TITLE: Isolating and growing fibroblast cells from threespine stickleback (Gasterosteus aculeatus)
AUTHORS: Gabrielle Q Le, Natalie Steinel, Lauren Fuess, Maria Rodgers 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol goes through the process of isolating and growing fibroblast cells from threespine stickleback.</div></div>

[STEPS]
?. [Obtaining tissue]
Prepare and sterile filter 30 mL of 5% FBS media.
?. [Obtaining tissue]
Prepare and sterile filter 10 mL of 20% FBS media.
?. [Obtaining tissue]
Prepare two petri dishes on ice for collecting the tissues. Fill the petri dish half of the way with the 5% FBS media.
?. [Obtaining tissue]
Prepare 2x 6 well culture dishes: 4 wells filled respectively with .2% iodine, 1% bleach, 0.9x PBS, and 0.9x PBS.
?. [Obtaining tissue]
Euthanize the fish following standard lab protocols and place body on ice.
?. [Obtaining tissue]
Using tweezers and mini dissecting scissors, cut as much of the pectoral, pelvic and caudal fins and place in the petri dish. (Note that this should be done outside the biosafety cabinet to avoid later contamination).  Place remaining fish back on ice and continue this step until all fins have been removed from all fish.
?. [Obtaining tissue]
Take petri dish to biosafety cabinet and wash with each of the 4 washes for 30 seconds-1 minute each.
?. [Obtaining tissue]
In the last PBS wash, use the scissors to cut the tissues as small as possible.
?. [Obtaining tissue]
Using a pipette, remove all liquid and tissue from last PBS wash well and transfer to sterile 15mL tube.
?. [Obtaining tissue]
Begin peritoneal tissue removal, using autoclaved dissecting tubes, open fish ventrally and pin body open. Wash inside of fish with iodine solution for 1 minute each.
?. [Obtaining tissue]
Remove peritoneal tissue using tweezers and mini dissecting scissors. Place tissue in second petri dish and repeat for each fish.  Once done with dissections, repeat steps f-h for peritoneal tissue.
?. [Digesting tissue]
Centrifuge tubes of PBS + tissue for 10 minutes at 90 g at 4C.
?. [Digesting tissue]
Prepare solution of .9x TrypsinLE in a small conical tube.
?. [Digesting tissue]
Aspirate the PBS from tissue tubes, leaving approximately 1.5 mL of liquid + tissue while avoiding disturbing the pellet.
?. [Digesting tissue]
Fill to 11mL (by adding approximately 10 mL) with TrypsinLE solution, and gently mix.
?. [Digesting tissue]
Let the suspension incubate on the vial’s side at ~17 degrees C for 20 minutes.
?. [Digesting tissue]
While incubating, prepare sterile collagenase and hyaluronidase solution.
?. [Digesting tissue]
Remove tubes from incubator and centrifuge at 90 g for 10 minutes at 4C.
?. [Digesting tissue]
Aspirate to remove the TrypsinLE solution, leaving leaving approximately 1.5 mL of liquid + tissue while avoiding disturbing the pellet.
?. [Digesting tissue]
Resuspend cells with the 5 mL of collagenase/hyaluronidase solution and gently mix.
?. [Digesting tissue]
Incubate for 2 hours at ~17 degrees C (17C).
?. [Digesting tissue]
Centrifuge again at 90g for 10 min at 4C.
?. [Digesting tissue]
Aspirate to remove the collagenase/hyaluronidase solution, leaving approximately 1.5 mL of liquid + tissue while avoiding disturbing the pellet.
?. [Digesting tissue]
Wash cells with 5% FBS solution; gently mix to resuspend pellet.
?. [Cell culture flask preparation and care]
Aspirate out the majority of the remaining liquid, leaving approximately 1.5 mL of liquid + tissue while avoiding disturbing the pellet.
?. [Cell culture flask preparation and care]
Resuspend pellet in ~5mL the 20% FBS media and gently mix.
?. [Cell culture flask preparation and care]
Pipette 5 mL of solution into each cell culture flask while they are positioned vertically. If there is any medium on the neck of the flask, aspirate it off.
