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

[DESCRIPTION]
This protocol goes through the process of isolating and growing fibroblast cells from threespine stickleback.

[BEFORE_START]
Recipes and Other Reagent Info

Reconstitution of cytokines and reagents
bFGF - 10ug/ml in sterile water. Do not filter. (stable up to 3 months at -80C; good for 1 week at 2-8C after being defrosted)
Recommended aliquoting is 25 uL
IGF-I - 100ug/ml in sterile water. Do not filter. (stable up to 3 months at -80C; good for 1 week at 2-8C after being defrosted)
Recommended aliquoting is 15 uL
Collagenase II - 100mg/ml in 0.9x PBS. Do not filter. (-20°C; do not use after defrosting)
Recommended aliquoting is 110 uL 
Hyaluronidase - 50,000 U/mL in 0.9x PBS. Do not filter. (-20°C; do not use after defrosting)
Recommended aliquoting is 15 uL
N-acetylglucosamine hydrochloride (NAG) -  10 mg/mL in 0.9x PBS. Sterile filter with media. (4°C, up to 2 weeks after added to PBS)
Recommended to just make 1 mL worth
Carboxymethyl-chitosan - 10 mg/mL in 0.9x PBS. Sterile filter with media.  (4°C, up to 2 weeks after added to PBS)
Recommended to just make 1 mL worth
0.9x PBS - 555 mL. Add 55 mL of water to each new bottle of 500 mL 1X PBS
0.9x L-15 - 555 mL. Add 55 mL of water to each new bottle of 500 mL 1X L-15

Media recipes
5% FBS media (30 mL)
1.5 mL FBS
28.5 mL L-15
100 uL of antibiotic/antimycotic
Needs to be sterile filtered
20% FBS initial media (~10 mL)
2 mL FBS
8 mL L-15
100uL antibiotic/antimycotic solution
Needs to be sterile filtered
20% FBS full media (~10 mL)
2 mL FBS
8 mL L-15
50 uL NAG
10 uL Carboxymethyl-chitosan
100 uL antibiotic/antimycotic solution 
10 uL bFGF
4 uL IGF-I
Needs to be sterile filtered

Digestion Recipes
TrypsinLE (10 mL)
9 mL 1X TrypsinLE 
1 mL 0.9x PBS
Needs to be sterile filtered
Hya + Coll. II (5 mL)
5 uL Hya
100 uL Coll. II
~5 mL 0.9x PBS
Needs to be sterile filtered

Tissue Sterilization Recipes
1% Bleach (50 mL)
.5 mL bleach/sodium hypochlorite
49.5 mL 0.9x PBS
.2% Iodine
.002 g IVP-Iodine
1 mL 0.9x PBS 

