Cryopreservation of hepatocyte monolayers

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

    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.

    Conference

    ConferenceHepatocyte Users Group Meeting
    CountryUnited Kingdom
    CityAberdeen
    Period28/03/0329/03/03

    Fingerprint

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

    Keywords

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

    Cite this

    Stevenson, D. J., Morgan, C., Goldie, E. I., Connel, G., & Grant, M. H. (2003). Cryopreservation of hepatocyte monolayers. Paper presented at Hepatocyte Users Group Meeting, Aberdeen, United Kingdom.
    Stevenson, D.J. ; Morgan, C. ; Goldie, E.I. ; Connel, G. ; Grant, M.H. / Cryopreservation of hepatocyte monolayers. Paper presented at Hepatocyte Users Group Meeting, Aberdeen, United Kingdom.
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    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.",
    keywords = "cryopreservation, monolayer hepatocyte cultures, carboxyfluorescein diacetate, cytochrome P-450 dependent monooxygenase activity, glucuronidation",
    author = "D.J. Stevenson and C. Morgan and E.I. Goldie and G. Connel and M.H. Grant",
    year = "2003",
    language = "English",
    note = "Hepatocyte Users Group Meeting ; Conference date: 28-03-2003 Through 29-03-2003",

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    Stevenson, DJ, Morgan, C, Goldie, EI, Connel, G & Grant, MH 2003, 'Cryopreservation of hepatocyte monolayers' Paper presented at Hepatocyte Users Group Meeting, Aberdeen, United Kingdom, 28/03/03 - 29/03/03, .

    Cryopreservation of hepatocyte monolayers. / Stevenson, D.J.; Morgan, C.; Goldie, E.I.; Connel, G.; Grant, M.H.

    2003. Paper presented at Hepatocyte Users Group Meeting, Aberdeen, United Kingdom.

    Research output: Contribution to conferencePaper

    TY - CONF

    T1 - Cryopreservation of hepatocyte monolayers

    AU - Stevenson, D.J.

    AU - Morgan, C.

    AU - Goldie, E.I.

    AU - Connel, G.

    AU - Grant, M.H.

    PY - 2003

    Y1 - 2003

    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 - monolayer hepatocyte cultures

    KW - carboxyfluorescein diacetate

    KW - cytochrome P-450 dependent monooxygenase activity

    KW - glucuronidation

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

    UR - http://hug.ncl.ac.uk/

    M3 - Paper

    ER -

    Stevenson DJ, Morgan C, Goldie EI, Connel G, Grant MH. Cryopreservation of hepatocyte monolayers. 2003. Paper presented at Hepatocyte Users Group Meeting, Aberdeen, United Kingdom.