?. [Cell culture flask preparation and care]
CRITICAL! Make sure there is no liquid remaining on the interior flask neck or cap in order to avoid contamination. If there is liquid, aspirate to remove.
?. [Cell culture flask preparation and care]
Carefully transfer flasks to incubator and lay them horizontally on the side that ensures the neck of the flask is facing up. Again, do not let liquid splash on the cap while in motion.
?. [Cell culture flask preparation and care]
Optional: check flasks under microscope for bacterial and fungal contamination.
?. [Cell culture flask preparation and care]
Incubate at ~17 C for 12-24 hours.
?. [Cell culture flask preparation and care]
Prepare the full 20% FBS media (with growth factors, chitosan, NAG, antibiotic- antimycotic). Make sure to sterile filter final solution.
?. [Cell culture flask preparation and care]
Remove flasks from the incubator, check for bacterial/fungal contamination, and transfer to biosafety cabinet.  Carefully remove cap and tilt flask mostly upright so that cells fall out of suspension to bottom of flask.
?. [Cell culture flask preparation and care]
Aspirate out as much media as possible without removing cells.
?. [Cell culture flask preparation and care]
Add 5 mL of full 20% FBS media to each flask making sure no liquid is left on the neck or cap of the flask. Aspirate any liquid which may have splashed on the neck of the flask.
?. [Cell culture flask preparation and care]
Incubate at ~17 degrees C for three-five days.
?. [Digesting tissue]
Centrifuge again at 90 g for 10 min at 4C.
?. [Cell culture flask preparation and care]
Monitor cells daily. Fibroblasts should be released 2-4 days after receiving growth factors. Replace full growth medium every 3-5 days and keep them incubated at ~17 C.
?. [Cell culture media change]
Remove flasks from the incubator, check for bacterial/fungal contamination, and transfer to biosafety cabinet.
?. [Cell culture media change]
Carefully remove cap and tilt flask mostly upright so that cells fall out of suspension to bottom of flask.
?. [Cell culture media change]
Aspirate out as much media as possible without removing cells.
?. [Cell culture media change]
Add 5 mL of full 20% FBS media to each flask making sure no liquid is left on the neck or cap of the flask.
?. [Cell culture media change]
Aspirate any liquid which may have splashed on the neck of the flask.
?. [Cell culture passaging]
Do not ever let the confluency reach over 80%.
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Once cells have reached 60-75% confluency, they are ready to be passaged.
?. [Cell culture passaging]
Prepare sterile .9x TrypsinLE (make 5 mL for each flask you passage), 20% FBS media with additives, and 20% FBS media (no additives).
?. [Cell culture passaging]
When transfering flasks into biosafety cabinet, always tilt the cap side away from yourself and then spray with ethanol to avoid spraying cap with ethanol.
?. [Cell culture passaging]
Carefully remove cap and tilt flask mostly upright so that cells fall out of suspension to bottom of flask. Aspirate old media and put in 5 mL of the TrypsinLE.
?. [Cell culture passaging]
For the very first passage, leave trypsin in for about 5 minutes. You may want to take flask from the biosafety cabinet and look under microscope while doing this to make sure most cells have dislodged from the flask. Gently move the flask side to side throughout the time frame. After about a minute, return it to upright position, and firmly hit the side of the flask to dislodge cells into suspension. Repeat as needed to resuspend most cells, but know there will probably still be hundreds of cells that will remain adhered. Don’t go past the 5 minute mark or else you risk further harming cells.
?. [Cell culture passaging]
In all subsequent passages, it may only take 2-3 minutes for cells to dislodge.
?. [Cell culture passaging]
Take flask back to the biosafety cabinet and place the flask upright.
?. [Cell culture passaging]
Add 8 mL of the 20% FBS media. Use the same pipette to thoroughly mix and resuspend any cells remaining adhered to the bottom of the flask.
?. [Cell culture passaging]
Place the media and the cells into a new 15mL conical tube and centrifuge at 90 g for 10 minutes at 4C.
