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

1µg/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 37°C)

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 37°C, 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 37°C 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