[STEPS]
SECTION: Obtaining tissue
1. Prepare and sterile filter 30 mL of 5% FBS media.
SECTION: Obtaining tissue
2. Prepare and sterile filter 10 mL of 20% FBS media.
SECTION: Obtaining tissue
3. Prepare two petri dishes on ice for collecting the tissues. Fill the petri dish half of the way with the 5% FBS media.
SECTION: Obtaining tissue
4. Prepare 2x 6 well culture dishes: 4 wells filled respectively with .2% iodine, 1% bleach, 0.9x PBS, and 0.9x PBS.
SECTION: Obtaining tissue
5. Euthanize the fish following standard lab protocols and place body on ice.
SECTION: Obtaining tissue
6. 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.
SECTION: Obtaining tissue
7. Take petri dish to biosafety cabinet and wash with each of the 4 washes for 30 seconds-1 minute each.
SECTION: Obtaining tissue
8. In the last PBS wash, use the scissors to cut the tissues as small as possible.
SECTION: Obtaining tissue
9. Using a pipette, remove all liquid and tissue from last PBS wash well and transfer to sterile 15mL tube.
SECTION: Obtaining tissue
10. 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.
SECTION: Obtaining tissue
11. 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.
SECTION: Digesting tissue
12. Centrifuge tubes of PBS + tissue for 10 minutes at 90 g at 4C.
SECTION: Digesting tissue
13. Prepare solution of .9x TrypsinLE in a small conical tube.
SECTION: Digesting tissue
14. Aspirate the PBS from tissue tubes, leaving approximately 1.5 mL of liquid + tissue while avoiding disturbing the pellet.
SECTION: Digesting tissue
15. Fill to 11mL (by adding approximately 10 mL) with TrypsinLE solution, and gently mix.
SECTION: Digesting tissue
16. Let the suspension incubate on the vial’s side at ~17 degrees C for 20 minutes.
SECTION: Digesting tissue
17. While incubating, prepare sterile collagenase and hyaluronidase solution.
SECTION: Digesting tissue
18. Remove tubes from incubator and centrifuge at 90 g for 10 minutes at 4C.
SECTION: Digesting tissue
19. Aspirate to remove the TrypsinLE solution, leaving leaving approximately 1.5 mL of liquid + tissue while avoiding disturbing the pellet.
SECTION: Digesting tissue
20. Resuspend cells with the 5 mL of collagenase/hyaluronidase solution and gently mix.
SECTION: Digesting tissue
21. Incubate for 2 hours at ~17 degrees C (17C).
SECTION: Digesting tissue
22. Centrifuge again at 90g for 10 min at 4C.
SECTION: Digesting tissue
23. Aspirate to remove the collagenase/hyaluronidase solution, leaving approximately 1.5 mL of liquid + tissue while avoiding disturbing the pellet.
SECTION: Digesting tissue
24. Wash cells with 5% FBS solution; gently mix to resuspend pellet.
SECTION: Digesting tissue
25. Centrifuge again at 90 g for 10 min at 4C.
SECTION: Cell culture flask preparation and care
26. Aspirate out the majority of the remaining liquid, leaving approximately 1.5 mL of liquid + tissue while avoiding disturbing the pellet.
SECTION: Cell culture flask preparation and care
27. Resuspend pellet in ~5mL the 20% FBS media and gently mix.
SECTION: Cell culture flask preparation and care
28. 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.
SECTION: Cell culture flask preparation and care
29. 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.
SECTION: Cell culture flask preparation and care
30. 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.
SECTION: Cell culture flask preparation and care
31. Optional: check flasks under microscope for bacterial and fungal contamination.
SECTION: Cell culture flask preparation and care
32. Incubate at ~17 C for 12-24 hours.
SECTION: Cell culture flask preparation and care
33. Prepare the full 20% FBS media (with growth factors, chitosan, NAG, antibiotic- antimycotic). Make sure to sterile filter final solution.
SECTION: Cell culture flask preparation and care
34. 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.
SECTION: Cell culture flask preparation and care
35. Aspirate out as much media as possible without removing cells.
SECTION: Cell culture flask preparation and care
36. 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.
SECTION: Cell culture flask preparation and care
37. Incubate at ~17 degrees C for three-five days.
SECTION: Cell culture flask preparation and care
38. 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.
SECTION: Cell culture media change
39. Remove flasks from the incubator, check for bacterial/fungal contamination, and transfer to biosafety cabinet.
SECTION: Cell culture media change
40. Carefully remove cap and tilt flask mostly upright so that cells fall out of suspension to bottom of flask.
SECTION: Cell culture media change
41. Aspirate out as much media as possible without removing cells.
SECTION: Cell culture media change
42. 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.
SECTION: Cell culture media change
