Translating Spermatogonial Stem Cell Transplantation to the Clinic - Male Germline Stem Cells Developmental and Regenerative Potential - page 234

Table 11.1 Yields and viability of fresh and cryopreserved/thawed rhesus macaque testis cells
Fresh cells a Cryopreserved cells b

Developmental
Stage

Duration
(mean days)
Neonate rhesus 754 ± 175 92.1 ± 0.9 Nd. Nd. Nd.
Juvenile rhesus 468 ± 42 96.8 ± 0.3 75.8 ± 3.7 80.8 ± 0.9 176 ± 33
Adult rhesus 189 ± 16 94.8 ± 0.6 71.7 ± 5.0 66.2 ± 2.8 228 ± 29
a Adult and juvenile testis cells were prepared from 26 and 25 individual animals, respectively.
Adult testis cell data are means of the four cell isolations reported in (Hermann et al . 2007 ),
together with 22 isolations performed subsequently. Juvenile testis cell yields and recovery are as
reported (Hermann et al. 2009 ). Neonatal rhesus testis cells were prepared from eight males (aver-
age 2.75 days postnatal)
b Recovery and viability of cryopreserved juvenile testis cells was performed in 37 individual
thaws from a total of 20 animals. From adults, data are from 29 thaws from 13 individual
animals

Cells/g
testis (×10 6 ) Viability (%) Recovery (%) Viability (%)

then digested with four enzymes to generate a single-cell suspension: collagenase
A (1.5 mg/ml, Roche), hyaluronidase type V (1.5 mg/ml, Sigma), trypsin (0.5 mg/ml,
Worthington) and DNase (10 U/ml. Invitrogen) at 37°C for 20 min with mild
agitation. Undigested tissue and cell clumps are removed by straining and isolated
cells are suspended in DMEM + 10% FBS. Cell yields using these approaches were
not reported, but a lower concentration of trypsin [fourfold; (Maki et al. 2009 )] or its
absence altogether (Schlatt et al. 2002a ) may be beneficial for characterizing cell
surface markers that are sensitive to trypsin cleavage (e.g., cKIT).

11.3.3 Cryopreservation

Autologous transplantation of SSCs in primates to study the regenerative capacity
of SSCs in infertile recipients (and eventual clinical application) requires storage of
cells between the times of collection and transplant. Several protocols have been
reported to cryopreserve primate testicular cells (Nagano et al. 2001b, 2002 ; Schlatt
et al. 2002a ; Hermann et al. 2007, 2009 ). Initial reports using baboon and human
testis cell suspensions employed methods developed for cryopreservation of rodent
SSCs (Avarbock et al. 1996 ). With this approach, 10% DMSO is used as the cryo-
protectant and freezing is performed in a controlled-rate freezing device. DMSO
penetrates cell membranes and acts as a cryoprotectant by preventing cellular dehy-
dration and formation of intracellular ice crystals (Lovelock and Bishop 1959 ).
Cells frozen with this approach and subsequently thawed were competent to colo-
nize mouse testes in xenotransplantation studies, however, cell recovery, viability,
and comparative phenotypic and functional attributes were not reported (Nagano
et al. 2001b, 2002 ).
The first protocol for rhesus monkey testis cell cryopreservation utilized 1.5 M
glycerol as a cell-permeant cryoprotectant in the presence of 4% autologous monkey
serum and freezing using a controlled-rate freezing machine (Schlatt et al. 2002a ).