Biomedical Engineering Reference
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characterize stem cell activity in any donor testis cell population. In order to translate
SSC transplantation to assess the fundamental biology and therapeutic application
of primate SSCs, it is necessary to optimize methods for primate testis cell isola-
tion, cryopreservation, and transplantation.
11.3.2
Primate Testis Cell Isolation
Studies of the fundamental biology and regenerative capacity of primate SSCs
require isolated suspensions of cells from primate testes. Several protocols have
been developed using enzymes to digest primate testicular parenchyma to a
single cell suspension (Nagano et al.
2001b, 2002
; Schlatt et al.
2002a
;
Hermann et al.
2007, 2009
; Muller et al.
2008
; Maki et al.
2009
). In general, a
two-step enzymatic digestion procedure is employed to liberate cells, although
an alternative protocol employs mechanical disruption and a single, extended
incubation with collagenase IV to generate a suspension of marmoset testis
cells (Muller et al.
2008
).
Nagano and colleagues were first to report a method for generating a single cell
suspension of nonhuman primate testis cells. In their protocol, fragments of
baboon seminiferous tubules were generated by digestion with collagenase II
(1 mg/ml, Worthington) at 33°C for 15-20 min, followed by digestion with trypsin
(0.5 mg/ml, Sigma) at 33°C for 15-20 min and addition of DNase I (1 mg/ml,
Sigma) with vigorous trituration to disrupt cell clumps and straining through a
nylon mesh to produce a single-cell suspension (Nagano et al.
2001b
). The resulting
testis cell suspension was resuspended in DMEM and provided cells suitable for
xenotransplantation. This protocol was later modified to produce a single-cell
suspension of rhesus macaque testicular cells (Hermann et al.
2007, 2009
), including
a first step digestion with collagenase type IV (1 mg/ml, Sigma), a second-step
digestion with trypsin (2 mg/ml) containing EDTA (1.04 mM) (Invitrogen) plus
DNase I (1.4 mg/ml, Sigma) and resuspension in minimum essential medium
alpha containing 10% FBS. Yields of rhesus testis cells using this protocol are
highly reproducible and result in suspensions of cells with high viability
[Table
11.1
; (Hermann et al.
2007, 2009
)]. Differences in cell yield between devel-
opmental stages are primarily attributed to the progressive increase in difficulty of
enzymatic digestion during postnatal testis development accompanying formation
of a basement membrane, increase in seminiferous cord-tubule diameter, and
increasing tissue complexity.
Different iterations of a two-step enzymatic digestion protocol have also been
reported for generation of cell suspensions from testes of rhesus macaques (Schlatt
et al.
2002a
; Maki et al.
2009
). Schlatt and colleagues generated rhesus testis cells by
digesting cultured testicular tissue fragments with collagenase I (Sigma), followed
digestion with hyaluronidase (Sigma) and DNase I (Sigma). In the protocol estab-
lished by Maki and colleagues, seminiferous tubules were finely minced and digested
with collagenase A (1 mg/ml, Roche) plus DNase (10 U/ml, Invitrogen) at 37°C for
15 min plus agitation (Maki et al.
2009
). The undigested seminiferous tubules are
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