Laser-assisted selection and passaging of human pluripotent stem cell colonies.
The derivation of somatic cell products from human embryonic stem cells (hESCs) requires a highly standardized production process with sufficient throughput. To date, the most common technique for hESC passaging is the manual dissection of colonies, which is a gentle, but laborious and time-consuming process and is consequently inappropriate for standardized maintenance of hESC. Here, we present a laser-based technique for the contact-free dissection and isolation of living hESCs (laser microdissection and pressure catapulting, LMPC). Following LMPC treatment, 80.6+/-8.7% of the cells remained viable as compared to 88.6+/-1.7% of manually dissected hESCs. Furthermore, there was no significant difference in the expression of pluripotency-associated markers when compared to the control. Flow cytometry revealed that 83.8+/-4.1% of hESCs isolated by LMPC expressed the surface marker Tra-1-60 (control: 83.9+/-3.6%). In vitro differentiation potential of LMPC treated hESCs as determined by embryoid body formation and multi-germlayer formation was not impaired. Moreover, we could not detect any overt karyotype alterations as a result of the LMPC process. Our data demonstrate the feasibility of standardized laser-based passaging of hESC cultures. This technology should facilitate both colony selection and maintenance culture of pluripotent stem cells.
Terstegge
Stefanie
S
Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn and Hertie Foundation, Bonn, Germany.
Rath
Barbara H
BH
Laufenberg
Iris
I
Limbach
Nina
N
Buchstaller
Andrea
A
Schütze
Karin
K
Brüstle
Oliver
O
D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D003114:Colony-Forming Units Assay; D006801:Humans; D007621:Karyotyping; D007834:Lasers; D042282:Microdissection; D039904:Pluripotent Stem Cells / Q000166:cytology*; D011312:Pressure