TITLE: Primary Human Fibroblast Cell Culture
AUTHORS: Aparna Kumar, Faria Zafar, Birgitt Schuele

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Growing and maintaining primary human skin fibroblast cell cultures.</div><div class = "text-block">Additional links for protocols can be found:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><a href="https://www.coriell.org/0/PDF/Fibroblast_Culture_FAQ.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.coriell.org/0/PDF/Fibroblast_Culture_FAQ.pdf</span></a></li><li style = "counter-reset:ol0;list-style-type:disc;"><a href="https://www.ncbi.nlm.nih.gov/pubmed/23852182" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.ncbi.nlm.nih.gov/pubmed/23852182</span></a></li></ul></div></div>

[STEPS]
?. [Preparing for Culturing]
Store cryovials in liquid nitrogen storage tank upon arrival. Place vials in dry ice when ready to work with cells (do not thaw immediately).
?. [Preparing for Culturing]
Warm up AmnioMax media in water bath, wipe bottle with 70% alcohol before placing in a biosafety cabinet. Aliquot  of the AmnioMax media into a 15 ml conical tube.
9 ml
?. [Culturing Cells]
Thaw the cells by holding the lower half of the vial in  water bath and carefully swirling the vial around until only a small amount of ice is left in the vial. Do not let cells thaw completely. Wipe vial with 70% alcohol, before placing in biosafety cabinet.
37 °C
?. [Culturing Cells]
Carefully remove the cap of vial and gently mix by triturating using a P1000 micropipette to resuspend the cells without causing foam.
?. [Culturing Cells]
Using a p1000 micropipette, take  of media from the 15 ml conical tube and add to the vial with cells drop by drop. Note: depending on the size of the croyvial adjust the volume. Standard cryovials hold 1.5-2.0 ml.
400 µl
?. [Culturing Cells]
Centrifuge the 15 ml conical tube containing the cells and the medium at  for . Note: always balance centrifuge.
?. [Culturing Cells]
Using a p20 micropipette, remove  from the vial for cell counting and add to a 1.5 ml microcentrifuge tube or onto a piece of parafilm.
10 µl
?. [Cell counting]
Take the  1.5 ml microcentrifuge tube or drop on parafilm from step 6 and add  of Trypan Blue and gently mix by pipetting up and down.
10 µl
?. [Culturing Cells]
Take the 15 ml conical tube from the centrifuge and aspirate Amniomax media until there is only a little left above the cell pellet.
?. [Preparing for Culturing]
Prepare culture dish to thaw fibroblasts by adding  of 0.1% gelatin (autoclaved) to a culture dish and incubate at room temperature for . Note: For plating density of human fibroblasts use 1.0-1.4 x 10^4 cells/cm^2, e.g. for T25 flask plate 2.5 x 10^5 – 3.5 x 10^5. If cells are plated too sparse the grow slows down and cells go into senescence.
2 ml
?. [Maintaining Cell Culture]
20 - 30% confluency should be reached 24 hours after the cells have been plated (the following day) 24hrs after plating. Figure Legend: Example of a human fibroblast cell culture
?. [Maintaining Cell Culture]
The whole media should be changed every 2 days until 90-100% confluency is reached, at which point the cells should be passaged.
?. [Preparing for Culturing]
Record the label of the vial (ID, date frozen, any other documentation) and document in inventory.
?. [Preparing for Culturing]
Aspirate gelatin and add  of AmnioMax media to the culture dish.
3 ml
?. [Culturing Cells]
Using a p1000 micropipette, aspirate solution from cryovial and slowly add to prepared conical tube with  Amniomax (Step 4) in a drop by drop fashion.
9 ml
?. [Cell counting]
Take  of the solution and fill cell counting slide.
10 µl
?. [Cell counting]
Insert the cell counting slide into the cell counter and count the cells. Record the number of cells and viability and then dispose of the cell counting slide. Based on cell counts and viability adjust the culture vessel size to plate 1.0-1.4x10^4 of viable cells per cm^2.
?. [Culturing Cells]
Add  of Amniomax media and gently triturate to resuspend the cell pellet.
1 ml
?. [Culturing Cells]
Add 1ml cell solution from step 16 to the culture dish and gentle rock the culture dish back and forth to evenly distribute the cell over entire surface. Place flask in , 5% CO2 humidified incubator.
37 °C
?. [Preparation for Passaging Cells]
Add  of 0.1% gelatin (autoclaved) to culture dishes (x2 T25) and incubate at room temperature for . Note: For plating density of human fibroblasts use 1.0-1.4 x 10^4 cells/cm^2, e.g. for T25 flask plate 2.5 x 10^5 – 3.5 x 10^5. If cells are plated too sparse the grow slows down and cells go into senescence.
