Apr 08, 2023

Schistosoma mansoni cercariae transformation (without needle) V.2

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5jp36l1b/v2
Schistosoma mansoni cercariae transformation (without needle)
  • Sarah Buddenborg1,
  • Geetha Sankaranarayanan1,
  • Magda E Lotkowska1,
  • Arporn Wangwiwatsin1,
  • Carmen L Dias Soria1,
  • Gabriel Rinaldi1,
  • Matt Berriman1
  • 1Wellcome Sanger Institute
  • Schistosoma mansoni
Sarah K Buddenborg
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DOI: https://dx.doi.org/10.17504/protocols.io.8epv5jp36l1b/v2
Protocol Citation: Sarah Buddenborg, Geetha Sankaranarayanan, Magda E Lotkowska, Arporn Wangwiwatsin, Carmen L Dias Soria, Gabriel Rinaldi, Matt Berriman 2023. Schistosoma mansoni cercariae transformation (without needle). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jp36l1b/v2Version created by Sarah K Buddenborg
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 03, 2023
Last Modified: April 08, 2023
Protocol  Integer ID: 79925
Keywords: schistosoma mansoni cercariae transformation, clonal cercariae from an individual snail, schistosoma mansoni cercariae, becoming schistosomula, schistosomula, called schistosomula, transformation of cercariae, clonal cercariae, schistosoma, mansoni cercariae transformation, centrifuging cercariae, individual snail, cercariae, number of cercariae, mansoni cercariae, epidermis to the dermi, epidermis, host skin, first intramammalian stage, lymphatic vessel, dermi
Funders Acknowledgements:
Wellcome Trust
Grant ID: 206194
Wellcome Trust
Grant ID: 098051
Abstract
Free-living aquatic S. mansoni cercariae transform into the first intramammalian stage, called schistosomula or somules, by burrowing in the host skin. Upon contact, cercariae begin to enter the skin and lose their tails, becoming schistosomula. Somules migrate through the epidermis to the dermis to find a small venule or lymphatic vessel to enter the vasculature.

Transformation of cercariae to schistosomula can be mimicked in the laboratory by centrifuging cercariae to remove tails and then culturing the somules in somule media. This method is particularly useful when the number of cercariae is low (i.e. clonal cercariae from an individual snail).

Somules can be cultured for several weeks with regular media changes.
Image Attribution
Images of somules taken by Dr. Gabriel Rinaldi
Guidelines
Media changes and opening of transformed somules to take place in tissue culture hood using sterile techniques
Materials
DMEM high glucose GlutaMAXGibco - Thermo Fisher ScientificCatalog #31966021
Lactalbumin Hydrolysate, powder (extra soluble)Thermo FisherCatalog #11800042
HypoxanthineMerck MilliporeSigma (Sigma-Aldrich)Catalog #H9636-1G
Serotonin HydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #H9523-25MG Insulin solution from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #I0516-5ML 33′5-Triiodo-L-thyronine sodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #T6397 MEM Vitamin Solution (100×)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M6895 Schneiders Insect MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0146 HEPES solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #H0887
Fetal Bovine SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #F4135
Antibiotic-Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062
Dulbecco′s Phosphate Buffered Saline 10XMerck MilliporeSigma (Sigma-Aldrich)Catalog #D1283
MilliQ water
Sterile Graduated Transfer PipetsFisher ScientificCatalog #13479108
Falcon 15 mL Conical Centrifuge TubesFisher ScientificCatalog #10773501
Nun Non-Treated 6-well plateThermo ScientificCatalog #10396482
1000 mL Vacuum Filter/Storage Bottle System 0.22 µm Pore 54.5cm² PES Membrane Sterile 12CorningCatalog #431098

Lamp or other light source
Chilling benchtop centrifuge with 15ml swing bucket rotor
Incubator at 37°C and 5% CO2
Reciprocating water bath
Class 2 Microbiological Safety Cabinet

1X DPBS + 2% ANTI-ANTI
50ml 10x DPBS
10ml 100x Antibiotic-Antimycotic (-20°C)
Fill to 500ml in vacuum filter unit with MilliQ water
Sterilise media with vacuum filter unit and store at 4°C. Use within 2 weeks.


