In vitro cultivation and cryopreservation of duck embryonic hepatocytes
Introduction
Hepatitis B virus (HBV) can be inactivated effectively by chemical disinfection procedures. However, Hepatitis B-virucidal efficacy has to be proven by the use of robust laboratory methods. With exception of Tupaia hepatocytes (Glebe et al., 2003), a convenient cell culture system for sensitive and practical in vitro detection of HBV infectivity is not available; therefore, an alternative procedure has to be adopted. Thus, the duck hepatitis B virus (DHBV), which is from the same virus family hepadnaviridae as HBV, has been recommended as the most promising and feasible agent for this purpose as DHBV can be propagated in primary duck embryonic hepatocytes (Marion et al., 1987, Tuttleman et al., 1986, Wang et al., 2002a). However, virucidal testing using the DHBV/duck hepatocyte system cannot be carried out easily since the virus obtained from serum of infected ducks is non-cytopathogenic, DHBV-free hepatocytes are required, and the preparation and cultivation of hepatocytes is complex.
Indeed, the critical points of hepatocyte cultivation include the attachment of prepared hepatocytes onto the surface of culture flasks or plates and maintaining survival of these cells in their differentiated stages. The successful attachment of hepatocytes depends mainly on the quality of the surface of cell culture vessels which can be improved by coating the surface with collagen 1 (Caron, 1990). The maintenance of hepatocyte differentiation seems to be essential for the susceptibility of cells to DHBV and the viral propagation (Galle et al., 1989, Pugh and Summers, 1989, Schorr et al., 2006, Tuttleman et al., 1986). This is a function of the intact microenvironment that can be produced by coating the growth surface with Matrigel™ (Becton Dickinson, Heidelberg, Germany) or molecules of the extracellular matrix (Hoshiba et al., 2007) and alternatively by the co-cultivation of hepatocytes with other cell types (Fourel et al., 1986, Kuri-Harcuch and Mendoza-Figueroa, 1989, Loréal et al., 1993, Morin and Norman, 1986). The dedifferentiation of hepatocytes may be associated with the change of the typical polygonal morphology into fibroblast-like morphology.
Sufficient cryopreservation would improve considerably the availability of primary duck embryonic hepatocytes since commercial eggs from Peking ducks are usually not continuously available throughout the year. There are many reports in the literature regarding cryopreservation of hepatocytes from different species like mouse, monkey, dog, rat, and also human (Desai and Hawksworth, 1990, Gomez-Lechon et al., 2006, Hengstler et al., 2000, Sun et al., 1990). By contrast, there is only one reference in relation to cryopreservation of duck hepatocytes (Wang et al., 2002b). Since hepatocytes are highly susceptible to freezing and thawing, the optimal conditions for cryoprotection are currently being reviewed. The most critical parameters seem to be the choice of cryoprotectant, the composition of freezing medium and the cooling/thawing rates. Hepatocytes have been reported to be cryopreserved as suspensions (Chesné et al., 1993, Gomez-Lechon et al., 2006, Innes et al., 1988), in spheroids (Aoki et al., 2005, Darr and Hubel, 2001, Guyomard et al., 1996, Hubel et al., 2000–2001), on microcarriers (Naik et al., 1997), in collagen sandwich configuration (Hubel et al., 1991, Koebe et al., 1990), and on collagen monolayers (Stevenson et al., 2004). Furthermore, many freezing media were tested in combination with dimethyl sulfoxide (ME2SO) or other cryoprotective agents including wheat extracts (Hamel et al., 2006), polyvinylpyrrolidone (Gomez-Lechon et al., 2006), arabinogalactin (Darr and Hubel, 2001), trehalose (Katenz et al., 2007) or other long-chain oligosaccharides (Miyamoto et al., 2006) to improve cryopreservation of the primary hepatocytes.
The objective of this current study was to improve the cultivation of primary duck embryonic hepatocytes for the use of DHBV propagation in virucidal experiments. Commercial culture plates with different growth surfaces were tested for the attachment of cells, the maintenance of hepatocyte differentiation, and the influence on DHBV infection. In addition, a practical and robust method for cryopreservation of duck primary hepatocytes in suspension was developed.
Section snippets
Cell preparation and DHBV infection
Primary duck embryonic hepatocytes were prepared from liver tissue as described previously (Sauerbrei et al., 2005, Sauerbrei et al., 2006). Briefly, fertilized Peking duck eggs obtained from the poultry farm Pötzsch (Ostrau, Germany) were incubated at 37 °C for 21 days. The liver tissue was harvested from 6 to 7 embryos under sterile conditions, pooled, minced and washed subsequently with phosphate-buffered saline (PBS) supplemented with 1 mg/ml glucose. The first wash fluids containing blood
Suitability of cell culture plates with different growth surfaces
The results of hepatocyte cultivation in culture plates containing different surfaces are demonstrated in Fig. 1. During the first 2 days after seeding cells, higher portions of hepatocytes were detectable in CELLCOAT plates (86.3 ± 8.9%) in comparison with the uncoated plates used (52.5 ± 3.5–76.3 ± 9.0%). On day 2 of cultivation, there were significant differences between CELLCOAT and CellBIND plates on the one hand and PRIMARIA, NUNCLON and TPP plates on the other one. However, after 5 days a
Discussion
An essential precondition for the cultivation of hepatocytes is their optimal preparation from liver tissue. This paper describes a method providing about 95% living cells. This method has the advantage that the preparation of hepatocytes is accompanied by PCR detection of DHBV since exclusively DHBV-free hepatocytes are usable for infectivity experiments. In a comparable study, 98% viability of cells has been reported (Wang et al., 2002b). Various preparation methods for adult hepatocytes of
Conclusions
In DHBV infectivity experiments for virucidal testing of biocides, cultivation of primary duck embryonic hepatocytes can be carried out using plates without collagen 1 such as CellBIND if cells are seeded with optimal density. Cryopreserved primary duck embryonic hepatocytes can be used as alternative when freshly isolated cells from the liver of duck embryos are not available. Growth medium supplemented with 10% FCS and 10% ME2SO, FCS supplemented with 10% ME2SO or cryosafe-1 are suitable
Acknowledgement
This work was supported by grants of the Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten e.V., Germany.
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