Mar 03, 2026
In vitro culture of Microsporidia species for genome sequencing with minimal host contaminant
Peer-reviewed method
- Anne Caroline Mascarenhas1,
- Pingdong Liang1,2,
- Oscar Juarez1,
- Jean-François Pombert1,
- Karina Tuz1
- 1Illinois Institute of Technology;
- 2Northwestern University
- PLOS ONE Lab ProtocolsTech. support email: [email protected]
External link: https://doi.org/10.1371/journal.pone.0344468
Protocol Citation: Anne Caroline Mascarenhas, Pingdong Liang, Oscar Juarez, Jean-François Pombert, Karina Tuz 2026. In vitro culture of Microsporidia species for genome sequencing with minimal host contaminant. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk1mz5g5r/v1
Manuscript citation:
Santos ACMd, Liang P, Juárez OX, Pombert J, Tuz K (2026) In vitro culture of human-infecting Encephalitozoon spp. for genome sequencing with minimal host contaminant. PLOS One 21(3). doi: 10.1371/journal.pone.0344468
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 27, 2026
Last Modified: March 03, 2026
Protocol Integer ID: 241873
Keywords: Microsporidia, Encephalitozoon, Propagation, Genomics, infecting microsporidian species, microsporidian genomic material suitable for advanced genomics application, producing microsporidian genomic material, microsporidian genome, minimal host contaminant microsporidia, culture of microsporidia species, microsporidia species, microsporidian species, host dna contamination, free host dna, host dna, obligate intracellular parasite, quality genomic dna with minimal host dna, reduction of host dna, minimal host dna, other intracellular pathogen, sufficient material for genomics study, spore, potential adaptability to other intracellular pathogen, advanced genomics application, human foreskin fibroblast, genomics study, sequencing experiment, quality genomic dna, spp, genome, severe infectious disease, immunocompromised individual, oxford nanopore, dnase
Funders Acknowledgements:
Jean-François Pombert
Grant ID: R15AI128627
Oscar Juarez and Karina Tuz
Grant ID: 1R01AI151152-01A1
Abstract
Microsporidia are obligate intracellular parasites infecting a wide range of hosts, including humans. They can cause severe infectious diseases if left untreated, particularly in immunocompromised individuals. The propagation of the human-infecting microsporidian species in vitro is essential for generating sufficient material for genomics studies, yet existing protocols often lack detail, accessibility, or strategies to minimize host DNA contamination. Here, we present an optimized, reproducible protocol for culturing Encephalitozoon spp. in human foreskin fibroblasts (HFF-1), designed to produce high-yield and high-quality genomic DNA with minimal host DNA for downstream sequencing experiments. In one month, our method yielded approximately one billion spores, which were purified using mechanical disruption, filtration, detergent treatment, and DNase I treatment to remove free host DNA. The reduction of host DNA was validated through PCR, and next-generation sequencing – with Illumina, PacBio and Oxford Nanopore - revealing that 83-97% of the reads mapped to microsporidian genomes. This protocol enabled the generation of the first telomere-to-telomere assemblies for E. cuniculi, E. hellem, and E. intestinalis. Our workflow provides a robust framework for producing microsporidian genomic material suitable for advanced genomics applications, with potential adaptability to other intracellular pathogens.
Materials
- DMEM (1X)
- 10% FBS
- Heat-inactivated FBS
- 1% PSQ (penicillin-streptomycin-glutamine)
- 2 mM L-glutamine
- FBS (fetal bovine serum)
- Gelatin from bovine skin (0.1%, autoclaved)
- PBS (1X)
- Trypsin/EDTA (0.05%)
- DMSO
- HFF-1 cells
- Microsporidia sample
- Bleach
- Tween 20
- DNase I (10 mg/mL)
- MgCl2 solution (1 M, filter sterilized)
- EDTA (0.5 M, pH 8, filter sterilized)
- Gelatin-coated petri dish
- Inverted microscope (phase contrast)
- Biosafety hood
- 15 mL conical tube
- 27-G needle
- 5 μm PVDF filter
- 10 mL syringe
- Centrifuge
- Hemocytometer
Table 1. Materials used for HFF-1 cell culture and microsporidian spore isolation.
