TITLE: Cryopreservation of Microalgae
AUTHORS: Lynn  Doran

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Cryopreservation protocol for the microalgae Ostreococcus tauri for long term storage.</div><div class = "text-block">Cell viability of O. tauri at -196 for x months = unknown</div><div class = "text-block">Regeneration growth rate of O. tauri at -196 for x months = unknown</div><div class = "text-block"><span style = "font-weight:bold;">Additional Information</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Abreu, Ligia & Borges, Lucélia & Marangoni, Juliano & Abreu, Paulo. (2012). Cryopreservation of some useful microalgae species for biotechnological exploitation. Journal of Applied Phycology. 24. 10.1007/s10811-012-9818-0. </li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Ben-Amotz, A. and A. Gilboa, Cryopreservation of Marine Unicellular Algae. I. A Survey of Algae with Regard to Size, Culture Age, Photosynthetic Activity and Chlorophyll-toCellRatio, MARINE ECOLOGY - PROGRESS SERIES, 2: 157-161. 1980.  </span><a href="http://www.int-res.com/articles/meps/2/m002p157.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">http://www.intres.com/articles/meps/2/m002p157.pdf</span></a><span></span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Buhmann, MatthiasT. ,John G. Day, Peter G. Kroth, Post-cryopreservation viability of the benthic freshwater diatomPlanothidiumfrequentissimumdepends on light levels, Cryobiology, Volume 67, Issue 1, 2013,Pages 23-29, ISSN 0011-2240, </span><a href="https://doi.org/10.1016/j.cryobiol.2013.04.005" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://doi.org/10.1016/j.cryobiol.2013.04.005</span></a><span style = ":;:UNDERLINE;"> </span><span>(https://www.sciencedirect.com/science/article/pii/S0011224013001235)</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Bui TVL, Ross IL, Jakob G, Hankamer B (2013) Impact of Procedural Steps and Cryopreservation Agents in the Cryopreservation of Chlorophyte Microalgae.PLoSONE 8(11): e78668.</span><a href="https://doi.org/10.1371/journal.pone.0078668" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://doi.org/10.1371/journal.pone.0078668</span></a></li><li style = "counter-reset:ol0;list-style-type:disc;">Crutchfield, Alexandra ,Kenneth Diller & Jerry Brand (1999) Cryopreservation ofChlamydomonasreinhardtii(Chlorophyta), European Journal of Phycology, 34:1, 43-52, DOI: 10.1080/09670269910001736072</li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Day, John G., Cryopreservation of marine microalgae employing a controlled rate cooler, Scottish Association for Marine Science (SAMS), Oban, UK. </span><a href="https://www.assemblemarine.org/assets/Uploads/Documents/tool-box/Controlled-rate-cooling-cryo-protocolSAMS.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.assemblemarine.org/assets/Uploads/Documents/tool-box/Controlled-rate-cooling-cryo-protocolSAMS.pdf</span></a></li><li style = "counter-reset:ol0;list-style-type:disc;">Fleck, Roland A., Roger W. Pickup, John G. Day, Erica E. Benson, Characterisationof cryoinjury in Euglenagracilisusing flow-cytometry andcryomicroscopy, Cryobiology, Volume 52, Issue 2, 2006, Pages 261-268, ISSN 0011-2240, https://doi.org/10.1016/j.cryobiol.2005.12.003</li><li style = "counter-reset:ol0;list-style-type:disc;"><span>OhnumaM, Kuroiwa T, Tanaka K. Optimization of cryopreservation conditions for the unicellular red alga Cyanidioschyzonmerolae. J Gen Appl Microbiol. 2011;57(3):137-43.doi: 10.2323/jgam.57.137. PMID: 21817825.(</span><a href="https://www.jstage.jst.go.jp/article/jgam/57/3/57_3_137/_pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://www.jstage.jst.go.jp/article/jgam/57/3/57_3_137/_pdf</span></a><span style = ":;:UNDERLINE;">)</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Piasecki, BrianP. ,Kenneth R. Diller, Jerry J. Brand, Cryopreservation of Chlamydomonasreinhardtii: A cause of low viability at high cell density, Cryobiology, Volume 58, Issue 1, 2009, Pages 103-109, ISSN 0011-2240, </span><a href="https://doi.org/10.1016/j.cryobiol.2008.11.001" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://doi.org/10.1016/j.cryobiol.2008.11.001</span></a><span style = ":;:UNDERLINE;"> </span><span>(https://www.sciencedirect.com/science/article/pii/S0011224008002782)</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Mortain‐Bertrand,Anne ,Freddy Etchart, and Marie‐Thérèse de Boucaud, A METHOD FOR THE CRYOCONSERVATION OF DUNALIELLA SALINA (CHLOROPHYCEAE): EFFECT OF GLYCEROL AND COLD ADAPTATION, Journal ofPhycology, Volume32, Issue2, April 1996,Pages 346-352.</span><a href="https://onlinelibrary.wiley.com/doi/abs/10.1111/j.0022-3646.1996.00346" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://onlinelibrary.wiley.com/doi/abs/10.1111/j.0022-3646.1996.00346</span></a><span>.