Cold storage and cryopreservation of tick cell lines

Background: Tick cell lines are now available from fifteen ixodid
and argasid species of medical and veterinary importance. However,
some tick cell lines can be difficult to cryopreserve, and improved
protocols for short- and long-term low temperature storage will
greatly enhance their use as tools in tick and tick-borne pathogen
research. In the present study, different protocols were evaluated
for cold storage and cryopreservation of tick cell lines derived
from Rhipicephalus (Boophilus) decoloratus, Rhipicephalus
(Boophilus) microplus, Ixodes ricinus and Ixodes scapularis. For
short-term cold storage, cells were kept under refrigeration at 6 C
for 15, 30 and 45 days. For cryopreservation in liquid nitrogen,
use of a sucrose-phosphate-glutamate freezing buffer (SPG) as
cryoprotectant was compared with dimethylsulfoxide (DMSO)
supplemented with sucrose. Cell viability was determined by the
trypan blue exclusion test and cell morphology was evaluated in
Giemsa-stained cytocentrifuge smears. Results: Cold storage at 6
degrees C for up to 30 days was successful in preserving R. (B.)
microplus, R. (B.) decoloratus, I. ricinus and I. scapularis cell
lines; lines from the latter three species could be easily
re-cultivated after 45 days under refrigeration. While cell lines
from all four tick species cryopreserved with 6% DMSO were
successfully resuscitated, the R. (B.) decoloratus cells did not
survive freezing in SPG and of the other three species, only the R.
(B.) microplus cells resumed growth during the observation period.
Conclusions: This constitutes the first report on successful
short-term refrigeration of cells derived from R. (B.) decoloratus,
R. (B.) microplus, and I. ricinus, and use of SPG as an alternative
to DMSO for cryopreservation, thus making an important contribution
to more reliable and convenient tick cell culture maintenance.