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.
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—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.
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 (<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 ‘mini-organs’ 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—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.
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 °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.
2. In Vitro Purification of Sporozoites from C. parvum Oocysts
3. In Vitro Culture of Human Intestinal and Lung Organoids for Microinjection
4. Microinjection of Oocysts/Sporozoites into the Organoid Lumen
5. Immunofluorescence Staining of Organoids
6. Isolation of Oocysts from Organoids
The protocols presented here result in the efficient purification of oocysts and sporozoites (Figure 1A) ready for microinjection. The excystation protocol results in the release of sporozoites from approximately 70–80% of the oocysts, therefore it is essential to filter out the remaining oocysts and shells through a 3 µm 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′,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.
Figure 1: Preparation and purification of Cryptosporidium oocysts and sporozoites. (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 µm. Please click here to view a larger version of this figure.
Figure 2: Microinjection of oocysts into the organoid lumen. 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. Please click here to view a larger version of this figure.
Figure 3: Immunofluorescence image of Cryptosporidium-infected airway organoid. 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. Please click here to view a larger version of this figure.
Figure 4: Immunofluorescence image of oocyst isolated from differentiated intestinal organoids. Oocyst wall is labeled with oocyst-specific antibody in green and the four sporozoite nuclei are visualized with DAPI (blue) Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
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.
Basement membrane extract (extracellular matrix) | amsbio | 3533-010-02 | |
Crypt-a-Glo antibody (Oocyst specific antibody) | Waterborne, Inc | A400FLR-1X | Final Concentration = Use 2-3 drops/slide |
Crypto-Grab IgM coated Magnetic beads | Waterborne, Inc | IMS400-20 | |
Dynamag 15 rack | Thermofisher Scientific | 12301D | |
Dynamag 2 rack | Thermofisher Scientific | 12321D | |
EMD Millipore Isopore Polycarbonate Membrane Filters- 3µm | EMD-Millipore | TSTP02500 | |
Fast green dye | SIGMA | F7252-5G | |
Femtojet 4i Microinjector | Eppendorf | 5252000013 | |
Glass capillaries of 1 mm diameter | WPI | TW100F-4 | |
Matrigel (extracellular matrix) | Corning | 356237 | |
Microfuge tube 1.5ml | Eppendorf | T9661-1000EA | |
Micro-loader tips | Eppendorf | 612-7933 | |
Micropipette puller P-97 | Shutter instrument | P-97 | |
Normal donkey Serum | Bio-Rad | C06SB | |
Penstrep | Gibco | 15140-122 | |
Sodium hypoclorite (use 5%) | Clorox | 50371478 | |
Super stick slides | Waterborne, Inc | S100-3 | |
Swinnex-25 47mm Polycarbonate filter holder | EMD-Millipore | SX0002500 | |
Taurocholic acid sodium salt hydrate | SIGMA | T4009-5G | |
Tween-20 | Merck | 8221840500 | |
Vectashield mounting agent | Vector Labs | H-1000 | |
Vortex Genie 2 | Scientific industries, Inc | SI0236 | |
Adv+++ (DMEM+Penstrep+Glutamax+Hepes) | Final amount | ||
DMEM | Invitrogen | 12634-010 | 500ml |
Penstrep | Gibco | 15140-122 | 5ml of stock in 500ml DMEM |
Glutamax | Gibco | 35050038 | 5ml of stock in 500ml DMEM |
Hepes | Gibco | 15630056 | 5ml of stock in 500ml DMEM |
INTESTINAL ORGANOID MEDIA-OME (Expansion media) | Final concentration | ||
A83-01 | Tocris | 2939-50mg | 0.5µM |
Adv+++ | make upto 100 ml | ||
B27 | Invitrogen | 17504044 | 1X |
EGF | Peprotech | AF-100-15 | 50ng/mL |
Gastrin | Tocris | 3006-1mg | 10 nM |
NAC | Sigma | A9125-25G | 1.25mM |
NIC | Sigma | N0636-100G | 10mM |
Noggin CM | In house* | 10% | |
P38 inhibitor (SB202190) | Sigma | S7076-25 mg | 10µM |
PGE2 | Tocris | 2296/10 | 10 nM |
Primocin | InvivoGen | ant-pm-1 | 1ml/500ml media |
RSpoI CM | In house* | 20% | |
Wnt3a CM | In house* | 50% | |
In house* – cell lines will be provided upon request | |||
INTESTINAL ORGANOID MEDIA-OMD (Differentiation media) | 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) | ||
LUNG ORGANOID MEDIA- LOM (Differentiation media) | Final concentration | ||
Adv+++ | make upto 100 ml | ||
ALK-I A83-01 | Tocris | 2939-50mg | 500nM |
B27 | Invitrogen | 17504044 | 0.0763888889 |
FGF-10 | Peprotech | 100-26 | 100ng/ml |
FGF-7 | Peprotech | 100-19 | 25ng/ml |
N-Acetylcysteine | Sigma | A9125-25G | 1.25mM |
Nicotinamide | Sigma | N0636-100G | 5mM |
Noggin UPE | U-Protein Express | Contact company directly | 10% |
p38 MAPK-I | Sigma | S7076-25 mg | 1µM |
Primocin | InvivoGen | ant-pm-1 | 1:500 |
RhoKI Y-27632 | Abmole Bioscience | M1817_100 mg | 2.5µm |
Rspo UPE | U-Protein Express | Contact company directly | 10% |
Reducing buffer (for resuspension of oocysts and sporozoites for injection) | Final concentration | ||
L-Glutathione reduced | Sigma | G4251-10MG | 0.5 μg/μl of OME/OMD/LOM |
Betaine | Sigma | 61962 | 0.5 μg/μl of OME/OMD/LOM |
L-Cysteine | Sigma | 168149-2.5G | 0.5 μg/μl of OME/OMD/LOM |
Linoleic acid | Sigma | L1376-10MG | 6.8 μg/ml of OME/OMD /LOM |
Taurine | Sigma | T0625-10MG | 0.5 μg/μl of OME/OMD/LOM |
Blocking buffer (for immunoflourescence staining) | Final concentration | ||
Donkey/Goat serum | Bio-Rad | C06SB | 2% |
PBS | Thermo-Fisher | 70011044 | Make upto 100ml |
Tween 20 | Merck | P1379 | 0.1% |
List of Antibodies used | |||
Alexa 568 goat anti-rabbit | Invitrogen | A-11011 | Dilution-1:500; RRID: AB_143157 |
Crypt-a-Glo Comprehensive Kit- Fluorescein-labeled antibody Crypto-Glo | Waterborne, Inc | A400FLK | Dilution- 1:200 |
Crypta-Grab IMS Beads- Magnetic beads coated in monoclonal antibody reactive | Waterborne, Inc | IMS400-20 | Dilution-1:500 |
DAPI | Thermo Fisher Scientific | D1306 | Dilution-1:1000; RRID : AB_2629482 |
Phalloidin-Alexa 674 | Invitrogen | A22287 | Dilution-1:1000; RRID: AB_2620155 |
Rabbit anti-gp15 antibody generated by R. M. O’Connor (co-author). | Upon request | Upon request | Dilution-1:500 |
Sporo-Glo | Waterborne, Inc | A600FLR-1X | Dilution- 1:200 |