| A | B | C | |
| Material | Manufacturer | Catalog number | |
| DMEM + 4.5 g/L D-Glucose + L-Glutamine + 110 mg/L Sodium pyruvate | Gibco | 11995-065 | |
| FBS | Cytiva | SH30070.03 | |
| L-Glutamine | Cytiva | SH40003.01 | |
| PSQ | Gibco | 10378016 | |
| DPBS (no CaCl2, no MgCl2) | Gibco | 14190 | |
| 0.05% Trypsin/EDTA | Gibco | 25300054 | |
| Gelatin from bovine skin Type B | Sigma-Aldrich | G9391 | |
| Tween 20 | Fisher scientific | BP337 | |
| DNase I from bovine pancreas | Sigma-Aldrich | DN25 | |
| Petri dishes | Thermo Scientific Nunc | 150350 |
Troubleshooting
1. Enriched Dulbecco’s Modified Eagle Media (DMEM) preparation
45m
DMEM (1X) + 10% FBS + 1% PSQ + 2 mM L-glutamine
Heat-inactivated FBS (fetal bovine serum) in water bath at 56 °C for00:45:00
45m
Thaw L-glutamine and PSQ (penicillin-streptomycin-glutamine) aliquots
Remove 60 mL of the DMEM (1X) media bottle
Add 50 mL of heat-inactivated FBS to DMEM bottle
Add 5 mL of PSQ (100X, filtered) to the DMEM bottle
Add 5 mL of L-glutamine (200 mM, filtered)
Mix bottle by inversion and make aliquots
Store at 4 °C
2. Human foreskin fibroblast cell culture infected with Microsporidia
1d 3h 7m
Coating plates with gelatin prior to HFF-1 cell culture
Coat petri dish with 10 mL of gelatin from bovine skin (0.1%, autoclaved)
Incubate petri dish at 37 °C , 5% CO₂ for at least 01:00:00
1h
Remove the gelatin solution from the petri dish by suction
Leave petri dish open in the biosafety hood until dry (max 00:20:00 )
Note
If gelatin is not dry after 20 min, quick use the suction to dry the wet spots; if petri dishes are open for too long it might damage the coating by over drying.
20m
Close dishes and start using it for cell culture now
Starting HFF-1 cell culture from frozen stocks
Add 10 mL of fresh enriched DMEM media to a gelatin-coated petri dish
Quickly thaw cell culture vial in water bath at 37 °C for00:02:00 ( 1-2 min)
2m
Resuspend cells in the vial by pipetting delicately
Transfer the entire content of the vial to the petri dish with media
Note
Cells should be added to the media and not to the bottom of the petri dish so they won’t get stuck and can spread throughout the plate.
Rock the petri dish to help cells distribute across the entire plate
Check if cells are evenly distributed in the plate under an inverted microscope
Incubate petri dish with cells 37 °C , 5% CO₂
Check if cells are attached to the plate after24:00:00 .
Note
If the cryo-media used to freeze the cells contains DMSO you may want to change half the media in the plate after 24h if cells are adhered to the petri dish.
