Dec 19, 2024

Public workspacePrimary cell culture of dorsal fin-derived fish fibroblasts in a CO2-independent environment

DOI
dx.doi.org/10.17504/protocols.io.rm7vzxd64gx1/v1
  • 1cE3c – Centre for Ecology, Evolution and Environmental Changes & CHANGE – Global Change and Sustainability Institute, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal;
  • 2MARE – Centro de Ciências do Mar e do Ambiente (MARE) & ARNET—Aquatic Research Network, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal
Joao M Moreno
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzxd64gx1/v1
Protocol Citation: Catarina Santos, Mariana Moço, Gabriela Rodrigues, Vitor C Sousa, Joao M Moreno 2024. Primary cell culture of dorsal fin-derived fish fibroblasts in a CO2-independent environment. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxd64gx1/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: January 25, 2024
Last Modified: December 19, 2024
Protocol Integer ID: 94126
Keywords: Cell culture, Fibroblasts, Fish cell culture, Primary cell culture
Funders Acknowledgements:
Fundação para a Ciência e a Tecnologia
Grant ID: SFRH/BD/143199/2019
Fundação para a Ciência e a Tecnologia
Grant ID: PTDC/BIA-EVL/4345/2021
Human Frontier Science Program Young Research
Grant ID: RGY0081/2020
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Abstract
A strong limitation to conduct experimental studies on non-model freshwater fish is related to the conservation status of about 20% of the species that are currently classified at least as vulnerable by the IUCN Red List of Threatened Species (Global Freshwater Fish Assessment 2020). To address this limitation, fin-derived cell cultures have emerged as a promising non-lethal tool for various assays, enabling the assessment of factors such as pollutants, environmental changes, and more. In this study, we present a protocol for obtaining primary cell cultures from dorsal fin samples of freshwater cyprinids belonging to the Squalius genus. This protocol could potentially be applied and adapted for use with a broad range of other freshwater fish species.
Materials
  • Sterilized dissection material (scissors, forceps, and bistoury)
  • 1.5 mL microcentrifuge tubes
  • 15 mL centrifuge tube
  • Petri Ø12mm dishes
  • 6-well tissue culture treated plates
  • Pipette (variable volumes) and respective filter tips



Solutions and reagents

Solution/ReagentBrandCatalog ID
Chlorhexidine gluconate 1% (w/v)Zoopan®1000000832
Sodium Hypochlorite solution 10% (w/v) technical gradePanReac AppliChem® 211921.1211
Ethanol, Absolute [100% (v/v)] (200 Proof), Molecular Biology GradeFisher BioReagents™ 16606002
Leibovitz's L-15 MediumGibco®11415064
Antibiotic-Antimycotic (100X) solution containing 10,000 units/mL of penicillin, 10,000 μg/mL of streptomycin, and 25 μg/mL of Amphotericin BGibco®15240062
Kanamycin Sulfate 10 mg/mLGibco®15160047
Nystatin, anti-fungal agentGibco®15340029
Donor Bovine Serum with IronGibco®10371029
Phosphate buffered saline (PBS) 1X Sigma-Aldrich®P4417
0.05% Trypsin - EDTA (1X) w: phenol red Gibco®25300062
Note: the PBS used in this protocol is sold in tablet form, so before used dissolve in distilled sterile water according to manufacturer's protocol and autoclave prior to use.
Before start
All materials should be sterilized in advance.
Tissue collection
Tissue collection
2h 30m
2h 30m
Prepare decontamination solution:

1X Earle's Balanced Salt Solution (EBSS) or Hanks' Balanced Salt Solution (HBSS) supplemented with Concentration2 % (v/v) Antibiotic-Antimycotic (100X), Concentration50 µg/mL Kanamycin Sulfate and 1:200 Nystatin suspension


Note
Make sure you prepare enough decontamination solution for the washing protocol described in section 2.

After washing the area surrounding the dorsal fin (and fin included) with chlorhexidine gluconate Concentration1 % (w/v) , collect the dorsal fin of the fish using sterilized dissecting scissors and put it in Amount1.5 mL of decontamination media. Leave it at room temperature for at least Duration00:30:00 and up to Duration02:00:00 .


Note
If you suspect severe contaminations use the following quarantine protocol: after collecting the tissue, put it in a 1.5 mL microtube with a mixture of 1X Earle's Balanced Salt Solution (EBSS) or Hanks' Balanced Salt Solution (HBSS) supplemented with 2% (v/v) Antibiotic-Antimycotic (100X),Concentration50 µg/mL Kanamycin Sulfate and 1:200 Nystatin suspension for Duration00:30:00 at room temperature. Next, transfer the tissue to a 1.5 mL microtube filled withAmount1.0 mL of culture medium supplemented withConcentration5 % (v/v) antibiotic/antimycotic (Concentration10000 mg/mL ), 1:200 Nystatin suspension, and Concentration100 mg/mL Kanamycin sulfate for another Duration00:30:00 at room temperature.



