Tissue collection, transport and isolation procedures required to optimize human hepatocyte isolation from waste liver surgical resections. A multilaboratory study.
BACKGROUND
The European Center for Validation of Alternative Methods (ECVAM) has funded a prevalidation study in three laboratories (France, USA and UK) on the use of human hepatocyte cultures to predict cytochrome P-450 induction.


OBJECTIVE
As first stage of this prevalidation study, the purpose of the present work was to set criteria for optimization and harmonization of hepatocyte isolation from human tissue among laboratories to establish a routine procedure. This was achieved by combining and/or comparing the data generated by the two independent European laboratories (France and UK).


RESULTS
The results confirmed that surgical waste material is a valuable source for obtaining high quality hepatocytes under certain pre-, intra- and post-operative conditions: cell yield of viable hepatocytes was not significantly affected by age and sex of patients, nor indications for resection, steatosis or cholestasis. Cold ischeamia up to 5 hours did not influence viable cell yield allowing transport of material.


CONCLUSIONS
The use of biopsy sizes between 50-100 g, cannulation with 2-4 cannulae, digestion with collagenase-containing digestion medium at a flow rate of 25 ml/cannula for 20 minutes, with cut surface being glued in order to reform Glisson's capsule, should optimize the total yield of viable human hepatocytes obtained per preparation of waste liver surgical resections.
Laboratoire de Biologie Cellulaire, Faculté de Médecine et de Pharmacie, Besançon, France. lysiane.richert@univ-fcomte.fr
Lysiane
L
Richert
Eliane
E
Alexandre
Tom
T
Lloyd
Samantha
S
Orr
Catherine
C
Viollon-Abadie
Rakhee
R
Patel
Shaun
S
Kingston
David
D
Berry
Ashley
A
Dennison
Bruno
B
Heyd
Georges
G
Mantion
Daniel
D
Jaeck
D000328:Adult; D001706:Biopsy; D002404:Catheterization; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D017364:Collagenases; D005060:Europe; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D008099:Liver / Q000166:cytology* / Q000601:surgery; D013525:Surgical Instruments; D020858:Tissue and Organ Harvesting / Q000379:methods*; D014186:Transportation
D017364:Collagenases