Apr 07, 2023

Public workspaceSchistosoma mansoni cercariae transformation (with needle)

DOI
dx.doi.org/10.17504/protocols.io.x54v9d4wqg3e/v1
  • Sarah K Buddenborg1,
  • Geetha Sankaranarayanan1,
  • Magda E Lotkowska1,
  • Arporn Wangwiwatsin1,
  • Carmen L Dias Soria1,
  • Anna V Protasio1,
  • Gabriel Rinaldi1,
  • Matt Berriman1
  • 1Wellcome Sanger Institute
Sarah K Buddenborg
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DOI: dx.doi.org/10.17504/protocols.io.x54v9d4wqg3e/v1
Protocol Citation: Sarah K Buddenborg, Geetha Sankaranarayanan, Magda E Lotkowska, Arporn Wangwiwatsin, Carmen L Dias Soria, Anna V Protasio, Gabriel Rinaldi, Matt Berriman 2023. Schistosoma mansoni cercariae transformation (with needle). protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9d4wqg3e/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: March 31, 2023
Last Modified: April 07, 2023
Protocol Integer ID: 79822
Funders Acknowledgement:
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 triturating cercariae to remove tails, eliminating tails in a percoll gradient, and then culturing the somules in somule media. This method is used when the number of cercariae is high as the percoll gradient results in loss of cercariae.

Somules can be cultured for several weeks with regular media changes.
Guidelines
Media changes and opening of transformed somules to take place in tissue culture hood using sterile techniques
Materials
ReagentDMEM high glucose GlutaMAXGibco - Thermo FischerCatalog #31966021
ReagentLactalbumin Hydrolysate, powder (extra soluble)Thermo FisherCatalog #11800042 ReagentHypoxanthineMerck MilliporeSigma (Sigma-Aldrich)Catalog #H9636-1G
ReagentSerotonin HydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #H9523-25MG
ReagentInsulin solution from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #I0516-5ML
Reagent33′5-Triiodo-L-thyronine sodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #T6397
ReagentMEM Vitamin Solution (100×)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M6895
ReagentSchneiders Insect MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0146
ReagentHEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #Sigma H0887
ReagentFetal Bovine SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #F4135
ReagentAntibiotic-Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062
ReagentDulbecco′s Phosphate Buffered Saline 10XMerck MilliporeSigma (Sigma-Aldrich)Catalog #D1283
ReagentMilliQ waterContributed by users
ReagentSterile Graduated Transfer PipetsFisher ScientificCatalog #13479108
ReagentFalcon 15 mL Conical Centrifuge TubesFisher ScientificCatalog #10773501
ReagentFalcon 50mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-49A
ReagentNun Non-Treated 6-well plateThermo ScientificCatalog #10396482
Reagent1000 mL Vacuum Filter/Storage Bottle System 0.22 µm Pore 54.5cm² PES Membrane Sterile 12CorningCatalog #431098
ReagentPercollMerck MilliporeSigma (Sigma-Aldrich)Catalog #P1644-500ML
ReagentMicro-Emulsifying Needles 22g x 2-7/8 With BarCatalog #7975
ReagentLaboratory pipetting needle with 90° blunt ends 22G L 1 1/2 in.Merck MilliporeSigma (Sigma-Aldrich)Catalog #CAD7931-12EA
ReagentBD PlastiPak Syringe with Luer LockFisher ScientificCatalog #15544835
Reagent5ml syringe luer lockgreiner bio-oneCatalog #SYR5LL
Reagent20ml syringe luer lockgreiner bio-oneCatalog #SYR20LL
ReagentCorning Cell Culture Treated Flasks with Vent CapFisher ScientificCatalog #10288990
Reagent3ml Sterile Graduated Transfer Pipets individually wrappedFisher ScientificCatalog #13469108

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.



