Biology Reference
In-Depth Information
attempts to culture PGCs themselves (and/or potentially progress them
toward gametogenesis) or to establish pluripotent EG cell lines from them.
Any modification that improves the survival or proliferation of PGCs in cul-
ture would be of potential benefit to both these ends. However, PGCs have
hitherto not been amenable to culture, and the limited success has largely
relied on the use of undefined components such as feeders and serum, as well
as the addition of further growth factors. (
de Felici & McLaren, 1983;
Donovan, 1994; Donovan & de Miguel, 2003
).
5.1. Requirement for feeders and serum
Although PGCs can survive and proliferate in serum-free medium (
Ffrench-
Constant, Hollingsworth, Heasman, &Wylie, 1991; Godin &Wylie, 1991
),
the use of serum is still routine. Irrespective of culture condition, PGCs do
not adhere readily to tissue culture plastic or gelatin (
de Felici & McLaren,
1983; Heath, 1978
), and although mouse and rat PGCs can survive for up to
a week in suspension, they exhibit only limited proliferation (
de Felici &
McLaren, 1983; Heath, 1978
). Robust proliferation of PGCs requires
adhesion to a feeder cell layer (
Dolci et al., 1991; Donovan, Stott,
Cairns, Heasman, &Wylie, 1986
). Various feeder cell lines have been shown
to support PGC proliferation including STO (
Donovan et al., 1986
), Sl4-
m220 (
Matsui et al., 1991
), and the Sertoli cell line TM
4
(
de Felici &
Dolci, 1991
). PGCs do adhere to a number of extracellular matrices includ-
ing fibronectin, laminin, collagen IV, and Matrigel (
de Felici & Dolci, 1989;
de Felici, Pesce, Giustiniani, & Di Carlo, 1998; Ffrench-Constant et al.,
1991; Garc´a-Castro et al., 1997
). However, cell fragmentation, consistent
with the occurrence of apoptosis, occurs within hours of culture on
fibronectin, laminin, and Matrigel (
de Felici et al., 1998
). Koshimizu
et al. reported limited cell survival of PGCs after 48 h of culture on
Matrigel (
Koshimizu et al., 1996
). As Matrigel is an undefined basement
membrane preparation extracted from Engelbreth-Holm-Swarm mouse
sarcoma cells, its use has little benefit over that of a feeder layer. In
the presence of serum and a cocktail of growth factors, PGCs do adhere
to and can survive for up to 5 days on a transwell membrane insert
(
Farini, Scaldaferri, Iona, La Sala, & de Felici, 2005
). However, there is
extensive growth of somatic cells in this system that may play a supportive
role during this period (
Farini et al., 2005
). Thus, the necessity of a feeder
layer remains a major obstacle in the development of fully defined culture
system for PGCs.