43. Aspirate any liquid which may have splashed on the neck of the flask.
SECTION: Cell culture passaging
44. Do not ever let the confluency reach over 80%.
SECTION: Cell culture passaging
45. Once cells have reached 60-75% confluency, they are ready to be passaged.
SECTION: Cell culture passaging
46. Prepare sterile .9x TrypsinLE (make 5 mL for each flask you passage), 20% FBS media with additives, and 20% FBS media (no additives).
SECTION: Cell culture passaging
47. 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.
SECTION: Cell culture passaging
48. 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.
SECTION: Cell culture passaging
49. 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.
SECTION: Cell culture passaging
50. In all subsequent passages, it may only take 2-3 minutes for cells to dislodge.
SECTION: Cell culture passaging
51. Take flask back to the biosafety cabinet and place the flask upright.
SECTION: Cell culture passaging
52. 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.
SECTION: Cell culture passaging
53. Place the media and the cells into a new 15mL conical tube and centrifuge at 90 g for 10 minutes at 4C.
SECTION: Cell culture passaging
54. 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.
SECTION: Cell culture passaging
55. Centrifuge again at 90 g for 10 minutes at 4C.
SECTION: Cell culture passaging
56. While waiting for centrifuge to finish, prepare and label new flasks (include date, name and passage #).
SECTION: Cell culture passaging
57. Add 4 mL of 20% FBS media with additives to each flask.
SECTION: Cell culture passaging
58. Once centrifuging is complete, aspirate each tube until there is about 2 mL left.
SECTION: Cell culture passaging
59. Using a pipette, thoroughly mix remaining liquid to dislodge the cells and resuspend.
SECTION: Cell culture passaging
60. Take 25%-50% of the 2mL and put them into a new flask.
SECTION: Cell culture passaging
61. Continue to change media every three-five days, and monitor until cells are ready for next passage.
SECTION: Cryopreservation
62. Ideally cells should be ready for passage (minimum 60% confluency); and have been through several passages prior to freezing.
SECTION: Cryopreservation
63. Prepare the Mr. Frosty using the steps listed on the container.
SECTION: Cryopreservation
64. Prepare sterile .9x TrypsinLE (make 5 mL for each flask you freeze), and 20% FBS media (no additives).
SECTION: Cryopreservation
65. 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.
SECTION: Cryopreservation
66. 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.
SECTION: Cryopreservation
67. Follow steps for passaging to dislodge cells from the flask.
SECTION: Cryopreservation
68. 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.
SECTION: Cryopreservation
69. Place the media and the cells into a new 15mL conical tube and centrifuge at 90 g for 10 minutes at 4C.
SECTION: Cryopreservation
70. 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.
SECTION: Cryopreservation
71. Centrifuge again at 90 g for 10 minutes at 4C.
SECTION: Cryopreservation
72. While waiting for centrifuge to finish, prepare and label new vials (include date, name and passage #).
SECTION: Cryopreservation
73. Fill vials carefully about halfway with DMSO.
SECTION: Cryopreservation
74. Once centrifuging is complete, aspirate each tube until there is about 2 mL left.
SECTION: Cryopreservation
75. Using a pipette, thoroughly mix remaining liquid to dislodge the cells and resuspend.
SECTION: Cryopreservation
76. Take 2 mL of suspension and pipette into vial.
SECTION: Cryopreservation
77. Transfer vial into Mr. Frosty and let freeze overnight in -80.
SECTION: Cryopreservation
78. Transfer all vials to vapor stage of a liquid nitrogen freezer as soon as possible for long term storage.
SECTION: Cell revival from cryopreservation
79. Prepare a 37C water bath.
SECTION: Cell revival from cryopreservation
80. Pipette 10mL 5% FBS into a sterile conical tube; place in incubator to warm to 18C.
SECTION: Cell revival from cryopreservation
81. Transport vial to lab on dry ice.
SECTION: Cell revival from cryopreservation
82. Thaw vial QUICKLY (<1 min) in an 18C water bath (hotter if necessary).
SECTION: Cell revival from cryopreservation
83. Transfer contents to warmed 18C conical tube.
SECTION: Cell revival from cryopreservation
84. Spin at 90G for 10 minutes.
SECTION: Cell revival from cryopreservation
85. Aspirate liquid.
SECTION: Cell revival from cryopreservation
86. Wash with 5mL 5% FBS.
SECTION: Cell revival from cryopreservation
87. Spin at 90G for 10 minutes.
SECTION: Cell revival from cryopreservation
88. Resuspend in 5mL full 20% media.
SECTION: Cell revival from cryopreservation
89. Transfer to new flask & incubate overnight.
SECTION: Cell revival from cryopreservation
90. Monitor cells daily for viability.