DK2733203T3 - Fremgangsmåder til ekspansion af embryonale stamceller i en suspensionskultur - Google Patents

Fremgangsmåder til ekspansion af embryonale stamceller i en suspensionskultur Download PDF

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DK2733203T3
DK2733203T3 DK13185969.6T DK13185969T DK2733203T3 DK 2733203 T3 DK2733203 T3 DK 2733203T3 DK 13185969 T DK13185969 T DK 13185969T DK 2733203 T3 DK2733203 T3 DK 2733203T3
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culture
embryonic stem
stem cells
medium
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Michal Amit
Joseph Itskovitz-Eldor
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Technion Res & Dev Foundation
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    • C12N5/0602—Vertebrate cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/20—Cytokines; Chemokines
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Claims (14)

1. Fremgangsmåde til ekspansion og opretholdelse af humane embryonale stamceller (hESC'er) i en udifferentieret tilstand, hvilken fremgangsmåde omfatter dyrkning af de humane embryonale stamceller i mindst 5 passager i en suspensionskultur under dyrkningsbetingelser uden adhærens til et eksternt substrat, og som tillader ekspansion af mindst 50 % af de humane embryonale stamceller i den udifferentierede tilstand, hvor det eksterne substrat omfatter komponenter af ekstracellulær matrix, en glasmikrobærer eller glasperler, og hvor dyrkningsbetingelserne omfatter et defineret, xenofrit og serumfrit dyrkningsmedium, der er valgt fra gruppen bestående af: et dyrkningsmedium, som omfatter basisk fibroblastvækstfaktor (bFGF), en opløselig interleukin-6-receptor (slL6R) i en koncentration på mindst 10 nanogram pr. milliliter (ng/ml) og opløseligt interleukin-6 (IL6), et dyrkningsmedium, som omfatter bFGF og mindst 1000 enheder pr. milliliter (u/ml) leukæmiinhibitorfaktor (LIF), et dyrkningsmedium, som omfatter bFGF og en IL6RIL6-kimære, og et dyrkningsmedium, der omfatter bFGF og en TGFp-isoform, og hvor dyrkningsbetingelserne omfatter dyrkning af de humane embryonale stamceller i en dyrkningsbeholder med en indvendig flade, som er udformet således, at de hESC'er, der dyrkes deri, ikke kan adhærere eller binde til fladen, hvorved de humane embryonale stamceller ekspanderes og holdes i den udifferentierede tilstand.
2. Fremgangsmåde til generering af cellelinjespecifikke celler ud fra humane embryonale stamceller, hvilken fremgangsmåde omfatter, at: (a) de humane embryonale stamceller dyrkes i en suspensionskultur ifølge fremgangsmåden ifølge krav 1, hvorved der opnås ekspanderede, udifferentierede humane embryonale stamceller; og (b) de ekspanderede, udifferentierede humane embryonale stamceller underkastes dyrkningsbetingelser, der er egnet til differentiering og/eller ekspansion af cellelinjespecifikke celler; hvorved de cellelinjespecifikke celler genereres ud fra de humane embryonale stamceller.
3. Fremgangsmåde til generering af embryoidlegemer ud fra humane embryonale stamceller, hvilken fremgangsmåde omfatter, at: (a) de humane embryonale stamceller dyrkes i en suspensionskultur ifølge fremgangsmåden ifølge krav 1, hvorved der opnås ekspanderede, udifferentierede humane embryonale stamceller; og (b) de ekspanderede, udifferentierede humane embryonale stamceller underkastes dyrkningsbetingelser, der er egnet til differentiering af de humane embryonale stamceller til embryoidlegemer; hvorved embryoidlegemerne genereres ud fra de humane embryonale stamceller.
4. Fremgangsmåde til generering af cellelinjespecifikke celler ud fra embryonale stamceller, hvilken fremgangsmåde omfatter, at: (a) de humane embryonale stamceller dyrkes i en suspensionskultur ifølge fremgangsmåden ifølge krav 1, hvorved der opnås ekspanderede, udifferentierede humane embryonale stamceller; (b) de ekspanderede, udifferentierede humane embryonale stamceller underkastes dyrkningsbetingelser, der er egnet til differentiering af de ekspanderede, udifferentierede humane embryonale stamceller til embryoidlegemer; og (c) celler af embryoidlegemerne underkastes dyrkningsbetingelser, der er egnet til differentiering og/eller ekspansion af cellelinjespecifikke celler; hvorved de cellelinjespecifikke celler genereres ud fra de humane embryonale stamcell
5. Fremgangsmåde ifølge krav 1, hvorTGF3-isoformen er en TGF3-isoform 1 (TGF3i).
6. Fremgangsmåde ifølge krav 1, hvorTGF3-isoformen er en TGF3-isoform 3 (TGF33).
7. Fremgangsmåde ifølge krav 1, hvor slL6R'en forekommer i en koncentration på 15-30 ng/ml.
8. Fremgangsmåde ifølge krav 1, hvor LIF'en forekommer i en koncentration på mindst 2000 enheder pr. milliliter (u/ml).
9. Fremgangsmåde ifølge et hvilket som helst af kravene 1 -8, hvor dyrkningen udføres under xenofrie betingelser.
10. Fremgangsmåde ifølge krav 5, hvorTGFPi'en forekommer i en koncentration på mindst 0,06 ng/ml.
11. Fremgangsmåde ifølge krav 6, hvor TGFPs'en forekommer i en koncentration på eller mindst 0,5 ng/ml.
12. Fremgangsmåde ifølge et hvilket som helst af kravene 1-11, hvor bFGF'en er i en koncentration på mindst 2 ng/ml.
13. Fremgangsmåde ifølge et hvilket som helst af kravene 1-11, hvor bFGF'en er i en koncentration på mindst 4 ng/ml.
14. Fremgangsmåde ifølge krav 1, hvor IL6RIL6-kimæren er i en koncentration på mindst 25 ng/ml.
DK13185969.6T 2006-08-02 2007-08-02 Fremgangsmåder til ekspansion af embryonale stamceller i en suspensionskultur DK2733203T3 (da)

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US83479506P 2006-08-02 2006-08-02
US84069206P 2006-08-29 2006-08-29
EP07790025.6A EP2059586B1 (en) 2006-08-02 2007-08-02 Methods of expanding embryonic stem cells in a suspension culture

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