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Laser-assisted selection and passaging of human pluripotent stem cell colonies

Paper ID Volume ID Publish Year Pages File Format Full-Text
24505 43520 2009 7 PDF Available
Title
Laser-assisted selection and passaging of human pluripotent stem cell colonies
Abstract

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.

Keywords
Human embryonic stem cells; Laser microdissection; Non-enzymatic passaging; Cell culture automation; Pluripotency
First Page Preview
Laser-assisted selection and passaging of human pluripotent stem cell colonies
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 143, Issue 3, 10 September 2009, Pages 224–230
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering