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US5399493A
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Methods and compositions for the optimization of human hematopoietic progenitor cell cultures
Assignee: University of Michigan (System)
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Filing: 1991-08-05
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Granted: 1995-03-21
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Expires: 2011-08-05
Abstract
Abstract Methods, including culture media conditions, which provide for ex vivo human stem cell division and stable genetic transformation and/or the optimization of human hematopoietic progenitor cell cultures and/or increasing the metabolism or GM-CSF secretion or IL-6 secretion of human stromal cells are disclosed. The methods rely on culturing human stem cells and/or human hematopoietic progenitor cells and/or human stromal cells in a liquid culture medium which is replaced, preferably perfused, either continuously or periodically, at a rate of 1 ml of medium per ml of culture per about 24 to about 48 hour period, and removing metabolic products and replenishing depleted nutrients while maintaining the culture under physiologically acceptable conditions. Optionally growth factors are added to the culture medium.
Inventors
- 👤 Stephen G. Emerson
- 👤 Michael F. Clarke
- 👤 Bernhard O. Palsson
- 👤 Richard M. Schwartz
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 09:35
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; A; A61; A61P; A61P25/00; A61P25/28; A61P3/00; A61P3/08; A61P3/10; A61P31/00; A61P31/12; A61P31/14; A61P31/18; A61P35/00; A61P7/00; A61P7/06; A61P9/00; A61P9/12; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/53; C07K14/535; C07K14/54; C07K14/5403; C07K14/5412; C07K14/705; C12M; C12M23/00; C12M23/34; C12M29/00; C12M29/04; C12M29/10; C12M35/00; C12M35/08; C12N5/00; C12N5/06; C12N5/0602; C12N5/0634; C12N5/0641; C12N5/0642; C12N5/0647; A61K; A61K48/00; C12N2501/00; C12N2501/10; C12N2501/125; C12N2501/14; C12N2501/20; C12N2501/22; C12N2501/23; C12N2501/30; C12N2501/38; C12N2501/39; C12N2502/00; C12N2502/13; C12N2502/1394; C12N2502/99; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/13011; C12N2740/13041; C12N2740/13043; C12N2830/00; C12N2830/001; C12N2830/002; C12N2830/80; C12N2830/85
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
140 words
A method for obtaining ex vivo human hematopoietic stem cell stable genetic transformation comprising the following steps: (a) obtaining a human cellular composition containing human hematopoietic stem cells; (b) culturing said cells in the presence of a liquid culture medium containing a retroviral vector containing a heterologous gene for two weeks, wherein two thirds of said culture medium is removed from each culture daily and said medium is replaced with an equivalent volume of fresh medium containing hematopoietic growth factors and said retroviral vector; (c) thereafter replacing said culture medium with culture medium free of said retroviral vector; (d) thereafter replacing said retroviral-vector-free culture medium, either periodically or continuously, at a rate of 50% to 100% daily replacement, while maintaining said culture under physiologically acceptable conditions; and, (e) obtaining a cellular composition containing stable, genetically transformed human hematopoietic stem cells.
Claim 2
Independent Claim
140 words
A method for obtaining ex vivo human hematopoietic stem cell stable genetic transformation comprising the following steps: (a) obtaining a human cellular composition containing human hematopoietic stem cells; (b) culturing said cells in the presence of a liquid culture medium containing a retroviral vector containing a heterologous gene for two weeks, wherein two thirds of said culture medium is removed from each culture daily and said medium is replaced with an equivalent volume of fresh medium containing hematopoietic growth factors and said retroviral vector; (c) thereafter replacing said culture medium with culture medium free of said retroviral vector; (d) thereafter replacing said retroviral-vector-free culture medium, either periodically or continuously, at a rate of 50% to 100% daily replacement, while maintaining said culture under physiologically acceptable conditions; and, (e) obtaining a cellular composition containing stable, genetically transformed human hematopoietic stem cells.
Claim 3
Dependent Claim
13 words
The method of claim 1 wherein said retroviral-vector-free culture medium is replaced continuously.
Claim 4
Dependent Claim
29 words
The method of claim 2, wherein replacement of said retroviral-vector-free culture medium comprises perfusing fresh medium through at least part of the mass of said human hematopoietic stem cells.
Claim 5
Dependent Claim
13 words
The method of claim 1, wherein said retroviral-vector-free culture medium is replaced periodically.
Claim 6
Dependent Claim
29 words
The method of claim 4, wherein replacement of said retroviral-vector-free culture medium comprises perfusing fresh medium through at least part of the mass of said human hematopoietic stem cells.
Claim 7
Dependent Claim
44 words
The method of claim 1, wherein said human cellular composition containing human hematopoietic stem cells is at least one member selected from the group consisting of human peripheral blood mononuclear cells, human bone marrow cells, human fetal liver cells, and human cord blood cells.
Claim 8
Dependent Claim
24 words
The method of claim 6, wherein said human cellular composition containing human hematopoietic stem cells have been enriched for said human hematopoietic stem cells.
Claim 9
Dependent Claim
26 words
The method of claim 1 wherein said stable, genetically transformed human hematopoietic stem cells obtained in step (d) produce stable, genetically transformed human hematopoietic progenitor cells.
Claim 10
Dependent Claim
26 words
The method of claim 6, wherein said stable, genetically transformed human hematopoietic stem cells obtained in step (d) produce stable, genetically transformed human hematopoietic progenitor cells.
Claim 11
Dependent Claim
31 words
The method of claim 1, wherein IL-3 and GM-CSF are added continuously to said retroviral-vector-free culture medium, each at a rate of 0.1 to 100 ng ml -1 day -1 .
Claim 12
Dependent Claim
31 words
The method of claim 1, wherein IL-3 and GM-CSF are added continuously to said retroviral-vector-free culture medium, each at a rate of 0.5 to 10 ng ml -1 day -1 .
Claim 13
Dependent Claim
31 words
The method of claim 1, wherein IL-3 and GM-CSF are added continuously to said retroviral-vector-free culture medium, each at a rate of 1 to 2 ng ml -1 day -1 .
Claim 14
Dependent Claim
51 words
The method of claim 10, wherein steel factor is added to said medium at a rate of from 1 to 100 ng ml -1 day -1 , or IL-1α is added to said medium at a rate of from 10 to 100 U ml -1 per 3 to 5 day period.
Claim 15
Dependent Claim
13 words
The method of claim 13, wherein steel factor is added to said medium.
Claim 16
Dependent Claim
12 words
The method of claim 13, wherein IL-1α is added to said medium.
Claim 17
Dependent Claim
16 words
The method of claim 13, wherein both steel factor and IL-α are added to said medium.
Claim 18
Dependent Claim
15 words
The method of claim 1, wherein said retroviral-vector-free culture medium comprises animal sera or plasma.
Claim 19
Dependent Claim
13 words
The method of claim 1, wherein said retroviral-vector-free culture medium comprises a corticosteroid.
Claim 20
Dependent Claim
55 words
The method of claim 1, comprising maintaining glucose concentration in said retroviral-vector-free culture medium in the range of from 5 to 20 mM, lactate concentration in said medium below about 35 mM, glutamine concentration in said medium in the range of from 1 to 3 mM, and ammonia concentration in said medium below 2.5 mM.
Claim 21
Dependent Claim
23 words
The method of claim 6, said culture comprising human stromal cells in an amount of 10 -3 to 10 -1 (stromal cells/total cells).
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