Apr 17, 2023
Isolation of Schistosoma mansoni eggs, miracidia, and sporocysts for in vitro cultivation
- Sarah K Buddenborg1,
- Geetha Sankaranarayanan1,
- Magda E Lotkowska1,
- Catherine McCarthy1,
- Gabriel Rinaldi1,
- Lisa Seymour1,
- Matt Berriman1
- 1Wellcome Sanger Institute
Protocol Citation: Sarah K Buddenborg, Geetha Sankaranarayanan, Magda E Lotkowska, Catherine McCarthy, Gabriel Rinaldi, Lisa Seymour, Matt Berriman 2023. Isolation of Schistosoma mansoni eggs, miracidia, and sporocysts for in vitro cultivation. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbye5yvpk/v1
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: April 11, 2023
Last Modified: April 17, 2023
Protocol Integer ID: 80333
Keywords: helminths, in vitro sporocysts, schistosoma mansoni
Funders Acknowledgement:
Wellcome Trust
Grant ID: 098051
Wellcome Trust
Grant ID: 206194
Abstract
The purpose of this procedure is to isolate eggs from livers collected from mice infected with Schistosoma mansoni. This protocol ensures a sterile prep of eggs to be used for culture of eggs and/or collection and culture of sporocysts, and snail infection.
Guidelines
Livers should be placed on ice, following mouse perfusion. This protocol is for up to 5 livers only. (i.e. for 20 livers 4 tubes containing 5 processed livers are used, and for 18 livers 2 tubes containing 5 livers each plus 2 tubes containing 4 livers each are used).
All steps should be performed in a sterile, cleaned tissue culture hood.
Materials
BIOMAT 2 Class 2 Microbiological Safety Cabinet
Rocking incubator at 37°C
Lamp or other light source
Pipette boy
Tweezers
Parafilm
2ml aspirating pipettes
50ml stripettes
25ml stripettes
250um sterile sieve
150μm sterile sieve
1L sterile beakers (x2)
Fetal Bovine SerumGibco - Thermo FischerCatalog #10270106
DMEM, high glucose, GlutaMAX™ SupplementThermo FisherCatalog #10566032
50ml Falcon tubesCorningCatalog #352070
Falcon™ 15mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-53A
70% Ethanol Contributed by users
1x DPBSGibco - Thermo FischerCatalog #14190144 CollagenaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #C5138
MilliQ waterContributed by users
SucroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #S7903
PercollMerck MilliporeSigma (Sigma-Aldrich)Catalog #P1644-500ML
Petri dishes sterileVWR InternationalCatalog #516-8029
Swann-Morton Stainless Steel Surgical Scalpels # 21Fisher ScientificCatalog #11748353
Antibiotic-Antimycotic 9100x0 [Anti-Anti]Thermo Fisher ScientificCatalog #15240062
Liver washing
Liver washing
In tissue culture hood, prepare three petri dishes with 1x DPBS+2% anti-anti, one with 70% ethanol, and one clean petri dish arranged in the following order:
Decant the livers into the first petri dish using ethanol-cleaned tweezers to submerge and continuously move them for 1 minute to ensure complete saturation of the tissue
Repeat step 3 for each petri dish with the exception of the 70% ethanol dish which should be submerged and rinsed for less than 10 seconds
Liver dissociation
Liver dissociation
Place all livers into a clean petri dish and using a sterile scalpel finely mince them
Transfer the minced livers to a 50ml falcon tube, re-suspend in 40ml of 1x DPBS + 2% anti-anti and label with necessary identifying information
Weigh out 0.05g of collagenase into a labelled 15ml falcon tube and add 10ml of dH20. Mix well (The amount of collagenase to prepare depends on the number of livers to be processed – collagenase is always prepared fresh)
Add 5ml of 0.5% collagenase solution to the liver suspension and mix well
- Optional: Add to the mix 500 ul of polymixin B (100K Units) (Sigma-Aldrich, P4932-1MU), a gram negative bactericidal antibiotic that reduce LPS contamination in the egg prep, in particular when SEA will be prepared from eggs and immunological studies or co-culture with cells, will be performed
Wrap securely in parafilm and secure the tube horizontally in a 37°C rocker overnight
Egg isolation
Egg isolation
The following day, centrifuge the liver suspension tube at 400g for 5 min (acceleration and deceleration 9)
Aspirate the supernatant and re-suspend in 50ml of 1x DPBS + 2% anti-anti.
