Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
2005-11-01
2005-11-01
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S377000, C435S378000, C435S384000, C435S386000, C435S387000, C435S392000
Reexamination Certificate
active
06960473
ABSTRACT:
We describe a new two-step culture method for mass production in vitro of erythroid cells from either CD34+(105cells/mL) or light-density (106cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method includes (i) culture of the cells in the presence of dexamethasone and estradiol (10−6M each) and (ii) the growth factors SCF (50 ng/mL), IL-3 (1 ng/mL), and EPO (1 U/mL). In their proliferative phase, these cultures generated about 1-2×107erythroblasts for each milliliter of blood collected from normal donors or thalassemic patients. They were composed mostly (90%) of CD45low/glycophorin (GPA)neg/CD71lowcells at day 7, 50-60% of which became CD45neg/GPA+/CD71highby days 15-20. However, when cells from days 7 to 12 of the proliferative phase were transferred in differentiation medium containing EPO and insulin, they progressed to mature erythroblasts (>90% benzidineposand CD45neg/GPA+/CD71medium) in 4 days. Because of the high number of erythroid cells that are generated from modest volumes of blood, this method will prove useful in donor-specific studies of erythroid differentiation.
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Franco Anna Rita
Migliaccio Giovanni
Dunston Jennifer
Istituto Superiore di Sanita
McKelvey Terry
Rothwell Figg Ernst & Manbeck
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