4 ml
?. [Preparation for Passaging Cells]
Warm up AmnioMax media in water bath, wipe bottle with 70% alcohol before placing in a biosafety cabinet.
?. [Preparation for Passaging Cells]
Aspirate gelatin and add  of AmnioMax media to the culture dish.
3 ml
?. [Passaging the Cells]
Remove Fibroblasts from incubator, place culture vessel in biosafety cabinet and aspirate media.
?. [Passaging the Cells]
Add  of PBS using 5ml serological pipette, swirl culture vessel, aspirate and discard PBS.
2 ml
?. [Passaging the Cells]
Add  of 0.25% Trypsin and incubate for  at  and 5% CO2. Firmly adherent cells can be detached quickly at 37°C.  Observe the cells under microscope. The detached cells appear rounded under microscope. Tap the culture vessel gently to detach all cells.Note: Prevent cell exposure to trypsin solution for longer periods (>=10 min)
2 ml
37 °C
?. [Passaging the Cells]
Add  of media to neutralize the trypsin. Note: add 2 volumes of pre-warmed complete growth media (containing serum) to inactivate trypsin.
4 ml
?. [Passaging the Cells]
Using a pipette, completely mix cells with no clumps, transfer the cell suspension to a 15 ml conical centrifuge tube.
?. [Passaging the Cells]
For cell counting, remove  of solution from the tube using a P20 micropipette and add to a 1.5 ml microcentrifuge tube, see steps under "Cell Counting".
10 µl
?. [Passaging the Cells]
Centrifuge the cells in the 15 ml conical tube for  at .
?. [Passaging the Cells]
Aspirate the supernatant until there is only a little left above the cell pellet.
?. [Passaging the Cells]
Resuspend the cell pellet in media by gently triturating.
?. [Passaging the Cells]
Remove 2.5 x 10^5 – 3.5 x 10^5 cells using a pipette (the amount of solution to pipette out is based on the number of cells which was determined by counting the cells) and add to each of the gelatin coated T25 flasks. Note: For plating density of human fibroblasts use 1.0-1.4 x 10^4 cells/cm^2, e.g. for T25 flask plate 2.5 x 10^5 – 3.5 x 10^5. If cells are plated too sparse the grow slows down and cells go into senescence.
?. [Passaging the Cells]
Place cells in incubator at  and 5% CO2 and follow protocol for "Maintaining Cell Culture."
37 °C
?. [Cryofreezing Cells]
If there are cells left in the 15 ml conical centrifuge tube that need to be cryofrozen, place the tube in the centrifuge and centrifuge for  at .
?. [Cryofreezing Cells]
Aspirate any supernatant until there is only a little left above the cell pellet and resuspend the pellet by tapping the side of the tube.
?. [Cryofreezing Cells]
Add  of pre-warmed complete growth media (containing serum) to the conical tube with the cells using a P1000 micropipette and gently triturate.
500 µl
?. [Cryofreezing Cells]
Add  of 20% DMSO (diluted in complete growth media) to the conical tube with the cells and gently triturate with a P1000 micropipette.
500 µl
?. [Cryofreezing Cells]
Using the pipette, transfer the cells to a Cryogenic Vial and label the vial. Note: If the time it will take between filling the vials and placing them in  will be quite long, keep the vials in ice as they are being filled and transfer them to the freezing container once you are at the  freezer.
-80 °C
-80 °C
?. [Cryofreezing Cells]
Place the Cryogenic Vial in a Cryogenic Vial freezing system container and place in . Note: Make sure all of the holes in the freezing conatiner are filled so that the temperature spreads evenly.
-80 °C
?. [Cryofreezing Cells]
Transfer the vials from the  to a Liquid Nitrogen tank no earlier than  after putting them in .
-80 °C
-80 °C
?. [Preparing the Media]
Thaw  Chang C supplement and wipe bottle with 70% alcohol before placing in a biosafety cabinet.
15 ml
?. [Preparing the Media]
Take the AmnioMax Basal Media and the Fungizone and wipe both bottles with 70% alcohol before placing in a biosafety cabinet.
?. [Preparing the Media]
Using a 50 ml pipette, aliquot  and using a 25 ml pipette, add the Chang C supplement to the media.
90 ml
15 ml
?. [Preparing the Media]
Add  of Fungizone to the media.
100 µl
?. [Preparing the Media]
Wrap the media container in foil to protect in the light and place in  until ready for use. Complete media can be stored at  for one month.
4 °C
4 °C
?. [Maintaining Cell Culture]
Aspirate the media the cells are in and then add new media (the media should be warmed up and the bottles should be wiped down with 70% alcohol before use).
?. [Results]