Two-stage culture protocol for isolating mesenchymal stem...

Details Two-stage culture protocol for isolating mesenchymal stem... Two-stage culture protocol for isolating mesenchymal stem...

2006-09-05

2006-09-05

McKelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of detaching cells, digesting tissue or establishing...

C435S366000, C435S383000, C435S384000, C435S395000, C435S091200, C435S007100

Reexamination Certificate

active

07101710

ABSTRACT:
A method of harvesting mesenchymal stem cells from human amniotic fluid uses a two-stage culture protocol comprising culturing human amniocytes and then culturing mesenchymal stem cells. For culturing human amniocytes, primary amniocyte cultures are set up using routine or standard culture protocol in a cytogenetic laboratory. Non-adherent human amniotic fluids cells in the supernatant medium are collected. For culturing mesenchymal stem cells (“MSC”), the non-adherent cells are centrifuged and then plated with an alpha-modified Minimum Essential Medium supplemented with fetal bovine serum. Incubate with humidified CO2for MSC growth. Reverse transcription polymerase chain reaction (“RT-PCR”) and immunocytochemical analyses reveal that Oct-4 mRNA and OCT-4 protein expression is detectable in the cultured amniotic fluid mesenchymal stem cells (“AFMSCs”). Under differentiation culture conditions, the AFMSCs can be induced to develop into multi-lineage cells, such as adipocytes, osteocytes, neuronal cells, etc.

REFERENCES:
patent: 2005/0042595 (2005-02-01), Haas
patent: 2005/0124003 (2005-06-01), Atala et al.
Kaviani et al. The amniotic fluid as a source of cells for fetal tissue engineering. Journal of Pediatric Surgery. 36(11):1662-1665, Nov. 2001.
Prusa et al. Amniotic fluid cells and human stem cell research—a new connection. Medical Science Monitor. 8(11):RA253-RA257, 2002.

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