Authors
Shiv Poojan and Sushil Kumar
Summary
A pure-population of keratinocyte stem cell culture is required for applications such as transgenic-mouse-stem cell preparation, stem cell therapy, tissue engineering, iPS technology, besides study of growth & differentiation, toxicology, and related mechanistic aspects. Protocols available in literature do not conform to desired purity and better yield in short span of time, culture-condition for rapid growth, and characterization methods1-24. We report here the improved, albeit comprehensive and replicable protocols to identify, isolate, characterize, culture, cryo-preserve, subculture, and grow quickly a pure population of multipotent stem cell from mouse epidermis. Protocol has been validated using modified Growth Promoting Medium; FACS-based LRC-detection and verification; FACSaria based LRCs sorting to improve stem-cell yield and re-culture; immunohistochemical & RT-PCR based verification of stem-cell biomarkers; low-Ca2+ culture-conditions; FACS based revalidation and repeated characterization of passaged LRCs. Cryopreservation and re-culture displayed cultivability of pure population of stem cell. FACS-generated results demonstrate the attainment of purity to the order of >90%. Key features of the present protocol are high yield of stem cell, rapid cell growth in 7d in contrast to 13-21d reported earlier, culture of pure population of stem cell as confirmed using FACS, and use of conditioned medium in place of feeder layer.Further details
The protocol was published on Protocol Exchange on 5 August 2010. To see the entire protocol, click on the source link.Advertisement
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