Sorting and expansion of murine embryonic stem cell colonies using micropallet arrays.
Isolation of cell colonies is an essential task in most stem cell studies. Conventional techniques for colony selection and isolation require significant time, labor, and consumption of expensive reagents. New microengineered technologies hold the promise for improving colony manipulation by reducing the required manpower and reagent consumption. Murine embryonic stem cells were cultured on arrays composed of releasable elements termed micropallets created from a biocompatible photoresist. Micropallets containing undifferentiated colonies were released using a laser-based technique followed by cell collection and expansion in culture. The micropallet arrays provided a biocompatible substrate for maintaining undifferentiated murine stem cells in culture. A surface coating of 0.025% gelatin was shown to be optimal for cell culture and collection. Arrays composed of surface-roughened micropallets provided further improvements in culture and isolation. Colonies of viable stem cells were efficiently isolated and collected. Colonies sorted in this manner were shown to remain undifferentiated even after collection and further expansion in culture. Qualitative and quantitative analyses of sorting, collection efficiency, and cell viability after release and expansion of stem cell colonies demonstrated that the micropallet array technology is a promising alternative to conventional sorting methods for stem cell applications.
Shadpour
Hamed
H
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Sims
Christopher E
CE
Thresher
Randy J
RJ
Allbritton
Nancy L
NL
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D002999:Clone Cells; D053595:Embryonic Stem Cells / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D051379:Mice; D046888:Tissue Array Analysis / Q000379:methods*
D000469:Alkaline Phosphatase