TITLE: Transfection of Atlantic salmon primary hepatocytes
AUTHORS: Alex K. Datsomor, Ragnhild Wilberg, Jacob S. Torgersen, Simen R. Sandve, Thomas N Harvey

[DESCRIPTION]
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.

[STEPS]
SECTION: Prepare buffers and plates
2. Coat wells of the culture plate with 1x PEI.
 

SECTION: Prepare buffers and plates
2.1. Dilute PEI stock 1:100 in 0.1 M borate buffer.
SECTION: Prepare buffers and plates
2.2. Add sufficient volume to empty wells. Enough to cover the bottom of the well.

SECTION: Prepare buffers and plates
2.3. Incubate for 60 min at room temperature.

SECTION: Prepare buffers and plates
2.4. Wash wells 2x with HBSS with Mg/Ca
SECTION: Prepare buffers and plates
1. Prepare the following:
 

Wash buffer: 1x HBSS without Ca/Mg, 1 mM EDTA, 10 mM HEPES, pH 7.4.
Mix reagents, adjust to pH 7.4 , increase volume to 500 mL using dH2O, filter sterilize and store at  4 °C.
400 mL 
50 mL 
1 mL 
5 mL 

Collagenase buffer: 1x HBSS with Ca/Mg, 10 mM HEPES, pH 7.5.
Mix reagents, adjust to pH 7.5 , increase volume to 200 mL using dH2O, filter sterilize and store at  4 °C.
100 mL 
20 mL 
2 mL 

Borate buffer: 0.1 M boric acid
Mix reagents, adjust to pH 8.4, increase volume to 400 mL using dH2O, filter sterilize and store at Room temperature
350 mL 
2.47 g 

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 
19 µL 

Cell culture media: L15 media, 5% fetal bovine serum, 1x penicillin-streptomycin

47 mL 
2.5 mL 
0.5 mL 

Collagenase: Resuspend collagenase powder in dH2O to a final concentration of 1*10^5 U/mL . Prepare 50 µL  and store at -20 °C 
SECTION: Dissection and perfusion
3. Dilute collagenase to a final concentration of 150 U/mL  in collagenase buffer.
45 µL 
30 mL 
 

SECTION: Digestion and washing
9. Pour the entire liver solution into a sterile Erlenmeyer flask containing a sterile magnetic stir bar. Incubate at 15 °C for 60 min with gentle stirring.

SECTION: Electroporation and plating
16. 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, 5 min, 4 °C 

SECTION: Dissection and perfusion
4. Kill the fish with a sharp blow to the head. Immediately record length and weight.
 

SECTION: Dissection and perfusion
5. 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. 
SECTION: Dissection and perfusion
6. Locate the portal vein and insert the needle. Perfuse liver with wash buffer for 5 min. The liver should turn from red to yellow/white.

 

SECTION: Dissection and perfusion
7. Without removing the needle switch the wash buffer tube to collagenase buffer from step 3. Perfuse with collagenase for 5 min.

SECTION: Dissection and perfusion
8. 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.
 

SECTION: Digestion and washing
10. 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.
SECTION: Digestion and washing
11. Centrifuge the cells at 100 x g, 5 min, 4 °C 
SECTION: Digestion and washing
12. Remove the supernatant and resuspend in 5 mL 

SECTION: Digestion and washing
13. Centrifuge again at 100 x g, 5 min, 4 °C 

SECTION: Digestion and washing
14. Remove the supernatant and resuspend in 5 mL 

SECTION: Digestion and washing
15. Mix cells 1:1 with trypan blue and count using a hemocytometer.
 

SECTION: Electroporation and plating
18. 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)
 


SECTION: Electroporation and plating
19. Insert the electroporation chamber into the Neon electroporator and add 3 mL buffer E.
SECTION: Electroporation and plating
17. Remove supernatant and resuspend cells in culture media.
 

SECTION: Electroporation and plating
18. Add the appropriate volume of cells suspension to the wells of a PEI coated culture plate.
SECTION: Electroporation and plating
19. Incubate cells at 15 °C under ambient atmosphere. Change media every 3-4 days. Cells remain viable for at least 3 weeks.

SECTION: Electroporation and plating
20. Add 1-5 ug of plasmid DNA or 1 µl RNP to cells.
 

 

SECTION: Electroporation and plating
17. 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.

SECTION: Electroporation and plating
21. Aspirate cell suspension into a 10 µl Neon tip, insert into the Neon electroporator, and electroporate at 1400 V, 20 ms, 2 pulses
SECTION: Electroporation and plating
22. Immediately add electroporated cells to media in prepared culture plate. Incubate cells at 15 °C.

SECTION: Electroporation and plating
23. 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.