SOMULE MEDIUM – BASCH MODIFIED MEDIUM (BMM)
  1. Mix the following reagents:
ABCDEF
Reagent1L500ml250ml[working]Storage
1x DMEM high glucose810.5ml405.25ml202.625ml1x4°C
1g/L Lactalbumin hydrolysate1g0.5g0.25g1g/L4°C
1mM Hypoxanthine500µl250µl125µl0.5µM-20°C
1mM Serotonin1ml500µl250µl1µM-20°C
Insulin1ml500µl250µl8 µg/ml4°C
1mM Hydrocortisone1ml500µl250µl1 µM-20°C
0.2mM Triiodo-L-thyronine1ml500µl250µl0.2 µM-20°C
100x MEM Vitamins5ml2.5ml1.25ml1x 4°C
1x Schneider's medium50ml25ml12.5ml5%4°C
1M HEPES10ml5ml2.5ml10mM4°C
1x Fetal calf serum inactivated100ml50ml25ml10%-20°C
100x PSF (add just before use)20ml10ml5ml2%-20°C

2. Sterilise media with vacuum filter unit and store at 4°C. Use within 2 weeks.
SEROTONIN STOCK 80mM (17mg/ml)
25mg serotonin hydrochloride (Sigma H9523-25MG) (4°C)
1.47ml H2O
Vortex well and store at -20°C in 350μl aliquots

1mM SEROTONIN
312.5μl of 80mM stock solution
24.6875ml NFW
Filter sterilize and store at -20°C in 1ml aliquots

3,3’5-TRIIODO-L-THYRONINE STOCK 10mM
100mg T3 (Sigma T6397-100MG)
14.86ml 0.2N NaOH
Vortex well and store at -20°C in 2ml aliquots

0.2mM 3,3’,5-TRIIODO-L-THYRONINE
5ml of 10mM stock solution
20ml NFW
Filter sterilize and store at -20°C in 1ml aliquots

HYPOXANTHINE STOCK 368mM (50mg/ml)
1g hypoxanthine (Sigma H9636-1G)
20ml 2:1 formic acid:H2O
Vortex well and store at -20°C in 1ml aliquots

1mM HYPOXANTHINE
34μl of 368mM stock solution
12.466ml sterile medium
Filter sterilize and store at -20°C in 500μl aliquots

HYDROCORTISONE STOCK 2.75mM (50μg/ml)
1mg hydrocortisone (H0135-1MG) (RT)
1ml absolute ethanol
Gently swirl to dissolve
19ml sterile medium
Swirl to mix and store in 9.5ml aliquots at -20°C

1mM HYDROCORTISONE
9.058ml of 2.76mM stock solution
15.942ml sterile medium
Filter sterilize and store at -20°C in 1ml aliquots

Safety warnings
Cercariae are infectious to humans. Please use proper PPE at all times, including a lab coat, waterproof over-gown, long-cuff gloves AQL <=1.5, and face shield.

  • All disposable materials should be placed in biohazardous waste bins
  • Glassware should be immersed in a klorsept solution of at least 50ppm for at least 2 hours, rinsed with diH2O and then autoclaved
  • Any liquids should be treated with klorsept solution of at least 50ppm for at least 2 hours
  • Liquids treated with klorsept should be diluted further and disposed in the drain

Schistosomula in suspension are not a risk to humans UNLESS they are injected directly into the blood stream.
Before start
Place 1 sterile 15ml falcon tube on ice per snail to be shed
Pre-chill benchtop centrifuge to 4°C
Prepare 1x DPBS+2% anti-anti and place in 37°C reciprocating water bath (see recipes in "MATERIALS" section)
Prepare somule media and place in 37°C reciprocating water bath (see recipes in "MATERIALS" section)
Cercariae collection
Shed cercariae from snails in a 6-well plate (see protocol "Schistosoma mansoni cercariae shedding"). The snails can be shed for up to 2 hrs by collecting cercariae and replacing with fresh water every 30 min
Using a sterile transfer pipette, dispense cercariae into sterile 15ml falcon tubes on ice

Note
IMPORTANT. All the following steps are carried out in the tissue culture biosafety cabinet. Keep a beaker containing 70% ethanol in the cabinet and before discarding any aspirating pipette or serological pipette aspirate ethanol to kill any contaminating cercariae in the pipette.

After collecting cercariae, adjust the volume in each 15ml falcon tube to 15ml with sterile water
Incubate tubes containing cercariae for 00:30:00 On ice

30m
Cercariae tail removal
Centrifuge the cercariae 2200 rpm, 4°C, 00:30:00 , Eppendorf 5810R centrifuge
30m
Quickly remove the supernatant and resuspend the pellet in 10ml pre-warmed 1x PBS + 2% PSF (see "MATERIALS" section for recipes) by gently inverting the tube (do not use pipet to mix)
Centrifuge the cercariae 1500 rpm, 4°C, 00:10:00 , Eppendorf 5810R centrifuge
10m
Repeat steps 6 and 7 six more times

Somules in culture
30m
Resuspend pellet of somules in ~5ml pre-warmed 37 °C somule media (see "MATERIALS" section for recipes)


Place somules in a 6-well plate and top up each well with pre-warmed 37 °C somule media so each has a total of approximately 4ml media
Somules on same day of transformation (Image credit: Dr. Gabriel Rinaldi)

Incubate at 37 °C , 5% CO2Overnight

30m
Optional: the following day, remove tails with transfer pipet (the tails will have floated to the top of the water column in the wells)
Somules the day after transformation, after removing tails (Image credit: Dr. Gabriel Rinaldi)