Cryopreservation of hepatocytes: the monolayer approach

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

    Research output: Contribution to conferencePaper

    Abstract

    The ability to cryopreserve hepatocytes would be useful both to the pharmaceutical industry and for bioartificial liver support systems. Unfortunately, suspension cryopreservation protocols typically result in low attachment efficiencies of cells upon thawing. To circumvent this problem, we have frozen rat hepatocytes as monolayers on collagen substrates, and attempted to optimise this cryopreservation protocol. A variety of parameters were measured in non-frozen and post-thaw frozen monolayer cultures, including viability, total protein and intracellular reduced glutathione (GSH) concentration, kaempherol glucuronidation, and testosterone hydroxylation. The effect of altering cryopreservation media composition (% of foetal calf serum varying between 0-90%) or freezing (0.4-3.8ºC/min) and thawing rates (26-128ºC/min) on these parameters was investigated. Under optimal conditions, post thaw cryopreserved cells maintained 72±4% viability, 65±4% total protein, 46±8% GSH, 48±8% kaempherol glucuronidation, and 16±11% testosterone hydroxylation of their corresponding non-frozen controls (mean ±SEM, n=3). Cryopreservation of hepatocyte monolayers as opposed to suspensions results in a more representative population of cells, with high viability, function, and recovery rates.
    Original languageEnglish
    Publication statusPublished - 2002
    EventRSC-DMG 2002: New Technologies in Drug Discovery - Edinburgh, Scotland
    Duration: 12 Dec 200213 Dec 2002

    Conference

    ConferenceRSC-DMG 2002: New Technologies in Drug Discovery
    CityEdinburgh, Scotland
    Period12/12/0213/12/02

    Fingerprint

    Cryopreservation
    Hepatocytes
    Hydroxylation
    Testosterone
    Suspensions
    Artificial Liver
    Recovery of Function
    Drug Industry
    Freezing
    Glutathione
    Proteins
    Collagen
    Serum
    Population

    Keywords

    • cryopreservation
    • hepatocytes
    • monolayer approach
    • biomaterials
    • monolayer hepatocyte cultures
    • carboxyfluorescein diacetate
    • cytochrome P-450 dependent monooxygenase activity
    • glucuronidation

    Cite this

    Stevenson, D. J., Grant, M. H., Goldie, E. I., Connel, G., & Morgan, C. (2002). Cryopreservation of hepatocytes: the monolayer approach. Paper presented at RSC-DMG 2002: New Technologies in Drug Discovery, Edinburgh, Scotland, .
    Stevenson, D.J. ; Grant, M.H. ; Goldie, E.I. ; Connel, G. ; Morgan, C. / Cryopreservation of hepatocytes: the monolayer approach. Paper presented at RSC-DMG 2002: New Technologies in Drug Discovery, Edinburgh, Scotland, .
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    title = "Cryopreservation of hepatocytes: the monolayer approach",
    abstract = "The ability to cryopreserve hepatocytes would be useful both to the pharmaceutical industry and for bioartificial liver support systems. Unfortunately, suspension cryopreservation protocols typically result in low attachment efficiencies of cells upon thawing. To circumvent this problem, we have frozen rat hepatocytes as monolayers on collagen substrates, and attempted to optimise this cryopreservation protocol. A variety of parameters were measured in non-frozen and post-thaw frozen monolayer cultures, including viability, total protein and intracellular reduced glutathione (GSH) concentration, kaempherol glucuronidation, and testosterone hydroxylation. The effect of altering cryopreservation media composition ({\%} of foetal calf serum varying between 0-90{\%}) or freezing (0.4-3.8ºC/min) and thawing rates (26-128ºC/min) on these parameters was investigated. Under optimal conditions, post thaw cryopreserved cells maintained 72±4{\%} viability, 65±4{\%} total protein, 46±8{\%} GSH, 48±8{\%} kaempherol glucuronidation, and 16±11{\%} testosterone hydroxylation of their corresponding non-frozen controls (mean ±SEM, n=3). Cryopreservation of hepatocyte monolayers as opposed to suspensions results in a more representative population of cells, with high viability, function, and recovery rates.",
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    author = "D.J. Stevenson and M.H. Grant and E.I. Goldie and G. Connel and C. Morgan",
    year = "2002",
    language = "English",
    note = "RSC-DMG 2002: New Technologies in Drug Discovery ; Conference date: 12-12-2002 Through 13-12-2002",

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    Stevenson, DJ, Grant, MH, Goldie, EI, Connel, G & Morgan, C 2002, 'Cryopreservation of hepatocytes: the monolayer approach' Paper presented at RSC-DMG 2002: New Technologies in Drug Discovery, Edinburgh, Scotland, 12/12/02 - 13/12/02, .

