NP460hTERT in KGM2

1. The trial of new medium KGM-2 ── comparing with medium DKSFM/EpiLife for culturing NP460hTert:

(1) Cells culturing in KGM-2 showed more like triangle shape rather than elongated morphology as seeding in DKSFM/EpiLife.

(2) Cells culturing in KGM-2 had slower proliferation rate (Doubling time:55.4hrs) than culturing in DKSFM/EpiLife medium (Doubling time:36hrs).

2.Different [Ca2+] treatments for 6 days in KGM-2 medium for NP460hTert

(1) Along the treatment, cells in 2 calcium free treatments showed poor adhesion to the dish (細胞沒有延伸或突觸等的貼附形態出現). *Mortality rate:32% and 35%.

(2) Cells in 0.1mM [Ca2+] showed good adhesion (細胞亦無層疊的現象) and had the lowest mortality rate (11%) among all treaments.

(3) Cells in 0.5mM,1mM and 1.5 mM Ca2+ showed the tendency to cluster and pile up together under high Ca2+ culture.(第四天後細胞堆疊明顯)

(4) Cells in 1.5mM Ca2+ showed the highest mortality rate (48%), almost 50% of the cells were dead at the measuring time. The mortality of 1mM Ca2+ treatment weas 24%, 0.5mM Ca2+ was27%.

(5) After trypisinization, cells didn’t look bigger when counting.(通常要準備走上senescence或分化的細胞會變較大)

討論結果:

(1) KGM-2和DKSFM/EpiLife下細胞生長形態的照片因為seeding不均勻的關係，要再重新試驗新種下比較均勻的10cm dish中再照一次。(8/10已重照完畢)

(2) 不同鈣離子濃度的試驗因為細胞種的不均勻可再做一次試驗(等下禮拜一母盤有夠多的細胞)，而其中一個無鈣離子的對照組可改為0.05mM來試驗(0.1mM still a little bit high or not?!)

(3) 下禮拜可以使用不同濃度的retinoic acid treatment!(in medium DKSFM/EpiLife or KGM-2)，可試試看曹老師用serum來促進differentiation的方法!

SFL 8/11/11, 11:21 AM
Yes,as Ingrid pointed out the “old” medium contains Ca2+ at a concentration of (60uM+<100um 0.1mm="" 30="" 50um="" a="" about="" after="" although="" and="" but="" can="" cells="" change="" chronic="" cut="" difference.="" does="" down="" far="" give="" good="" gradually="" if="" ingrid="" it="" make="" may="" natures="" p="" s="" seems="" so="" some="" that="" their="" to="" try.="" um="" we="" while="" why="" worried="" xxum="">

Start: 08/10/2011

Timezone: Asia/Taipei