Maintaining a HFF-1 cell culture:
Media renewal
Cell culture media should be renewed every 2-3 days
Check if cells are adhered to the petri dish and if confluence is increasing using an inverted microscope
Remove 5 mL of media from the petri dish
Add 5 mL of fresh media to the culture
Place plates back in the incubator ( 37 °C , 5% CO₂)
HFF-1 cell culture passage
Passage is recommended when cells reach a 90% plate confluence
Note
This will ensure the cells grow in a monolayer
Remove and discard media from the petri dish
Wash cells with 3 mL of PBS (1X, 7.4 ) twice
Add 3 mL of trypsin/EDTA (0.05%) to cells and incubate ( 37 °C , 5% CO₂ ) for 00:01:30
1m 30s
Check if cells are detached under an inverted microscope
Add3 mL of fresh media to the petri dish to inactivate trypsin
Incubate cells at RT for00:02:30
2m 30s
Flush cells from plate, collect them and transfer to a conical tube
Centrifuge cells at 1000 x g, Room temperature, 00:05:00
10m
Discard supernatant and resuspend cells in 1 mL of fresh media by gently pipetting
Split cells in a 1:4 or 1:6 ratio (recommend by ATCC protocol)
Transfer the selected volume of cell suspension to gelatin-coated plates (protocol 2) with 10 mL of fresh enriched DMEM media
Rock petri dish and check if cells are evenly distributed under a microscope
Incubate plates (37 °C and 5% CO2), after 01:00:00 check if cells are attached to the plates.
Note
Follow the media renewal protocol until passage is required again
1h
Infecting HFF-1 cells with Microsporidia spores from ATCC
HFF-1 cell culture should be between 90-100% confluent at the time of infection
Quickly thaw vial with microsporidia sample in a water bath at 37 °C for 00:02:00 (1-2 min)
2m
Gently resuspend the content of the vial by pipetting
Transfer the contents of the vial to the plate with the HFF-1 cells
Note
Make sure you add the spores to the media and not to the bottom of the dish
Add media to the vial to recover most of the spores and transfer it to the petri dish
Rock the plate to ensure that microsporidian spores are properly distributed
Incubate petri dish with infected HFF-1 cells at 37 °C at 5% CO₂
Media should be renewed 24:00:00 post-infection (Protocol 7 "Maintaining a HFF-1 cell culture infected with Microsporidia: Media renewal ")
Note
ATCC cryogenic media may contain DMSO, which should be removed from the cell culture to avoid HFF-1 cell toxicity
1d
Maintaining a HFF-1 cell culture infected with Microsporidia:
Media renewal
Media should be renewed when it gets cloudy or the color changes to orange
Check if confluence of infected cells is lower than 90-100%
Note
Microsporidia are observable live in the cell culture under phase contrast microscopy
Remove all the10 mL of media from the culture and transfer to a conical tube
Note
Follow protocol 8 "Harvesting microsporidian spores from supernatant" to harvest spores
Add 10 mL of fresh enriched DMEM media to the cell culture
Check culture under microscope for confluence and adherence of infected cells
Return petri dish to the incubator
Note
Cell culture should be checked daily to determine the need of media renewal
Harvesting microsporidian spores from supernatant
Collect cell culture supernatant into a 15 mL conical tube
Centrifuge supernatant-containing tube at 1500 x g, Room temperature, 00:20:00
20m
Keep spore pellet. Discard supernatant in a waste tube with 1:4 bleach
Resuspend pellet in 10 mL of PBS (1X)
Store spore suspension at 4 °C for over a year
Microsporidia infected HFF-1 cell culture passage
Passage is recommended when infected cells reach 90% confluence
Remove all media from petri dish and transfer to a conical tube (to harvest spores later, protocol 8)
Wash cells with 3 mL of PBS (1X) two times and transfer to a conical tube (to harvest spores later, protocol 8)
Add 3 mL of trypsin/EDTA (0.05%) to cells and incubate (37 °C , 5% CO₂) for 00:01:30
1m 30s
Add 3 mL of fresh enriched DMEM to the cell culture
