Apr 28, 2022

Public workspaceHuman fibroblast culturing

DOI
dx.doi.org/10.17504/protocols.io.rm7vzy542lx1/v1
  • 1Department of Clinical and Movement Neurosciences, Queen Square Institute of Neurology, University College London, University of London
Laura Smith
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.rm7vzy542lx1/v1
Protocol Citation: Laura Smith, David C 2022. Human fibroblast culturing. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzy542lx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 28, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 61598
Keywords: ASAPCRN
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.
Safety warnings
SAFETY:-
Biological Hazards:
·The culturing of human cells is a class II activity and all appropriate safety issues need to be addressed.
·If using GMOs read the Laboratory Codes of practice for working with GMOs

Characteristics
Characteristics
Characteristics
  • Obtained /developed from human adult dermal biopsies
  • Proliferative until p20
  • Doubling time ~ 24h
  • Maintain between 60 – 90% confluent
Complete growth medium
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
Subculturing by trypsinisation, and cell counting
  1. Wash cells grown onto 10 cm Ø plate with PBS.
  2. 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.
  3. Tap the plates and add 2 ml complete medium to stop trypsination.
  4. To a 50 ul sample, add equal vol of 0.4% Trypan blue (Sigma # T8154 or LT # 15250-061); fed it to a hemacytometer.
  5. Optional -- to pellet cells and resuspend into fresh medium.
  6. Replate the cells accordingly; typically 10 ml medium is used for 10 cm Ø plate.
  7. Subcultivation ratio of 1:4 is recommended; medium renewal every 2-3d.

Freezing and thawing
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.