Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation

D.J. Stevenson, C. Morgan, E. Goldie, G. Connel, M.H. Grant

    Research output: Contribution to journalArticle

    24 Citations (Scopus)

    Abstract

    Little work in the literature focuses on the cryopreservation of primary hepatocytes as monolayer cultures, yet this technique offers many distinct advantages over other cryopreservation systems, including high recovery, high post-thaw nutrient penetration, and low numbers of trapped dead cells. This article investigates the cryopreservation of primary rat hepatocytes at −78 °C attached as monolayers to collagen coated culture dishes, and describes efforts to increase post-thaw viability and function through manipulation of the freeze/thaw protocol. Different concentrations of foetal calf serum (FCS) with 10% (v/v) dimethyl sulphoxide (ME2SO) were tested as cryopreservation media, and high cryoprotectant serum levels were found to be important in maintaining membrane integrity and function in the cryopreserved rat hepatocyte monolayer cultures. Cultures cryopreserved with 90% (v/v) FCS plus 10% (v/v) ME2SO maintain 79.7 ± 6.5% of the monolayer area as viable cells with normal morphology (by image analysis), 112.7 ± 14.2% protein concentration, 55.4 ± 4.2% carboxyfluorescein diacetate de-acetylation, 27.2 ± 7.5% kaempherol glucuronidation (a measure of UDP-glucuronosyl transferase activity), and 39.3 ± 7.3% testosterone hydroxylation (a measure of cytochrome P-450 activity) compared with non-cryopreserved controls. This method of cryopreservation may provide a simple, convenient means of long-term storage of hepatocytes for in vitro metabolism studies.
    LanguageEnglish
    Pages97-113
    Number of pages16
    JournalCryobiology
    Volume49
    Issue number2
    DOIs
    Publication statusPublished - 2004

    Fingerprint

    Cryopreservation
    cryoprotectants
    blood serum
    Metabolism
    cryopreservation
    hepatocytes
    testosterone
    Testosterone
    Hepatocytes
    Monolayers
    metabolism
    Serum
    fetal bovine serum
    Rats
    Acetylation
    Hydroxylation
    Uridine Diphosphate
    Transferases
    Dimethyl Sulfoxide
    Culture Techniques

    Keywords

    • cryopreservation
    • monolayer hepatocyte cultures
    • carboxyfluorescein diacetate
    • cytochrome
    • glucuronidation
    • bioengineering

    Cite this

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    title = "Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation",
    abstract = "Little work in the literature focuses on the cryopreservation of primary hepatocytes as monolayer cultures, yet this technique offers many distinct advantages over other cryopreservation systems, including high recovery, high post-thaw nutrient penetration, and low numbers of trapped dead cells. This article investigates the cryopreservation of primary rat hepatocytes at −78 °C attached as monolayers to collagen coated culture dishes, and describes efforts to increase post-thaw viability and function through manipulation of the freeze/thaw protocol. Different concentrations of foetal calf serum (FCS) with 10{\%} (v/v) dimethyl sulphoxide (ME2SO) were tested as cryopreservation media, and high cryoprotectant serum levels were found to be important in maintaining membrane integrity and function in the cryopreserved rat hepatocyte monolayer cultures. Cultures cryopreserved with 90{\%} (v/v) FCS plus 10{\%} (v/v) ME2SO maintain 79.7 ± 6.5{\%} of the monolayer area as viable cells with normal morphology (by image analysis), 112.7 ± 14.2{\%} protein concentration, 55.4 ± 4.2{\%} carboxyfluorescein diacetate de-acetylation, 27.2 ± 7.5{\%} kaempherol glucuronidation (a measure of UDP-glucuronosyl transferase activity), and 39.3 ± 7.3{\%} testosterone hydroxylation (a measure of cytochrome P-450 activity) compared with non-cryopreserved controls. This method of cryopreservation may provide a simple, convenient means of long-term storage of hepatocytes for in vitro metabolism studies.",
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    Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation. / Stevenson, D.J.; Morgan, C.; Goldie, E.; Connel, G.; Grant, M.H.

    In: Cryobiology, Vol. 49, No. 2, 2004, p. 97-113.

    Research output: Contribution to journalArticle

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    T1 - Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation

    AU - Stevenson, D.J.

    AU - Morgan, C.

    AU - Goldie, E.

    AU - Connel, G.

    AU - Grant, M.H.

    PY - 2004

    Y1 - 2004

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    KW - cryopreservation

    KW - monolayer hepatocyte cultures

    KW - carboxyfluorescein diacetate

    KW - cytochrome

    KW - glucuronidation

    KW - bioengineering

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    U2 - 10.1016/j.cryobiol.2004.05.006

    DO - 10.1016/j.cryobiol.2004.05.006

    M3 - Article

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    SP - 97

    EP - 113

    JO - Cryobiology

    T2 - Cryobiology

    JF - Cryobiology

    SN - 0011-2240

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    ER -