Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model.
BACKGROUND
To evaluate embryonic stem cell (ESC) harvesting methods with an emphasis on derivation of ESC lines without feeder cells or sera. Using a murine model, laser-assisted blastocyst dissection was performed and compared to conventional immunosurgery to assess a novel laser application for inner cell mass (ICM) isolation.


METHODS
Intact blastocysts or isolated ICMs generated in a standard mouse strain were plated in medium with or without serum to compare ESC harvesting efficiency. ESC derivation was also undertaken in a feeder cell-free culture system.


RESULTS
Although ICM growth and dissociation was comparable irrespective of the media components, an enhanced ESC harvest was observed in our serum-free medium (p < 0.01). ESC harvest rate was not affected by ICM isolation technique but was attenuated in the feeder cell-free group.


CONCLUSIONS
Achieving successful techniques for human ESC research is fundamentally dependent on preliminary work using experimental animals. In this study, all experimentally developed ESC lines manifested similar features to ESCs obtained from intact blastocysts in standard culture. Cell/sera free murine ESC harvest and propagation are feasible procedures for an embryology laboratory and await refinements for translation to human medical research.
Tanaka
Noriko
N
Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY 10021, USA. not2003@med.cornell.edu
Takeuchi
Takumi
T
Neri
Queenie V
QV
Sills
Eric Scott
ES
Palermo
Gianpiero D
GD