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 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 80 °C 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 3500. Characterizing the inhibitory substance and the pathway that leads to its formation may explain why most multicellular algae remain recalcitrant to cryopreservation, and lead to protocols that allow the cryopreservation of a broader range of organisms.