TITLE: Thawing, Expanding, and Freezing Human Fibroblasts
AUTHORS: Jacob Marsh, Rj Martinez, Celeste Karch

[STEPS]
?. [Thawing Human Fibroblasts]
Prior to thawing cells, prepare culture media
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hFibroblast Media: DMEM/F12, 10-20%FBS1% L-Glut (or Glutamax)1% Antibiotic
?. [Thawing Human Fibroblasts]
Remove cells from liquid nitrogen storage and thaw quickly by placing vial in a  water bath for approximately
37 °C
Be sure to monitor cells during thaw and make sure they are removed from water bath just prior to completely thawing
?. [Thawing Human Fibroblasts]
Quickly transfer thawed cells into conical tube containing  of room temperature DMEM/F12
9 ml
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Centrifuge: 750 33, 3 min
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Aspirate supernatant, be careful not to distrub cell pellet
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Re-suspend cells in  of hFibroblast Media by pipetting cells up and down 2-3 times to achieve a single cell suspension
5 ml
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Transfer cell suspension to T25 cell culture flask
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Incubate at  and 5% C02 overnight
37 °C
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Replace hFibroblast Media every 2-3 days
?. [Expanding Human Fibroblasts]
Upon reaching 80-90% confluency, fibroblasts will create a swirling pattern. This is the appropriate time to split the cells
Confluent fibroblasts can be split from one T25 cell culture flask into one T75 cell culture flask OR two T25 cell culture flasks.
?. [Expanding Human Fibroblasts]
Aspirate hFibroblast Media from T25 cell culture flask
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Wash cells with  1X PBS
5 ml
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Add  of 0.25% Trypsin to cells. Incubate at  for  to allow cells to dissociate from cell culture flask
1 ml
37 °C
After incubation period, check cells under a microscope to determine the extent to which cells have detached from the flask. If many cells remain attached, incubate at   for an additional   .
?. [Expanding Human Fibroblasts]
Re-suspend cells in  of hFibroblast Media. Pipet cells up and wash flask 4-6 times to collect all cells and to achieve a single cell suspension.
5 ml
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Add cell suspension to  of fresh hFibroblast Media and transfer to T75 cell culture flask
7 ml
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Incubate at  at 5% C02 overnight
37 °C
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Change media every 2-3 days until fibroblasts become confluent
At this point fibroblasts can be passaged again, used for experiments, or frozen down and banked
?. [Freezing Human Fibroblasts]
Upon reaching 80-90% confluency, fibroblasts will create a swirling pattern. This is the appropriate time to split the cells
It is important to keep fibroblast cultures at a low passage number, thus when thawing cells it should be a priority to expand cells for freezing over expanding cells for experiments.
One T75 cell culture flask will produce enough cells to freeze 2-3   aliquots.
?. [Freezing Human Fibroblasts]
Aspirate hFibroblast Media from T25 cell culture flask
?. [Freezing Human Fibroblasts]
Wash cells with  1X PBS
5 ml
?. [Freezing Human Fibroblasts]
Add  of 0.25% Trypsin to cells. Incubate at  for  to allow cells to dissociate from cell culture flask
2 ml
37 °C
?. [Freezing Human Fibroblasts]
Re-suspend cells in  of hFibroblast Media. Pipet cells up and wash flask 4-6 times to collect all cells and to achieve a single cell suspension.
7 ml
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Transfer cell suspension to conical tube for centrifugation
?. [Freezing Human Fibroblasts]
Centrifuge: 500 33, 5 min
?. [Freezing Human Fibroblasts]
Aspirate supernatant, be careful not to distrub cell pellet
?. [Freezing Human Fibroblasts]
Re-suspend cell pellet in  hFibroblast Media.
1.5 ml
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Add  2X Freezing Media (FBS, 10% DMSO) to cell suspenion from Step 26.
1.5 ml
This should result in a final volume of   which can be equally distributed into   aliquots across three cryovials.
?. [Freezing Human Fibroblasts]
Transfer  to each pre-labeled cryovial
1 ml
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Immediately place cells in  freezer for
-80 °C
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Transfer cells to long-term liquid nitrogen storage