Repeat steps 9-10 three more times
After the final aspiration, re-suspend the pellet in 25ml of 1x DPBS + 2% anti-anti
Using a 50ml stripette, pass the suspension through the 250uM sieve into a 1L sterile beaker
Pass this filtrate through the 150uM sieve into a second 1L sterile beaker
Wash the beaker with 5ml of 1x DPBS + 2% anti-anti to collect any remaining eggs and add this to the filtrate by passing through the 150uM sieve
Decant the filtrate into a 50ml falcon tube and centrifuge (400g, 5 minutes, acceleration and deceleration 9)
Aspirate the supernatant and resuspend in 10ml 1x DPBS + 2% anti-anti
Prepare a Percoll gradient (one percoll gradient per 5 livers):
Prepare a 0.25M sucrose solution (4.27g sucrose + 50ml diH2O) and filter the solution through 0.22um syringe filter
In 50ml falcon tube mix 8ml Percoll and 32ml of the 0.25M sucrose solution. Invert 5 times
Very carefully pipette the resuspended eggs onto the surface of the Percoll gradient around the circumference to create a defined layer
Centrifuge the gradient at 800g for 10 minutes (acceleration 2 and deceleration 1)
Aspirate the supernatant and re-suspend in 10ml of 1x DPBS + 2% anti-anti. Transfer to a 15ml falcon tube
Centrifuge at 400g for 5 min (acceleration and deceleration 9)
Repeat steps 21-22 two more times
IMPORTANT. Check the eggs under microscope, if the prep is still ‘dirty’ or ‘contaminated with liver debris’ proceed with a second percoll gradient
Resuspend the pellet in 10ml 1x DPBS + 2% anti-anti and count 12 aliquots of 5µl to estimate total number of collected eggs
Eggs in culture
Eggs in culture
Centrifuge eggs in falcon tube at 400g for 5 mins (acceleration and deceleration 9)
Resuspend eggs in adult media (DMEM + 10% FBS + 2% anti-anti) and transfer to 6 well plates (5-6ml of media per well)
Keep eggs in culture at 37°C and 5% CO2
- Eggs can be kept in culture for up to ~10 days and retain the ‘hatchability’ however, the hatching rate will drop over time
Hatching eggs and collecting miracidia
Hatching eggs and collecting miracidia
Centrifuge eggs in falcon tube at 400g for 5 mins (acceleration and deceleration 9)
In the culture hood, aspirate supernatant and re-suspend in 6ml diH2O
Aliquot 1ml each in a 24 well plate
- It is important to use 24 well plate given the miracidia get more diluted in 12 or 6 well plate and more egg shells are picked up when collecting the miracidia)
Rinse the original falcon tube with 6 ml of water and distribute 1 ml to each well containing 1ml of eggs (i.e. the eggs will be in 2ml of water)
Place under light for hatching
At 30-40 min intervals over ~3 hrs, gently remove the top 1ml of water containing the miracidia into a 50ml falcon tube and top up the wells with 1ml of diH2O
Count miracidia and proceed with snail infections dx.doi.org/10.17504/protocols.io.36wgqjkkxvk5/v1 or sporocyst transformation
Sporocyst transformation
Sporocyst transformation
Place the tube containing miracidia on ice for ~20 min
Centrifuge at 800g for 15min (acceleration and deceleration 9)
Quickly aspirate the water and resuspend the pellet of miracidia in complete sporocyst media
- Sporocysts can be kept in culture at 28°C with malaria gas (90-92% N, 5% CO2, 3-5% O2) in a sealed container changing the media once to twice a week
- IMPORTANT. To avoid contamination always replace the media with fresh complete media the day after transformation