Incubate dish at Room temperature for 00:02:30
2m 30s
Transfer detached HFF-1 cells/microsporidian spores to a tube
Centrifuge sample at 1000 x g, Room temperature, 00:05:00
5m
Discard supernatant in a tube containing 1:4 bleach
Resuspend cell suspension in 1 mL of fresh enriched DMEM
Transfer 100 µL of suspension per each gelatin-coated plate (total of 10 plates) containing 10 mL of fresh enriched DMEM media for a 1:10 split ratio
Note
The 1:10 split ratio is customized and could be adjusted accordingly
Incubate plates at 37 °C at 5% CO₂
Note
Check cell culture daily for media renewal and passage needs
3. Purification of microsporidia spores
21m 30s
From infected HFF-1 cells (protocol per petri dish)
Remove media from plate and transfer to 15 mL conical tube (may have spores for harvesting, protocol 8)
Wash cells with 3 mL of PBS (1X, 7.4 ) twice
Discard PBS in 1:4 bleach waste
Add 3 mL of trypsin/EDTA (0.05%) to cells and incubate ( 37 °C at 5% CO₂) for 00:01:30
1m 30s
Check under inverted microscope to ensure cells are detached
Add 3 mL of fresh media to the plate to inactivate trypsin
Pass sample through a 27-G needle 3 times (up and down) with a 10 mL syringe
Pass the syringe-lysed cell suspension through a 5 μm PVDF filter onto a sterile tube
Pellet spores by centrifugation at1500 x g, Room temperature, 00:20:00
Remove supernatant and discard in a waste tube containing 1:4 bleach
Resuspend pellet with 1 mL of PBS-Tween 20 (0.3%, filter sterilized)
Centrifuge sample at1500 x g, Room temperature, 00:20:00 and discard supernatant in 1:4 bleach waste
Repeat steps 10.10 – 10.12
Resuspend pellet in 1 mL of PBS (1X)
Centrifuge tube at1500 x g, Room temperature, 00:20:00
Remove supernatant and discard in a waste tube containing 1:4 bleach
Repeat steps 10.14 – 10.16 for 7 times
Resuspend spores in 1 mL of PBS (1X)
Perform a spore count using a hemocytometer by transferring 10 µL of a 1:100 diluted cell suspension aliquot
Store spore suspension at 4 °C for over a year
From cell culture supernatant
Collect cell culture supernatant into a 15 mL conical tube
Replace media collected from cell culture with 10 mL of fresh media and place plate back in incubator ( 37 °C at 5% CO₂)
Centrifuge the 15 mL tube containing media with spores at 1500 x g, Room temperature, 00:20:00
20m
Discard supernatant in a waste tube containing 1:4 bleach
Resuspend spores in 10 mL of PBS (1X)
Pass sample through a 27-G needle 3 times (up and down) with a 10 mL syringe
Pass the syringe-lysed cell suspension through a 5 μm PVDF filter onto a sterile tube
Pellet spores by centrifugation at1500 x g, Room temperature, 00:20:00
Remove supernatant and discard in a waste tube containing 1:4 bleach
Resuspend pellet with 1 mL of PBS-Tween 20 (0.3%, filter sterilized)
Centrifuge tube at1500 x g, Room temperature, 00:20:00
Remove supernatant and discard in a waste tube containing 1:4 bleach
Repeat steps 11.10 – 11.12
Resuspend pellet in 1 mL of PBS (1X)
Centrifuge tube at1500 x g, Room temperature, 00:20:00
Remove supernatant and discard in a waste tube containing 1:4 bleach
Repeat steps 11.14 – 11.16 for 7 times
Resuspend spores in 1 mL of PBS (1X)
Count spores with the hemocytometer and 10 µL of the 1:100 diluted suspension
Store sample at 4 °C
4. DNase I treatment of microsporidia purified spores
20m
Remove host-DNA contaminant
Add 10 µL of DNase I (10 mg/mL ) to the resuspended spores
Resuspend the 1 mL of spores/cell suspension in PBS by vortexing
Add 15 µL of MgCl₂ solution (1 Molarity (M) , filter sterilized) to the sample
Incubate at Room temperature for 00:15:00 under rotation
Add60 µL of EDTA (0.5 Molarity (M) , 8 , filter sterilized) and mix by inversion
Centrifuge sample at1500 x g, Room temperature, 00:20:00 and discard supernatant
Resuspend pellet in 1 mL of PBS (1X) and centrifuge at1500 x g, Room temperature, 00:20:00
20m
Repeat step 12.7 5 times for a total of 6 washes
Resuspend spores in 1 mL of PBS (1X) and store sample at 4 °C .