Nov 30, 2022
Transfection of Atlantic salmon primary hepatocytes
- Alex K. Datsomor1,
- Ragnhild Wilberg1,
- Jacob S. Torgersen2,
- Simen R. Sandve1,
- Thomas N Harvey1
- 1Department of Animal and Aquacultural Sciences, Faculty of Biosciences, Centre for Integrative Genetics (CIGENE), Norwegian University of Life Sciences, Ås, Norway;
- 2AquaGen AS, P. O. 1240, 7462, Trondheim, Norway
- Thomas N Harvey: Corresponding author;
External link: https://doi.org/10.1093/g3journal/jkad039
Protocol Citation: Alex K. Datsomor, Ragnhild Wilberg, Jacob S. Torgersen, Simen R. Sandve, Thomas N Harvey 2022. Transfection of Atlantic salmon primary hepatocytes. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw4p1l5r/v1
Manuscript citation:
Datsomor AK, Wilberg R, Torgersen JS, Sandve SR, Harvey TN, Efficient transfection of Atlantic salmon primary hepatocyte cells for functional assays and gene editing. G3: Genes|Genomes|Genetics 13(4). doi: 10.1093/g3journal/jkad039
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: October 13, 2022
Last Modified: November 30, 2022
Protocol Integer ID: 71297
Keywords: Transfection, Electroporation, Primary cell, Hepatocyte, Atlantic salmon, CRISPR
Funders Acknowledgements:
The role of whole genome duplication in vertebrate adaptation
Grant ID: 274669
Abstract
This protocol is for isolation and transfection of primary hepatocytes from Atlantic salmon. We have confirmed plasmid transfection efficiency of up to 46% and RNP cutting efficiency up to 60%. Primary cells can survive in culture for at least three weeks, with transgene expression 48 hours post transfection. We find that coating of culture plates with polyethylenimine (PEI) is essential to cell adhesion.
Materials
- 10x Hanks balance salt solution (HBSS) -Mg/-Ca ThermoFisher, 14175095
- 10x Hanks balance salt solution (HBSS) +Mg/+Ca ThermoFisher, 14025092
- 1 M HEPES Sigma, H3537
- Boric acid Sigma, B6768
- L-15 medium ThermoFisher, 11415064
- Fetal bovine serum (FBS) Sigma, F7524
- Penicillin-Streptomycin ThermoFisher, 15140122
- Polyethyleneimine (PEI, branched) Sigma, 408719
- Collagenase ThermoFisher, 17018029
- Trypan blue Bio-rad, 1450021
- Cell culture plates (6, 12, 24 well, or 35 mm)
- Paristaltic pump
- Erlenmeyer flask with stir bar
- 70 µM cell strainer
- Neon electroporator ThermoFisher, MPK5000
- Neon electroporation tip, 10 ul ThermoFisher, MPK1025
- Neon electroporation buffer R ThermoFisher, MPK1025
- Neon electroporation buffer E ThermoFisher, MPK1025
Safety warnings
See MSDS for safety information.
Prepare buffers and plates
Prepare buffers and plates
Prepare the following:
Note
This can be done beforehand to save time.
Wash buffer: 1x HBSS without Ca/Mg, 1 mM EDTA, 10 mM HEPES, pH 7.4.
Mix reagents, adjust to 7.4 , increase volume to 500 mL using dH2O, filter sterilize and store at 4 °C .
400 mL dH2O
50 mL 10x HBSS without Ca/Mg
1 mL 0.5 M EDTA
5 mL 1 M HEPES
Collagenase buffer: 1x HBSS with Ca/Mg, 10 mM HEPES, pH 7.5.
Mix reagents, adjust to 7.5 , increase volume to 200 mL using dH2O, filter sterilize and store at 4 °C .
100 mL dH2O
20 mL 10x HBSS with Ca/Mg
2 mL 1 M HEPES
Borate buffer: 0.1 M boric acid
Mix reagents, adjust to 8.4 , increase volume to 400 mL using dH2O, filter sterilize and store at Room temperature
350 mL dH2O
2.47 g Boric acid
100x Polyethylenimine (PEI) stock: 1 mg/mL polyethylenimine, branched
Mix and filter sterilize. Polyethylenimine is very viscous, take care when pipetting. Store at 4 °C for up to 3 months. Heat at 37 °C if a precipitate forms.
20 mL 0.1 M borate buffer
19 µL polyethylenimine, branched
Cell culture media: L15 media, 5% fetal bovine serum, 1x penicillin-streptomycin
47 mL L15 media
2.5 mL Fetal bovine serum
0.5 mL 100x penicillin-streptomycin
Collagenase: Resuspend collagenase powder in dH2O to a final concentration of 1*10^5 U/mL . Prepare 50 µL aliquots and store at -20 °C
Coat wells of the culture plate with 1x PEI.
