The effect of the controlled release of nerve growth factor from collagen gel on the efficiency of neural cell culture.
In this study, we tested the hypothesis that the amount of nerve growth factor (NGF) required for pheochromocytoma (PC12) cell culture can be dramatically reduced by controlled release of NGF from a collagen gel coating on the culture surface. Cells were cultured on collagen gels loaded with various amounts of NGF. As a control, PC12 cells were cultured on collagen gels with daily addition of various amounts of NGF to the culture medium. After an initial 12h burst, NGF was steadily released from the gels for 4 days. Apoptotic activity and cell viability were determined using terminal uridine nick end labeling and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Neuronal differentiation was determined using immunocytochemistry and Western blot analysis. Compared to 100 ng NGF daily addition (300 ng over 3 days), 1 ng NGF daily addition showed dramatically decreased cell viability and neuronal differentiation and increased apoptotic activity. In contrast, collagen gels loaded with 10 ng NGF yielded cell viability, apoptotic activity, and neuronal differentiation similar to those of culture with 100 ng NGF daily addition. Our method reduced the amount of NGF required for PC12 cell culture to 1/3th of that used in daily addition without affecting cell viability, apoptosis, or differentiation. This method could economize large-scale culture of stem cells by reducing the amount of costly growth factors needed.
Bhang
Suk Ho
SH
Department of Bioengineering, Hanyang University, 17 Hangdang-dong, Sungdong-gu, Seoul 133-791, Republic of Korea.
Lee
Tae-Jin
TJ
Lim
Jae Min
JM
Lim
Jung Su
JS
Han
Ah Mi
AM
Choi
Cha Yong
CY
Kwon
Yun Hee Kim
YH
Kim
Byung-Soo
BS
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D003094:Collagen / Q000378:metabolism*; D003470:Culture Media; D005782:Gels; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D013499:Surface Properties / Q000187:drug effects
D003470:Culture Media; D005782:Gels; D003094:Collagen; D020932:Nerve Growth Factor