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><a href="https://ncma.bigelow.org/cryopreservation" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">NCMA at Bigelow Laboratory</span></a><span>, </span><a href="https://ncma.bigelow.org/cryopreservation" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Cryopreservation</span></a></li><li style = "counter-reset:ol0;list-style-type:disc;"><a href="http://www.roscoff-culture-collection.org/protocols/cryopreservation" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Roscoff Culture Collection</span></a><span>, </span><a href="http://www.roscoff-culture-collection.org/protocols/cryopreservation" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Cryopreservation protocol </span></a></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Tanaka, Jocelyn Y., John R. Walsh, Kenneth R. Diller, Jerry J. Brand, Shanti J. Aggarwal, Algae Permeability to Me2SO from −3 to 23°C, Cryobiology, Volume 42, Issue 4, 2001, Pages 286-300, ISSN 0011-2240, https://doi.org/10.1006/cryo.2001.2334 (</span><a href="https://www.sciencedirect.com/science/article/pii/S001122400192334X" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.sciencedirect.com/science/article/pii/S001122400192334X</span></a><span>)</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Taylor, R., Fletcher, R.L. Cryopreservation of eukaryotic algae – a review of methodologies.Journal of AppliedPhycology10,481–501 (1998).</span><a href="https://doi.org/10.1023/A:1008094622412" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://doi.org/10.1023/A:1008094622412</span></a><span style = ":;:;:UNDERLINE;:UNDERLINE;">(</span><a href="https://link.springer.com/article/10.1023/A:1008094622412#citeas" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://link.springer.com/article/10.1023/A:1008094622412#citeas</span></a><span>)</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><a href="https://utex.org/pages/cryopreservation#liquid" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">UTEX Culture Collection of Algae</span></a><span>, Cryopreservation protocol</span></li><li style = "counter-reset:ol0;list-style-type:disc;">Youn, J.Y. ,& Hur, S. B.( 2009). Cryopreserved Marine Microalgae Grown Using Different Freezing Methods. ALGAE, 24 (4) The Korean Society of Phycology. doi:10.4490/ALGAE.2009.24.4.257</li></ul></div></div>

[STEPS]
?. [Cryopreservation]
Verify culture concentration daily.  Cryopreservation should be made towards the end of the log phase or 1-2 days after the beginning of the stationary phase.
?. [Cryopreservation]
In a laminar flow hood, add  filter sterilized DMSO (Diméthylsulfoxyde) to a final concentration to of culture and transfer to a 2mL cryogenic tube.
1 mL
DMSO abosrbs rapidly through skin.  Wear appropriate PPE including nitrile gloves, lab coat, and safety eyewear.
The best cryoprotectant varies by species, strain, storage condition, and storage length.  The three most common cryoprotectants for microalgae are DMSO, methanol, and glycerol.Typical ranges found that 12% was typically toxic. (Abreu et al.) 5% and 10% are the most commonly used concentrations for methanol and DMSO. Rate of DMSO was found to be optimized between 6-7% for multiple strains. (Bui et al. 2013)Methanol may induce light sensitivity. (Alexandra et al. 2009)
?. [Cryopreservation]
Progressively freeze the algae at a target rate of -1oC/min starting at   by placing the samples in an isopropanol filled, pre-chilled () Mr. Frosty freezing container and moving to a  freezer for 2-4 hours.  Do not move the freezing container during progressive freezing. Sloshing or spilling of isopropanol will cause an inconsistent cooling rate.
20 °C
4 °C
-80 °C
Ideal end point for progressive freezing varies across the literature from -30C to -55C and occurs in freezers ranging from -40 to -80C.  Results in these ranges seemed comparable.  -80C was selected based on our available equipment.2-3 hour passive cooling led to higher cell survival than 1hr cooling or overnight cooling in C.Merolae. (Ohnumaet al.)Optimal freezing rate may be closer to 0.5oC/min but this rate is only achievable using a programmable progressive freezer. (Fleck et al. 2003) In general slower cooling rates (25µm diameter). (Day, SAMS)Best practice is to use a programmable progressive freezer.  Cost of a base model progressive freezer is $26000. A Mr. Frosty freezing container is about $100.
?. [Cryopreservation]
When the cycle is finished, immediately plunge the cryogenic tubes into liquid nitrogen. It is essential that the cryovials are not allowed to warm up prior to plunging into liquid nitrogen.
Cryogens and liquid nitrogen pose a safety hazard.  Wear appropriate thermal gloves, safety goggles, lab coat, and close toed shoes.  Evaporate from liquid nitrogen can pose an asphyxiation risk.  Never transfer or use liquid nitrogen in an enclosed space.
