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 karyorype 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. (C) 2009 Elsevier B.V. All rights reserved.