title: Development of a modified vitrification strategy suitable for subsequent scale-up for hepatocyte preservation.
abstract: This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VS(basic) (40% (v/v) ethylene glycol 0.6M sucrose, i.e. 7.17 M ethylene glycol 0.6M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VS(basic) and increase the solution's total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5 days. Results indicated Ficoll as the least toxic additive. Within 60 min, the exposure of hepatocytes to a solution composed of 9% Ficoll+0.6M sucrose (10⁻³ M Ficoll+0.6 M sucrose) sustained attachment efficiency of 95%, similar to control. Furthermore, this additive did not cause any detriment to the attachment of these cells when supplementing the base vitrification solution VS(basic). The addition of 9% Ficoll, raised the total solute concentration to 74.06% (w/v) with a negligible 10⁻³ M increase in molarity of the solution. This suggests main factor in inducing detriment to cells was the molar contribution of the additive. Vitrification protocol for scale-up condition sustained hepatocyte suspension attachment efficiency and albumin production. We conclude that although established approach will permit scaling-up of vitrification of hepatocyte suspension, vitrification of hepatocytes which are attached prior to vitrification is more effective by comparison.
authors.affiliation: Low Temperature Preservation Unit, National University Medical Institutes, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Xianwei
authors.initials: X
authors.lastname: Wang
authors.forename: Raquel
authors.initials: R
authors.lastname: Magalhães
authors.forename: Yingnan
authors.initials: Y
authors.lastname: Wu
authors.forename: Feng
authors.initials: F
authors.lastname: Wen
authors.forename: Sok Siam
authors.initials: SS
authors.lastname: Gouk
authors.forename: Paul F
authors.initials: PF
authors.lastname: Watson
authors.forename: Hanry
authors.initials: H
authors.lastname: Yu
authors.forename: Lilia L
authors.initials: LL
authors.lastname: Kuleshova
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000378:metabolism; D019855:Ethylene Glycol / Q000378:metabolism; D005362:Ficoll / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology*; D010539:Permeability; D051381:Rats; D013395:Sucrose / Q000378:metabolism; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D005362:Ficoll; D013395:Sucrose; D019855:Ethylene Glycol