?. [Cryopreservation]
Place cryogenic tubes in cryoboxes and store in a  freezer and/or in a liquid nitrogen container for long-term storage.
-150 °C
Storage temperature is critical and should be  . It is advisable that replicate samples are stored in at least two separate cold storage locations.  Samples can be stored at   in a mechanical freezer, at   in the vapor phase of a liquid nitrogen dewar/cabinet, or at   submerged directly into liquid nitrogen.  All three methods have advantages and disadvantages.  Best practice would be to store replicate samples in two different cold storage locations and using two different types of cold storage.
?. [Culture Regeneration]
Allow vials to warm at  suspended in the air for exactly  then plunge tube in a  water bath just until melted.
-195 Room temperature
40 °C
For most marine taxa it is important not to prolong their incubation at 40o C. Alternative, slower warming  in a 25o C water-bath may be beneficial for some strains, but in general rapid warming is optimal as it avoids/ minimises ice crystal regrowth. (Day, SAMS)A one minute warming in the air before immersion in the 40oC water bath improved viability by 60% over immediate immersion for diatoms. A preliminary slow thaw is likely to have permitted slow glass relaxation avoiding potentially lethal fractures and recrystallisation. The second stage of rapid warming to ambient temperature, avoids ice crystal growth that could cause further damage to the algal cells. (Fleck 2013)Roscoff recommends thawing at 25-30 C.
?. [Culture Regeneration]
As soon as ice in the tube has melted, quickly and thoroughly clean the outside of the tube with  ethanol and in a laminar fume hood aseptically transfer the cells into a prepared flask containing at least  of fresh sterilized culture medium.
20 mL
?. [Culture Regeneration]
Surround the flasks with aluminum foil to keep them in the dark and put them at the optimal temperature for the culture.
?. [Culture Regeneration]
After 24 hours, remove the aluminum foil. Grow them in a reduced light intensity (8 µE m−2 s−1) for two weeks. Alternatively SAMS places cultures in complete dark for 24 hours, then allows half light for 96 hours (partially removes foil), before returing to full light (Day, SAMS).
During the recovery phase lower light intensities ( ∼8 µE m−2s−1) for two weeks were found to be optimal for growth. The light is beneficial for recovery even in the presence of a heterotrophic carbon source, but above ∼8 µE m−2s−1the cells in the recovery phase are very sensitive to photodamage. It is hypothesized there is a compromise between the beneficial effect of light (particularly for ATP production) on cell viability and the harmful effect of light-mediated damage on photosynthetic membranes where integrity and repair mechanisms may be compromised by freeze-thaw damage. (Bui et al. 2013)Both Roscoff and SAMS recommend only 24 hours in the dark but for benthic diatoms recovery under lower light levels (3–8μmol photons m−2s−1) resulted in increased levels of post-thaw viability (4.5±2.1%). The highest post-thaw viability levels were observed for samples that had been incubated in complete darkness for 4–7days (59±8.3%). It is hypothesized that recovery was significantly enhanced by removing the potentially injurious effects of uncoupled photosynthetic activity and photooxidative stress. (Buhmann et al.)
?. [Culture Regeneration]
Observe the cultures every other day (by colour and/or by flow cytometry and/or by microscopy).
?. [Culture Regeneration]
Culture recovery can take 2-8 weeks for normal culture that was healthy at time of cryopreservation (Day, SAMS).
?. [Cryopreservation]
Allow algae sample with cryoprotectant  to equilibrate for  at room temperature ()
4 Room temperature
Both DMSO and MeOH permeability of algal membranes and cell absorption is higher at warmer temperatures. (Tanka et al. 2001)
?. [Cryopreservation]
Add filter sterilized cryoprotectant additives to cultures, if using.
In some species, using a combination of cryoprotectants or cryoprotectant additives, such as sugars or antioxidants, has shown positive results for thawed cell viability.  The positive additive affect is not well studied and is highly variable by species, strain, and protocol.
?. [Cryopreservation]
In laminar flow hood, dilute algal cell concentration to 1×106 cell mL−1 in  of filter sterilized medium prepared with selected cryoprotectant and additives.
1 mL
It is theorized that the diminished viability of microalgal cells cryopreserved at high cell densities is caused by the enzymatic release of a cell-wall component.  Of the tested cell densities in several species, the highest recovery was seen at 1×10^6 cell mL−1. There is the possibility that further dilution could further improve recovery rates. Although improved viability at lower cell concentrations appears advantageous, lower cell concentrations mean a substantial reduction in the absolute numbers of colonies, even with an improved survival, and so is of limited interest. (Bui et al. 2013) (Piasaki et al. 2009)
?. [Cryopreservation]
Optional: Induce cold adaptation by placing algal cultures at  for 3 days.
4 °C
Freezing resistance of some algae has been shown to increase with a decrease in ambient culture temperature, this phenomenon being analogous to the cold-hardening of higher plants during the cooler autumn months (Taylor et al.) ((Mortain-Bortrandet al.)