DK2173863T3 - Automatiseret fremgangsmåde og apparatur til embryonisk stamcellekultur - Google Patents

Automatiseret fremgangsmåde og apparatur til embryonisk stamcellekultur Download PDF

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DK2173863T3
DK2173863T3 DK08781187.3T DK08781187T DK2173863T3 DK 2173863 T3 DK2173863 T3 DK 2173863T3 DK 08781187 T DK08781187 T DK 08781187T DK 2173863 T3 DK2173863 T3 DK 2173863T3
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cells
cell
automated
media
population
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DK08781187.3T
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Nathaniel Beardsley
Veit Bergendahl
Megan Fitzgerald
Christine Daigh
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Fujifilm Cellular Dynamics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/50—Means for positioning or orientating the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
    • C12M33/06—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles for multiple inoculation or multiple collection of samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
    • C12M33/07—Dosage or metering devices therefore
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Claims (13)

  1. PAT E N T K RAV
    1. Fremgangsmåde til automatiseret ekspansion af pluripotente stamceller under definerede mediebetingelser, omfattende: (a) tilvejebringelse af en første population af pluripotente celler i et defineret vækstmedie; (b) separering af de pluripotente celler med et automatiseret separationssystem, som anvender kemisk separation af celler eller ved at bringe cellerne i kontakt med et proteolytisk enzym, hvor det proteolytiske enzym er trypsin, rekombinant trypsin, en trypsin-lignende proteinase, eller TRYPLE, og hvor den kemiske separation af cellerne anvender cheleringsmolekyler, fortrinsvis EDTA, EGTA eller citrat; og (c) suspendering af de separerede celler i frisk defineret vækstmedie for tilvejebringelse af en ekspanderet population af pluripotente celler, hvor den automatiserede ekspansion omfatter automatiseret fodring og splitning.
  2. 2. Fremgangsmåde ifølge krav 1, hvor de pluripotente celler er ES-celler eller inducerede pluripotente celler (iPS), eller fortrinsvis humane ES-celler.
  3. 3. Fremgangsmåde ifølge krav 1, hvor ingen eller i det væsentlige ingen differentiering forekommer i i det mindste 97% af de dyrkede ekspanderede pluripotente ES-celler.
  4. 4. Fremgangsmåde ifølge krav 1, hvor vækstmediet omfatter TeSR-medie.
  5. 5. Fremgangsmåde ifølge krav 1, hvor den første population af pluripotente celler er imellem 50% og omkring 99% confluente på tidspunktet for celleseparation, fortrinsvis omkring 60%, 70%, 80% eller 90% confluente på tidspunktet for celleseparation.
  6. 6. Fremgangsmåde ifølge krav 1, hvor suspendering af de separerede celler i frisk vækstmedie omfatter såning af cellerne i en eller flere nye cellekulturplade(r), fortrinsvis hvor overfladearealet af den eller de nye cellekulturplader er imellem fra omkring 5 til 35, eller fortrinsvis fra omkring 10 til omkring 35 gange større end overfladearealet af pladen omfattende den første population af ES-celler.
  7. 7. Fremgangsmåde ifølge krav 1, hvor det friske vækstmedie omfatter en inhibitor for et proteolytisk enzym, fortrinsvis en inhibitor for det proteolytiske enzym, som er anvendt til celleseparation, mere foretrukkent hvor den proteolytiske enzyminhibitor er en trypsin inhibitor eller en sojabønnetrypsininhibitor.
  8. 8. Fremgangsmåde ifølge krav 1, hvor separationssystemet er automatiseret med en væskehåndteringsrobot, fortrinsvis hvor det automatiserede separationssystem omfatter: (i) fjernelse af mediet fra den første pluripotente cellepopulation; (ii) at bringe de pluripotente celler i kontakt med et proteolytisk enzym; og (iii) inkubering af cellerne med et proteolytisk enzym for at separere celle-clustere.
  9. 9. Fremgangsmåde ifølge krav 8, hvor et defineret medie, omfattende en proteolytisk enzyminhibitor og en Rho-associeret kinase (ROCK)-inhibitor, tilføres til opløsningen efter trin (iii).
  10. 10. Fremgangsmåde ifølge krav 8, hvor det automatiserede separationssystem yderligere omfatter: (iv) underkastning af de inkuberede celler for mekanisk omrøring for yderligere at separere celle-clustere.
  11. 11. Fremgangsmåde ifølge krav 1, hvor populationen af pluripotente celler er fri for eller i det væsentlige fri for ikke-pluripotente celler, fortrinsvis hvor populationen af pluripotente celler er humane ES-celler, og hvor populationen af humane ES-celler er fri for eller i det væsentlige fri for ikke-humane celler.
  12. 12. Fremgangsmåde ifølge krav 1, yderligere defineret som en fremgangsmåde til automatiseret seriel ekspansion af embryoniske stamceller (ES)-celler omfattende: (a) tilvejebringelse af en første population af ES-celler i et vækstmedie; (b) separering af ES-cellerne med et automatiseret separationssystem; (c) suspendering af de separerede celler i frisk vækstmedie for tilvejebringelse af en ekspanderet population af ES-celler; (d) inkubering af den ekspanderede ES-cellepopulation under betingelser, som understøtter cellevækst; og (e) gentagelse af trinene b-d én eller flere gange for tilvejebringelse af en serielt ekspanderet population af ES-celler.
  13. 13. Fremgangsmåde ifølge krav 1, hvor vækstmediet omfatter en effektiv mængde af en Rho-associeret kinase (ROCK)-inhibitor, fortrinsvis hvor den Rho-associerede kinase (ROCK)-inhibitor er HA-100 eller H-1135.
DK08781187.3T 2007-06-29 2008-06-30 Automatiseret fremgangsmåde og apparatur til embryonisk stamcellekultur DK2173863T3 (da)

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PCT/US2008/068814 WO2009006422A1 (en) 2007-06-29 2008-06-30 Automated method and apparatus for embryonic stem cell culture

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EP (2) EP3293256B1 (da)
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KR (2) KR20160005142A (da)
AU (1) AU2008272949B2 (da)
CA (1) CA2691793A1 (da)
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