2h 30m
Critical
Establishing primary cell culture
Establishing primary cell culture
Prepare enough volume of complete L-15 media:

L-15 media supplemented withConcentration15 % (v/v) Donor bovine serum with Iron and Concentration1 % (v/v) Antibiotic-Antimycotic (100X).


Note
If you suspect strong contaminations add: Concentration50 µg/mL Kanamycin Sulfate (for bacterial contamination) and/or 1:200 Nystatin suspension (for fungal contamination). Note that both Kanamycin and Nystatin are toxic for the cells and should be avoided or their presence in the media should be minimal and as reduced as possible.

Dilute sodium hypochlorite solution toConcentration1 % (w/v) and absolute ethanol toConcentration65 % (v/v) .
Prepare six 1.5 mL tubes according to the following scheme:

  • Tube 1: Amount1.0 mL Concentration65 % (v/v) ethanol
  • Tube 2:Amount1.0 mL L-15 withConcentration2 % (v/v) antibiotic/antimycotic, 1:200 Nystatin suspension andConcentration50 µg/mL Kanamycin
  • Tube 3:Amount1.0 mL Concentration1 % (v/v) sodium hypochlorite
  • Tube 4:Amount1.0 mL L-15 withConcentration2 % (v/v) antibiotic/antimycotic, 1:200 Nystatin suspension andConcentration50 µg/mL Kanamycin
  • Tube 5:Amount1.0 mL Concentration65 % (v/v) ethanol
  • Tube 6:Amount1.0 mL L-15 withConcentration2 % (v/v) antibiotic/antimycotic, 1:200 Nystatin suspension and Concentration50 µg/mL Kanamycin



Use sterilized forceps to take the tissue out of the 1.5 mL microtube and proceed with the following washing protocol using the tubes prepared in the previous step:
  • 2 to 5 seconds in the Tube 1.
  • 5 to 8 seconds in the Tube 2.
  • 5 seconds in the Tube 3.
  • 5 seconds in Tube 4.
  • 6 to 8 seconds in the Tube 5.
Transfer the tissue to tube 6 and leave it there for at least 45 seconds (ideally up to 2 – 5 min).
Transfer the tissue to a Ø12mm petri dish containing 1X PBS supplemented withConcentration2 % (v/v) antibiotic/antimycotic. Wash the tissue for around Duration00:00:30 .

30s
Transfer the tissue to a Ø12mm petri dish containingAmount750 µL trypsin-EDTAConcentration0.05 % (w/v) . Cut the tissue into tiny pieces with a sterile pair of tweezers or bistoury.
Incubate the pieces of tissue in the trypsin forDuration00:10:00 at room temperature (shake occasionally).

Note: trypsin is toxic to cells and will cause cells to die off if incubated for longer periods.
10m
AddAmount2.5 mL complete media (see step 3) to the petri dish.


Note
If you suspect severe contamination, supplement the complete media with 1:200 Nystatin suspension in case of fungus and/orConcentration50 µg/mL Kanamycin in case of bacteria.

Transfer the media (with remaining chunks of tissue) to a 6-well plate.
Note: plates with visible chunks of tissue seem to grow the most cells, but are also most prone to infection. It is possible to divide the previous suspension into more than one plate to plate each well with various amounts of visible tissue and assess the most manageable ratio of cell and bacterial growth.
AfterDuration18:00:00 toDuration24:00:00 , all chunks of tissue that didn't adhere to the bottom of the plate must be removed, and the media must be changed. For that, all the media should be removed, and Amount3.0 mL fresh media should be added to each well.

Note: If you observe small debris in the media, wash the well with complete media (preferably) or 1X PBS, and then addAmount3.0 mL fresh complete media.

1d 18h
Critical
Maintenance and establishment of cell lines
Maintenance and establishment of cell lines
Replace all of the media every 2-3 days, or whenever you notice a significant change in its color (towards a more yellow/orange or purple tone).
When cells reach around 80% confluency, trypsinize them and transfer all the cells to a different well. It is important that, in this step, no chunks of fin go into the new well.

Note:Amount150 µL ofConcentration0.05 % (w/v) Trypsin - EDTA (1X) per well (6-well plate) is used for trypsinization.

After cells reach confluency in the new well, pass them into a new well or T25 flask to proliferate them, or use them for further experiments.
Protocol references
Rábová, M., R. Monteiro, M. J. Collares-Pereira, e P. Ráb. «Rapid Fibroblast Culture for Teleost Fish Karyotyping». In Fish Cytogenetic Techniques: Ray-Fin Fishes and Chondrichthyans. CRC Press, 2015. https://www.taylorfrancis.com/books/9781482211993