SOMULE WASH
5ml 1M Hepes
10ml antibiotic-antimycotic
To 500ml with 1x DMEM in vacuum filter unit
Sterilize media with vacuum filter unit and store at 4°C for up to 2 weeks

Percoll Gradient - 15ml
6.5ml percoll (shake before use)
1.2ml 1M NaCl
2.5ml Somule Wash
Keep percoll solutions on ice until use
Invert the mixture several times to establish a good percoll gradient
Avoid bubbles in the percoll (remove any if found)


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)
Prepare somule wash and place in 37°C reciprocating water bath (see recipes in "MATERIALS" section)
Cercariae collection
Cercariae collection
30m
30m
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 an autoclaved 50ml beaker
Fill the beaker containing cercariae to 45-50ml with diH2O and incubate for Duration00:30:00 TemperatureOn ice

30m
Carefully transfer the cercariae to a 50ml Falcon tube and centrifuge at Centrifigation500 x g, 4°C, 00:20:00
20m

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.
Discard the supernatant and add 5ml 1x PBS + 2% Anti-Anti to the pellet. Gently resuspend by flicking the tube (avoid using pipette because the cercariae are quite sticky and get lost)
Critical
Add 45ml 1x PBS + 2% Anti-Anti and centrifuge at Centrifigation500 x g, 4°C, 00:10:00
10m
Repeat this steps 6-7 once more with 1x PBS + 2% Anti-Anti and then twice with somule wash
During the centrifugations in Steps 7 and 8, prepare 2 percoll gradient tubes (2 tube for less than ~150K cercs, 4 tubes every ~150K cercs)
Following centrifugation of the cercariae, remove the supernatant and add 5ml of somule wash per percoll gradient you will use
Vortex for 1 min at max speed. This will start to break off the tails.
Cercariae tail removal with micro-emulsifying needle trituration
Cercariae tail removal with micro-emulsifying needle trituration
Open 8 x 5ml luer-lock syringes
Of 8 syringes, take out the syringe pump of 4 syringes. Place 4 syringe pumps on 100mm or 150mm petri dish
Lock one intact syringe and one syringe with the pump removed to a sterile 22G micro-emulsifying needle on each side
Aliquot cercariae equally into the connected needles. Each syringe should hold ~3ml of cercariae
Place syringe pumps back into the filled syringes and push the pump all the way. The cercariae should pass through the needle to the opposite syringe * Check for any leakage from the locks. If there is a leak, try tightening the lock or do not use that needle
Unlock the empty syringe from the connected needle, place the needle and syringe with cercariae in the 15ml tubes containing the Percoll solution

Push the cercariae through the needle, into the tube. Do this for all four needles with max ~5ml per percoll tube


Push the syringes back and forth 12-13 more times
Remove the empty syringe from the connected needle and place the needle and syringe with cercariae in the 15ml tubes containing the Percoll solution
Alternative tail removal: 20G needle trituration
Alternative tail removal: 20G needle trituration
Pass the cerariae suspension thought a 22G blunt-end needle in a luer lock syringe as follows: If 5ml solution – 15 times using 10ml luer lock syringe If 10 ml solution – 30 times using 20ml luer lock syringe
Take a drop of cercariae in a petri dish to check under the microscope if the passages have separated the tails. If there are still many cercariae (more than ~20%), continue with ~5 more passages
Percoll gradient
Percoll gradient
Place now-headless cercariae carefully on top of Percoll solution tube using a plastic pasteur pipette (5 ml per Percoll solution tube) or directly into the tube of Percoll solution from the syringe
Centrifuge at Centrifigation350 x g, 4°C, 00:05:00 , acceleration and deceleration =1
5m
Remove from centrifuge carefully. Discard supernatant and the white interface of cercaria tails
Washing somules
Washing somules
Take schistosomula “pellets” and pool them together in a 15ml falcon tube. Top to 15ml with somule wash
Centrifuge at Centrifigation500 x g, 4°C, 00:05:00
5m
Discard supernatant and add 15ml somule wash
Repeat washing steps 3 times in total
After the last wash, remove as much supernatant as possible and add 6ml of somule media
Somules in culture
Somules in culture
Dispense 2ml of somules into each well of a 6-well plate and add 4ml somule media to each
Incubate at 37°C in 5% CO2 incubator
The following day observe the somules to look for contamination, and replace media daily
Protocol references

CITATION
Paul F. Basch (1981). Cultivation of Schistosoma mansoni In vitro. I. Establishment of Cultures from Cercariae and Development until Pairing. The Journal of Parasitology.
LINK
https://doi.org/10.2307/3280632

Citations
Paul F. Basch. Cultivation of Schistosoma mansoni In vitro. I. Establishment of Cultures from Cercariae and Development until Pairing
https://doi.org/10.2307/3280632