?. [Cell culture passaging]
Aspirate as much of the liquid as possible, leaving just enough to ensure that the pellet is not disturbed. Add ~10mL of L-15 media to thoroughly rinse the cells.
?. [Cell culture passaging]
Centrifuge again at 90 g for 10 minutes at 4C.
?. [Cell culture passaging]
While waiting for centrifuge to finish, prepare and label new flasks (include date, name and passage #).
?. [Cell culture passaging]
Add 4 mL of 20% FBS media with additives to each flask.
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Once centrifuging is complete, aspirate each tube until there is about 2 mL left.
?. [Cell culture passaging]
Using a pipette, thoroughly mix remaining liquid to dislodge the cells and resuspend.
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Take 25%-50% of the 2mL and put them into a new flask.
?. [Cell culture passaging]
Continue to change media every three-five days, and monitor until cells are ready for next passage.
?. [Cryopreservation]
Ideally cells should be ready for passage (minimum 60% confluency); and have been through several passages prior to freezing.
?. [Cryopreservation]
Prepare the Mr. Frosty using the steps listed on the container.
?. [Cryopreservation]
Prepare sterile .9x TrypsinLE (make 5 mL for each flask you freeze), and 20% FBS media (no additives).
?. [Cryopreservation]
When transfering flasks into biosafety cabinet, always tilt the cap side away from yourself and then spray with ethanol to avoid spraying cap with ethanol.
?. [Cryopreservation]
Carefully remove cap and tilt flask mostly upright so that cells fall out of suspension to bottom of flask. Aspirate old media and put in 5 mL of the TrypsinLE.
?. [Cryopreservation]
Follow steps for passaging to dislodge cells from the flask.
?. [Cryopreservation]
Add 8 mL of the 20% FBS media. Use the same pipette to thoroughly mix and resuspend any cells remaining adhered to the bottom of the flask.
?. [Cryopreservation]
Place the media and the cells into a new 15mL conical tube and centrifuge at 90 g for 10 minutes at 4C.
?. [Cryopreservation]
Aspirate as much of the liquid as possible, leaving just enough to ensure that the pellet is not disturbed. Add ~10mL of 20% FBS media to thoroughly rinse the cells.
?. [Cryopreservation]
Centrifuge again at 90 g for 10 minutes at 4C.
?. [Cryopreservation]
While waiting for centrifuge to finish, prepare and label new vials (include date, name and passage #).
?. [Cryopreservation]
Fill vials carefully about halfway with DMSO.
?. [Cryopreservation]
Once centrifuging is complete, aspirate each tube until there is about 2 mL left.
?. [Cryopreservation]
Using a pipette, thoroughly mix remaining liquid to dislodge the cells and resuspend.
?. [Cryopreservation]
Take 2 mL of suspension and pipette into vial.
?. [Cryopreservation]
Transfer vial into Mr. Frosty and let freeze overnight in -80.
?. [Cryopreservation]
Transfer all vials to vapor stage of a liquid nitrogen freezer as soon as possible for long term storage.
?. [Cell revival from cryopreservation]
Prepare a 37C water bath.
?. [Cell revival from cryopreservation]
Pipette 10mL 5% FBS into a sterile conical tube; place in incubator to warm to 18C.
?. [Cell revival from cryopreservation]
Transport vial to lab on dry ice.
?. [Cell revival from cryopreservation]
Thaw vial QUICKLY (
?. [Cell revival from cryopreservation]
Transfer contents to warmed 18C conical tube.
?. [Cell revival from cryopreservation]
Spin at 90G for 10 minutes.
?. [Cell revival from cryopreservation]
Aspirate liquid.
?. [Cell revival from cryopreservation]
Wash with 5mL 5% FBS.
?. [Cell revival from cryopreservation]
Spin at 90G for 10 minutes.
?. [Cell revival from cryopreservation]
Resuspend in 5mL full 20% media.
?. [Cell revival from cryopreservation]
Transfer to new flask & incubate overnight.
?. [Cell revival from cryopreservation]
Monitor cells daily for viability.