Note
This can be done the day before to save time. Coated plates can safely be stored in the incubator overnight.
Dilute PEI stock 1:100 in 0.1 M borate buffer.
Add sufficient volume to empty wells. Enough to cover the bottom of the well.
Incubate for 01:00:00 at room temperature.
1h
Wash wells 2x with HBSS with Mg/Ca
Dissection and perfusion
Dissection and perfusion
Dilute collagenase to a final concentration of 150 U/mL in collagenase buffer.
45 µL collagenase
30 mL collagenase buffer
Note
This should be done the day of the experiment
Kill the fish with a sharp blow to the head. Immediately record length and weight.
Note
We have tested the protocol on 100-400g fish (freshwater stage, parr and freshwater smolt). Larger fish and saltwater stage should be possible
Open the fish from the side with a scalpal by cutting in an arc from the gill to the anus. Try to cut just below the kidney to open the body cavity.
Locate the portal vein and insert the needle. Perfuse liver with wash buffer for 00:05:00 . The liver should turn from red to yellow/white.
Note
It is important to insert the needle into the portal vein rather than the bile duct which is very close. If the needle is in the bile duct the gull bladder will begin to enlarge. If the needle is inserted into the portal vein then the liver will immediately begin to change color. If it is too difficult to find the portal vein then the needle can be inserted further into the liver in the vicinity of the portal vein for a less efficient perfusion.
5m
Without removing the needle switch the wash buffer tube to collagenase buffer from step 3. Perfuse with collagenase for 00:05:00 .
5m
Excise the liver and move to an ice cold petri dish. Move to a LAF bench and gently rip the liver apart with tweesers until few large chunks remain. Shake the pieces to loosen the cells.
Note
Ripping rather than cutting produces jagged edges, increasing the suface area exposed to collagenase.
Digestion and washing
Digestion and washing
Pour the entire liver solution into a sterile Erlenmeyer flask containing a sterile magnetic stir bar. Incubate at 15 °C for 01:00:00 with gentle stirring.
1h
Filter the digested cells through a 70 µm cell strainer and collect in 50 mL conical tube. Rinse the cells with L-15 media until the tube is nearly full. From this point on keep cells on ice at all times.
Centrifuge the cells at 100 x g, 4°C, 00:05:00
5m
Remove the supernatant and resuspend in 5 mL HBSS without Mg/Ca
Centrifuge again at 100 x g, 4°C, 00:05:00
5m
Remove the supernatant and resuspend in 5 mL HBSS without Mg/Ca
Mix cells 1:1 with trypan blue and count using a hemocytometer.
Note
Cells may need to be diluted before counting. Typically a 1:10 dilution is sufficient. Try to only count hepatocytes which are large and round. Blood cells are oval shaped and should not be counted, but can indicate the efficiency of the perfusion.
Divide appropriate number of cells into 1.5 mL eppendorf tubes (electroporation) or conial tubes (direct plating) for the number of conditions in the experiment. Centrifuge at 100 x g, 4°C, 00:05:00
5m
Electroporation7 steps
If cells will be used for electroporation proceed here. This protocol is for 10 µl Neon electroporation tips.
Prepare culture plate by adding the appropriate amount of culture media without antibiotic to each well. Keep in the incubator at 15 °C until needed.
Remove supernatant completely and resuspend cells in buffer R at 1*10^7 cells/mL to 4*10^7 cells/mL . (i.e. 1*10^5 cells to 4*10^5 cells per 10 µl electroporation)
Note
Storing the cell suspension in buffer R for more than 15-30 minutes can reduce cell viability and transfection efficiency. Keep on ice.
Insert the electroporation chamber into the Neon electroporator and add 3 mL buffer E.
Add 1-5 ug of plasmid DNA or 1 µl RNP to cells.
Note
Do not add more than 10% of the cell volume. Amount of plasmid depends on the number of cells. See Neon electroporation manual for guidelines.
Note
If using RNPs, prepare according to the IDT protocol "Delivery of ribonucleoprotein complexes into Jurkat T cells using the Neon™ Transfection System".
Aspirate cell suspension into a 10 µl Neon tip, insert into the Neon electroporator, and electroporate at 1400 V, 20 ms, 2 pulses
Immediately add electroporated cells to media in prepared culture plate. Incubate cells at 15 °C overnight .
The next day, change antibiotic free media with culture media containing antibiotics. Cells can be kept at 15 °C for several weeks, changing media every 3-4 days. Transgene expression (GFP, luciferase, etc...) can be detected after 48 hours.