Culture Conditions HMEC-1[1] 12.02.2003

Medium (original)

MCDB 131 Gibco 10372

10 mM L-Glutamine

alternative

Endothelial basal Medium Clonetic cc3121

 

10-15% fetal bovine serum

Medium (my lab)

MEM Earle w NEA, w L-Glutamine Biochrom T437-05

10ml/l MEM Vitamines (100x) Biochrom K0373

10ml/l N-Acetyl L-Alanyl L-Glutamine Biochrom K0202
(200mM)

20ml/l Amino Acids (50x) Biochrom K0363

10% FBS

Medium Supplement (non essential for growth)

10ng/ml Epidermal Growth Factor

1g/ml Hydrocortisone

Culturing Procedures

aspirate spent medium

wash monolayer sheet with Trypsin/EDTA (1.5ml/75cm2 flask)

aspirate Trypsin/EDTA wash

add 1.5ml Trypsin/EDTA and allow the cells to detach (RT or 37C)

add 4ml growth medium to stop Trypsin/EDTA activity and dispense cells

count cells and determine viability using Trypan Blue

inoculate new flask at 5 x 104 to 1 x 105 cells/ml

incubate at 37C, 5% CO2

if feed between splits, cell monolayer may require scraping to disperse and sub-culture

Thawing Procedure

Initiate thawing as soon as possible upon receipt

Thaw by rapid agitation in 37C water bath

Dilute contents of vial 1:25 with growth media

Notes

Cells usually will become confluent in 2-3 days

Cells tend to senesce at ~ passage 27

 



[1] HMEC-1 cells from Edwin W Ades were received from the Centers of Disease Control and Prevention.

Revised by Michael Dillon, approved by Francis Candal from CDC, 04/07/99

adapted by Manfred Maitz