    Cryopreservation of hepatocytes: the monolayer approach. / Stevenson, D.J.; Grant, M.H.; Goldie, E.I.; Connel, G.; Morgan, C.

    2002. Paper presented at RSC-DMG 2002: New Technologies in Drug Discovery, Edinburgh, Scotland, .

    Research output: Contribution to conferencePaper

    TY - CONF

    T1 - Cryopreservation of hepatocytes: the monolayer approach

    AU - Stevenson, D.J.

    AU - Grant, M.H.

    AU - Goldie, E.I.

    AU - Connel, G.

    AU - Morgan, C.

    PY - 2002

    Y1 - 2002

    N2 - The ability to cryopreserve hepatocytes would be useful both to the pharmaceutical industry and for bioartificial liver support systems. Unfortunately, suspension cryopreservation protocols typically result in low attachment efficiencies of cells upon thawing. To circumvent this problem, we have frozen rat hepatocytes as monolayers on collagen substrates, and attempted to optimise this cryopreservation protocol. A variety of parameters were measured in non-frozen and post-thaw frozen monolayer cultures, including viability, total protein and intracellular reduced glutathione (GSH) concentration, kaempherol glucuronidation, and testosterone hydroxylation. The effect of altering cryopreservation media composition (% of foetal calf serum varying between 0-90%) or freezing (0.4-3.8ºC/min) and thawing rates (26-128ºC/min) on these parameters was investigated. Under optimal conditions, post thaw cryopreserved cells maintained 72±4% viability, 65±4% total protein, 46±8% GSH, 48±8% kaempherol glucuronidation, and 16±11% testosterone hydroxylation of their corresponding non-frozen controls (mean ±SEM, n=3). Cryopreservation of hepatocyte monolayers as opposed to suspensions results in a more representative population of cells, with high viability, function, and recovery rates.

    AB - The ability to cryopreserve hepatocytes would be useful both to the pharmaceutical industry and for bioartificial liver support systems. Unfortunately, suspension cryopreservation protocols typically result in low attachment efficiencies of cells upon thawing. To circumvent this problem, we have frozen rat hepatocytes as monolayers on collagen substrates, and attempted to optimise this cryopreservation protocol. A variety of parameters were measured in non-frozen and post-thaw frozen monolayer cultures, including viability, total protein and intracellular reduced glutathione (GSH) concentration, kaempherol glucuronidation, and testosterone hydroxylation. The effect of altering cryopreservation media composition (% of foetal calf serum varying between 0-90%) or freezing (0.4-3.8ºC/min) and thawing rates (26-128ºC/min) on these parameters was investigated. Under optimal conditions, post thaw cryopreserved cells maintained 72±4% viability, 65±4% total protein, 46±8% GSH, 48±8% kaempherol glucuronidation, and 16±11% testosterone hydroxylation of their corresponding non-frozen controls (mean ±SEM, n=3). Cryopreservation of hepatocyte monolayers as opposed to suspensions results in a more representative population of cells, with high viability, function, and recovery rates.

    KW - cryopreservation

    KW - hepatocytes

    KW - monolayer approach

    KW - biomaterials

    KW - monolayer hepatocyte cultures

    KW - carboxyfluorescein diacetate

    KW - cytochrome P-450 dependent monooxygenase activity

    KW - glucuronidation

    UR - http://dmg.nott.ac.uk/dmg/1202/1202.pdf

    UR - http://dx.doi.org/10.1016/j.cryobiol.2004.05.006

    M3 - Paper

    ER -

    Stevenson DJ, Grant MH, Goldie EI, Connel G, Morgan C. Cryopreservation of hepatocytes: the monolayer approach. 2002. Paper presented at RSC-DMG 2002: New Technologies in Drug Discovery, Edinburgh, Scotland, .