.jove.com/v/59610">Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection.</a> J. Vis. Exp. (2019)
In this video, we demonstrate an in vitro method to purify sporozoites from thick-walled oocysts of the pathogenic parasite Cryptosporidium parvum. The isolated sporozoites can be used for infection studies.

1. In Vitro Purification of Sporozoites from C. parvum Oocysts

	Prepare Cryptosporidium oocysts for infecting cells (Figure 1A).
	NOTE: Cryptosporidium ...

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

Summary

Explore More Videos

Chapters in this video

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

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

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

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions

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

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

Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria

CRC Organoid Culture: A Method to Obtain 3D Organoids from Colorectal Cancer Cells

In Vitro Sporozoite Isolation from Cryptosporidium parvum Oocysts