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Oligodendrocyte precursor cells and method of obtaining and culturing the same

A technology of oligodendrocytes and astrocytes, applied in the field of cells, can solve problems such as difficult to separate oligodendrocyte progenitor cell populations

Inactive Publication Date: 2006-09-13
OTSUKA PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is difficult to isolate a phenotypically homogeneous population of primary oligodendrocyte progenitor cells with the same developmental stage

Method used

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  • Oligodendrocyte precursor cells and method of obtaining and culturing the same
  • Oligodendrocyte precursor cells and method of obtaining and culturing the same
  • Oligodendrocyte precursor cells and method of obtaining and culturing the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

Example 1 : Homogeneous Population of Purified Rat Oligodendrocyte Progenitor Cells

[096] First, by the sequential separation method using a Petri dish, or by indirect immunization described in Gard et al., Neuroprotocols 2:209-218, 1993, and McCarthy et al., J. Cell Biol. A2B5(+)O4(-) or A2B5(+)O4(+) cells were obtained from rat embryonic spinal cord (E14-E19) by adherence dissociation. Cells were then cultured on 10 cm dishes (Falcon) pre-coated with 0.001% poly-L-ornitine (Sigma) at a density of approximately 20,000-50,000 cells / cm 2 , in medium A (DMEM / N2 (Gibco); 25 ng / ml PDGF (R&D); 15 ng / ml bFGF (R&D); 5 ng / ml NT-3 (R&D); 0.05% bovine serum (Sigma)). Medium A was replaced every other day and bFGF was supplemented daily.

[097] After about a week, when the plates became sub-confluent, they were cultured with medium B (0.125% trypsin; 0.26mM EDTA; Ca(-)Mg(-) Hank's buffered saline (Hank's Buffered Saline Solution (Gibco)) trypsinized cells at 37°C for 20 minutes. Tr...

Embodiment 2

Example 2 : a homogeneous population of purified human oligodendrocyte progenitor cells

[0101] First, A2B5(+)O4(-) or A2B5(+)O4(+ )cell. Cells were then cultured on 0.001% poly-L-ornithine and 0.01% laminin pre-coated 10 cm dishes (Falcon) at a density of approximately 20,000-50,000 cells / cm 2 , in medium A (DMEM / N2 (Gibco); 25 ng / ml PDGF; 5 ng / ml NT-3). Medium A was changed daily.

[0102] After approximately one week, when the plates become confluent, trypsinize with medium B (0.125% trypsin; 0.26 mM EDTA; Ca(-)Mg(-) Hank's buffered saline (Gibco)) at 37°C cells for 20 minutes. Trypsinized cells were replated in medium C (DMEM / B27 (Gibco); 10 μM 3,3′,5′-triiodothyronine (T3); 10 ng / ml bFGF) for approximately one moon.

[0103]During one month of incubation in medium C, the cells began to change from the bipolar morphology (A2B5(+)O4(-)) to the multipolar morphology that is characteristic of A2B5(+)O4(+) cells. When almost all the cells acquired sun-like multipolar ...

Embodiment 3

Example 3: Oligodendrocyte progenitor cells can be freeze-thawed

[0105] Rat and human oligodendrocyte progenitor cells obtained according to Examples 1 and 2, respectively, were frozen in 5-10% DMSO in DMEM / B27 medium supplemented or not supplemented with 15 ng / ml of bFGF . When cells were thawed and cultured in Medium D, cells were recovered with an average 90% viability, with a maximum viability ranging from 97-99% in five independent experiments. Furthermore, the cells did not show any significant changes in their physical or functional characteristics, such as homogeneity, morphology, proliferative capacity, differentiation capacity and dedifferentiation capacity. When cultured in Medium D, the cells maintained their homogeneity and continued to proliferate without differentiation.

[0106] Oligodendrocyte progenitor cells obtained according to Examples 1 and 2 can also be frozen and maintained in the medium taught herein in the substantial absence of growth factors or ...

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Abstract

The invention describes a self-renewing, phenotypically homogeneous population of oligodendrocyte precursor cells having a synchronized developmental stage and methods of obtaining a self-renewing phenotypically homogeneous population of oligodendrocyte precursor cells. Other methods include methods of maintaining and storing a homogeneous population of oligodendrocyte precursor cells for a prolonged period of time without change in the characteristics of the cells and methods of dedifferentiating oligodendrocyte precursor cells. The self-renewing, phenotypically homogeneous population of oligodendrocyte precursor cells or homogeneous population of oligodendrocytes may be useful for treating a patient having a CNS disorder or condition.

Description

[0001] related application [0002] [001] This application claims the benefit of U.S. Provisional Application No. 60 / 487,933, filed July 18, 2003. technical field [0003] [002] The present invention relates to methods of obtaining self-renewing, phenotypically homogeneous populations of oligodendrocyte progenitor cells having synchronized developmental stages, and to cells obtained by the methods of the invention. [0004] [003] The invention further relates to methods of maintaining a self-renewing, phenotypically homogeneous population of oligodendrocyte progenitor cells with synchronized developmental stages for extended periods of time without altering cell identity. [0005] [004] The oligodendrocyte progenitor cells of the invention can be dedifferentiated back to an earlier developmental stage by sequential application of cell separation (by digestion reagents such as trypsin) followed by defined culture conditions. Acco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00C12N5/02C12N5/06C12N5/08A61K35/12C12NC12N5/079C12N5/0797
CPCC12N2501/13C12N2501/155C12N2501/115C12N2503/02C12N5/0622A61K35/12C12N2501/135C12N2501/395C12N5/0623A61P21/04A61P25/00A61P25/14A61P25/16A61P25/18A61P25/28A61P27/02A61P3/02A61P31/06A61P31/12A61P31/18A61P31/22A61P35/00A61P37/02A61P39/02A61P43/00A61P9/10A61P3/10C12N5/0618
Inventor H·冈崎
Owner OTSUKA PHARM CO LTD
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