Biomedical Engineering Reference
In-Depth Information
We subsequently adapted the baboon, human and rodent protocol for cryopreservation
of rhesus testis cells at high concentrations (20 × 10 6 /ml) in medium containing 10%
DMSO by freezing 1-2 ml aliquots in isopropanol controlled-rate freezing devices
[−1°C/min, Nalgene-Nunc International; (Hermann et al. 2009, 2007 )]. Larger cryovials
(5 ml) can also be used when frozen using electronic controlled-rate freezing devices
(e.g., CryoMed, ThermoFisher). Importantly, these methods result in high recovery
and viability of cells that retain normal phenotypic and functional characteristics
[Table 11.1 ; (Hermann et al. 2007, 2009 )].
After storage in liquid nitrogen, cryopreserved cells are thawed rapidly at
37°C and the cell suspension is diluted ~5- to 10-fold by the drop-wise addition
of excess medium (MEMa + 10% FBS). Dilution with medium is performed in
a drop-wise fashion to reduce osmotic damage due to addition of the relatively
hypotonic medium. Cells are then washed several times in medium to eliminate
DMSO prior to experimentation. Viability of cryopreserved testis cells after
thawing varies with the developmental stage from which they were isolated
(Table 11.1 ), but is generally good. In our hands, testis cells from prepubertal
animals typically survive a freeze-thaw cycle better than those from adult testes
(Table 11.1 ), likely due to the absence of relatively fragile differentiating germ
cells. Rhesus-to-nude mouse xenotransplantation was used to assess the coloni-
zation potential of cryopreserved/thawed rhesus testis cells compared to freshly
isolated cells (Hermann et al. 2007 ) and no statistically significant difference
was observed following cryopreserved of rhesus testis cells. Furthermore, cryo-
preserved rhesus testis cells exhibited similar phenotypic profiles for the cell
surface marker THY-1 (CD90) before and after freezing based on FACS analysis
(Hermann et al. 2009 ). Thus, rhesus SSCs appear to retain normal phenotypic
and functional attributes after cryogenic storage. A similar approach was used to
ship and store baboon testis cells prior to xenotransplantation, but recovery,
viability, phenotype, and function of frozen-thawed testis cells were not reported
(Nagano et al. 2001b ).
While the data obtained to date suggest that surviving nonhuman primate SSCs
retain normal functional attributes following cryopreservation (using standard 10%
DMSO as a cryoprotectant and controlled-rate cooling), not all cells survive the
freeze-thaw process, and thus, some of the starting regenerative pool is lost (see
recovery and viability results, Table 11.1 ). To address this potential drawback of
freezing cells, additional experiments are ongoing in rodents, monkeys, and humans
to evaluate alternative cryoprotectants and additives. For example, inclusion of
long-chain oligosaccharides or trehalose (a disaccharide of glucose), in addition to
DMSO, results in improved cell recovery after cryopreservation of various cell
types (Buchanan et al. 2004 ; Miyamoto et al. 2006 ; Katenz et al. 2007 ). Trehalose
is a non-permeant cryoprotectant that is thought to safeguard lipid membranes from
freeze-thaw damage and protect labile cell-surface proteins, although the most
pronounced effects of trehalose are obtained with intracellular loading (Katenz
et al. 2007 ). Thus, there is potential for increased recovery and viability of frozen-
thawed primate testis cells with optimization of the cryopreservation medium
(e.g., addition of Trehalose).
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