Cryopreservation of Chlamydomonas reinhardtii (Chlorophyta)
A L E X A N D RA L. M. C R U T C H F I E L D1, K E N N E T H R. D I L L ER2 A N D J E R R Y J. B R A N D1
" Department of Botany, The University of Texas at Austin, Austin, Texas 78713–7640, USA # Biomedical Engineering Program, The University of Texas atAustin, Austin, Texas 78712–1084, USA (Received 5 June 1998 ; accepted 23 October 1998) Cryopreserved Chlamydomonas reinhardtii cultures remained viable when frozen by cooling slowly to k55 mC, then plunging into liquid nitrogen for at least 1 day of storage. High viability ( 40 %) was retained when cultures contained 2–10 % (v\v) methanol as a cryoprotective agent prior to freezing, while dimethylsulphoxide was ineﬀective. However, methanol was lethal to cells in the presence of light. Frozen cultures became non-viable within 24 h when stored at k80 mC, whereas those stored below k130 mC remained viable for at least several months. Highest viability was attained in cultures that were frozen and stored at a low cell density. High viability also required that frozen cultures be warmed rapidlyand the cryoprotective agent removed immediately thereafter in preparation for culturing in liquid or on solid medium. Individual cell viability was determined by measurements of colony counts after cell plating and by the penetration of Evans blue dye into non-viable cells. Viability in bulk culture was conveniently measured by comparing the rates of photosynthetic oxygen evolution, corrected fordark respiration, in previously frozen cultures with the corresponding rates in unfrozen controls. Cultures that had lost viability as measured by a decline in whole-cell photosynthetic oxygen evolution after freezing and thawing retained functional Photosystem II activity for some time thereafter, indicating that the loss in photosynthetic activity was due to some process other thanphotoinhibition of Photosystem II. Key words : algal cryopreservation, Chlamydomonas reinhardtii, freezing damage, methanol cryoprotection, photosynthetic oxygen evolution
Introduction Cryopreservation, the storage of living cells or organisms at very low (typically liquid nitrogen (LN )) temperature, # is an eﬀective method for preserving valuable genetic stock indeﬁnitely. However, most cells are killedduring freezing or thawing unless special precautions are taken. Especially important considerations for cryopreservation are the addition of a cryoprotective agent (CPA) prior to freezing and the rates at which cells are cooled and warmed. Dimethyl sulphoxide (Me SO) is the most frequently # used CPA for cryopreserving algal species (Beaty & Parker, 1992 ; Daggett & Nerad, 1992 ; Kuwano et al.,1994), although methanol (MeOH) is also reported to be an eﬀective CPA for some algae (Morris & Canning, 1978 ; Morris et al., 1979 ; Canavate & Lubian, 1994). Glycerol 4 has been used successfully as a CPA for animal cells and a few eukaryotic algal cells (Hwang & Horneland, 1965), but Morris (1976) found glycerol to be toxic to Chlorella. Preliminary work in this laboratory demonstrated that anyconcentration of glycerol over 3 % (w\v) was toxic to Chlamydomonas UTEX F89. Most successful cryopreservation procedures utilize a step-wise cooling process such as cooling the culture at a
Correspondence to : J. J. Brand. Fax :j1 (512) 471 3878. e-mail : jbrand!utxvms.cc.utexas.edu.
relatively slow and constant rate (0.2–1.0 mC min−") to some sub-zero temperature (typically somewhere betweenk30 mC and k75 mC), then plunging it into LN (Beaty & # Parker, 1992 ; Day & Fenwick, 1993 ; Bodas et al., 1995). Frozen cultures are usually thawed very rapidly (Canavate 4 & Lubian, 1997) in order to prevent recrystallization of intracellular ice and to avoid excessive cell dehydration during the thawing process. Limited success has been reported in cryopreserving Chlamydomonas reinhardtii...