PIASECKI, BRIAN1*, YANG FAN1, KENNETH R. DILLER2, and JERRY J. BRAND1. 1Molecular Cell and Developmental Biology, University of Texas at Austin, Austin, TX 78712, U.S.A.; 2Department of Biomedical Engineering, University of Texas at Austin, Austin, TX 78712, U.S.A.. - Survival of Chlamydomonas subsequent to cryopreservation is prevented by a substance released from damaged cells.
Storage of algae in liquid nitrogen offers a convenient way to
permanently preserve many cultures with minimum genetic drift. We have
successfully cryopreserved over 1400 strains in the Culture Collection
of Algae at the University of Texas (UTEX), but over one-fourth of
UTEX cultures remain recalcitrant. Our previous studies demonstrate
that unicellular algae frozen at a high cell density (> 2,000,000
cells/ml) do not survive cryopreservation well. We now show that
viability during cryopreservation of Chlamydomonas reinhardtii
UTEX #89 at high cell densities is not affected by the physical
proximity of cells. Instead, when cells are damaged, a small organic
molecule(s) is produced and released into the medium preventing
cryopreservation of other cells in the culture. Healthy C.
reinhardtii cultures were treated with extracts prepared from
damaged cells or culture medium separated from damaged cells, then
cryopreserved by a standard method (Crutchfield, et al. 1999. Eur. J.
Phycol. 34: 43-52). Viability was measured after thawing by the
ability of a cell wall to preclude Evan's blue dye, and was confirmed
by quantitative agar plating. The inhibitory substance from these
fractions appears to affect living cells only during cryopreservation,
but can be produced from cells by a variety of treatments. The
inhibitor is heat-stable, but heating cells to 80C precludes its
production. The substance is stable when separated from cellular
particulate matter, but gradually becomes inactive in the presence of
cell membrane fractions. The inhibitor is a water-soluble and
heat-stable organic compound(s), with a molecular mass less than or
equal to 3,500. Characterizing the inhibitory substance and the
pathway that leads to its formation may explain why most
multi-cellular algae remain recalcitrant to cryopreservation, and lead
to protocols that allow the cryopreservation of a broader range of
organisms.
Key words: algal cryopreservation, Chlamydomonas, freezing damage