Human fibroblast culturing

Abstract

Fibroblasts are cultured in Dulbecco’s modified eagle media (DMEM) 4500 (mg/L) growth medium supplemented with Glutamax (Gibco), 10% foetal bovine serum (FBS), non-essential amino acids (NEAA: 0.1 mM of: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine) and penicillin/streptomycin antibiotic cocktail (50ng/ml) at 37°C and 5% CO₂.

Steps

Characteristics

Characteristics

Obtained /developed from human adult dermal biopsies
Proliferative until p20
Doubling time ~ 24h
Maintain between 60 – 90% confluent

Complete growth medium

DMEM (LT # 61965-059; 4500 mg/L and no pyruvate) Glutamax medium* Fetal bovine serum (FBS, 10% final) – 1 / 10 dilution

Pen / Strep (LT # 15140-122, 50 U/ml and 50 ng/ml final) -- 1 / 200 dilution
MEM / NEAA (LT # 11140-035) -- 1 / 100 dilution
As required -- Sodium pyruvate (Sigma # S8636, 1 mM final) – 1 / 100 dilution
As required -- Uridine (Sigma # U3003, stock of 25 mg/ml in H2O filter-sterilised; 50 ug/ml final) – 1 / 500 dilution
As required, as permitted by DSO -- Fungizone (LT # 15290-026; original of 250 ug/ml) – 1 / 100 dilution

Subculturing by trypsinisation, and cell counting

Wash cells grown onto 10 cm Ø plate with PBS.1. Add 1.5 ml diluted trypsin (4 ml of 2.5%, LT # 15090-046, into 100 ml Versene LT # 15040-033) and leave at 37’C for several mins.

Tap the plates and add 2 ml complete medium to stop trypsination.
To a 50 ul sample, add equal vol of 0.4% Trypan blue (Sigma # T8154 or LT # 15250-061); fed it to a hemacytometer.
Optional -- to pellet cells and resuspend into fresh medium.
Replate the cells accordingly; typically 10 ml medium is used for 10 cm Ø plate.
Subcultivation ratio of 1:4 is recommended; medium renewal every 2-3d.

Freezing and thawing

1.After centrifugation to pellet cells, resuspend in Freezing medium (90% FBS / 10% DMSO filter sterilised) and aliquot into freezing vials; typically ¼ of a 50% confluent 10 cm Ø plate of 125K cells in 0.5ml.Freeze cells in a stepwise fashion from -80’C to liq N.

2.Thaw cells at 37’C.