Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of detaching cells – digesting tissue or establishing...
Reexamination Certificate
1999-02-26
2001-05-29
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of detaching cells, digesting tissue or establishing...
C435S381000, C435S378000, C435S368000
Reexamination Certificate
active
06238922
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to a production or manufacturing process for the in vitro proliferation and culture of neural stem cell cultures using collagenase to dissociate the cultured cells.
BACKGROUND OF THE INVENTION
The development of the mammalian central nervous system (CNS) begins in the early stage of fetal development and continues until the post-natal period. The mature mammalian CNS is composed of neuronal cells (neurons), and glial cells (astrocytes and oligodendrocytes). The first step in neural development is cell birth, which is the precise temporal and spatial sequence in which stem cells and stem cell progeny (i.e. daughter stem cells and progenitor cells) proliferate.
One identifying feature of a stem cell is its ability to exhibit self-renewal or to generate more of itself. A definition of a stem cell is provided by Potten & Loeffler, 110 Development 1001 (1990) who have defined stem cells as “undifferentiated cells capable of (a) proliferation, (b) self-maintenance, (c) the production of a large number of differentiated functional progeny, (d) regenerating the tissue after injury, and (e) a flexibility in the use of these options.” The role of stem cells is to replace cells that are lost by natural cell death, injury or disease.
U.S. Pat. Nos. 5,750,376, 5,851,832 (both naming Weiss) and 5,753,506 (Johe), each incorporated herein by reference, refer to in vitro cultures containing neural stem cells. The Weiss patents refer to both suspension and adherent culture, while Johe refers to particular adherent cultures. When the cells are propagated as neurospheres in suspension culture, within 3-4 days in the presence of a proliferation-inducing growth factor, a multipotent neural stem cell begins to divide giving rise to a cluster of undifferentiated cells referred to as a “neurosphere”. The cells of a single neurosphere are clonal in nature because they are the progeny of a single neural stem cell. In the continued presence of one or more proliferation-inducing growth factors, such as EGF, bFGF, or the like (and combinations thereof), cells within the neurosphere continue to divide resulting in an increase in the size of the neurosphere and the number of undifferentiated cells. The cells within the neurosphere are immunoreactive for nestin, an intermediate filament protein found in many types of undifferentiated CNS cells. In contrast, mature differentiated cell types derived from the neural stem cell progeny are predominantly negative for nestin.
In the prior art, the cells in the cluster were mechanically dissociated by trituration to produce single cells between passages. Trituration, because it is a mechanical process, exerts shear forces on the cells, that may reduce cell viability between passages. The object of the present invention is to provide an improved culture and manufacture process that increases cell viability from passage to passage and maintains more of the most primitive cells (with the greatest differentiation potential and self-renewal capability).
SUMMARY OF THE INVENTION
The invention provides a method for the in vitro proliferation of multipotent neural stem cell culture using collagenase to dissociate cells in neurospheres between passages. According to the method of this invention, use of collagenase results in improved neural stem cell culture viability, increased number of proliferated cells in those cultures over time, and improved maintenance of cell cultures, as compared with dissociation by trituration or other enzymatic treatments such as trypsinization.
REFERENCES:
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patent: 5061620 (1991-10-01), Tsukamoto et al.
patent: 5270191 (1993-12-01), McKay et al.
patent: 5342777 (1994-08-01), Cole et al.
patent: 5411883 (1995-05-01), Boss et al.
patent: 5750376 (1998-05-01), Weiss et al.
patent: 5753506 (1998-05-01), Johe
patent: WO 94/09119 (1994-04-01), None
patent: WO 96/04368 (1996-02-01), None
patent: WO 96/15226 (1996-05-01), None
patent: WO 97/41209 (1997-11-01), None
Kalyani et al. Developmental Biology. 1997. vol. 186. pp. 202-223.
Afremova Vera
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
Saucier Sandra E.
StemCells, Inc.
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