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Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

Fifteen years of coral cryopreservation
Chiahsin Lin1,2* and Sujune Tsai3
1National Museum of Marine Biology and Aquarium, Checheng, Pingtung, Taiwan
2National Dong-Hwa University, Checheng, Pingtung, Taiwan
3Mingdao University, Peetow, Chang Hua, Taiwan*Corresponding author. Email: chiahsin@nmmba.gov.tw

Abstract
Over the past decade, coral cryopreservation research has advanced rapidly due tothe field’s inter-disciplinary approach, whereby concepts and technologies fromcryogenic biology, nano-engineering, and cellular biochemistry have been combined.
Cryopreservation of coral gametes, larvae, and their dinoflagellate endosymbionts has
been under continuous development, and the resulting publications have gathered theattention of conservationists across the globe. Herein we have reviewed progress in thisfield, with a particular emphasis on 1) understanding the factors required for successful
cryopreservation and 2) optimizing cryopreservation protocols for different cell/tissue
types. We advocate, at a minimum, 1) reducing cryo-injury (i.e., ice crystal formation)and 2) optimizing freezing techniques, for developing successful cryopreservation
protocols for coral gametes, tissues, larvae, and the endosymbiotic dinoflagellates that
reside within the tissues of all reef-building corals.
Keywords: Coral reef, Conservation, Freezing, Symbiodiniaceae, Cryobank

Introduction

endosymbiotic relationship between corals

Coral reefs (Fig. 1) are unique, high-and dinoflagellates is mutually beneficial,biodiversity ecosystems that are definedas the former provide shelter and nutrientsby both their biological (“coral”) andtogeological(“reef”)

components

dinoflagellates nourish their hosts with

(Buddemeieret2004).

The

photosynthetically fixed carbon. Coral

owe their

reefs are valuable not only to the plethora

photosyntheticof marine organisms that call reefs home,

dinoflagellates (family Symbiodiniaceae)

but also to humans, as they providethat inhabit their gastrodermal cells. The

nurseries for commercially important fish,al.,framework-building corals
success

to

the

53

their

endosymbionts,

while

the

Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

Fig. 1. A healthy coral reef near Bandaneira, Maluku, Indonesia. Photo
credit: Anderson Mayfield.
shelter coastlines from wave damage, aremass coral “bleaching” events since thesources of pharmaceuticals, and are

1980s (reviewed in Donner et al., 2017),

common tourist destinations. Indeed, thewith bleaching events now occurring

economies of many small island nations,annually in many parts of the world. Suchas well as the welfare of their citizens, arebreakdowns occur because most coral-

intrinsically linked with healthy coral reefs.

dinoflagellate endosymbioses exist near

Unfortunately, climate change and morethelocalized human activities (e.g., seawater

thermotolerance (Mayfield & Gates, 2007).

pollution) have resulted in mass coralThis dramatic loss of coral necessitates

decline across the globe (Hoegh-Guldberg

proactive conservation and preservation

et al., 2007). Climate change-associatedstrategies (Gattuso et al., 2015), including

temperature increases have been causing(non-exhaustively)
54

upper

thresholdofenvironmental

their

and

Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

climate mitigation, coral transplantation,

Since then, coral sperm (Hagedorn et al.,coral stress-hardening, assisted evolution,

2012, 2017; Viyakarn et al., 2017),

and even cryopreservation for when/if theoocytes (Fig. 2; Tsai et al., 2011, 2016;

aforementioned approaches fail (Tsai et al.,Tsai & Lin, 2012), larvae (Daly et al., 2018;2010b).

Cirino et al., 2019), “tissue balls” (coral

Most studies on marine invertebratetissues that have detached from theircryopreservation have focused on oysters

calcium(Hughes, 1973; Yankson & Movse, 1991;

Feuillassier

Tervit et al., 2005) and sea urchins (Adams

Symbiodiniaceae (Chong et al., 2016c; Linet al., 2003, 2006). Studies on the

et al., 2019a; Di Genio et al., 2020) havecryopreservation of sea cucumbers (Shaoall been studied, with cryopreservation

et al., 2006), shrimp (Tsai and Lin, 2009;

effectsLin

mollusks

mitochondrial DNA content (Tsai et al.,(Odintsova et al., 2001, 2009) have also

2014a, 2015a), gene expression (Chong et

been

coral

al., 2017), protein structure (Lu. 2019),

cryopreservation began in earnest only 10-

and other means (discussed in detail

15 years ago (Hagedorn et al., 2006a; Tsaibelow). Different coral tissues and life

et al., 2010b); our knowledge of coral

history stages require their own optimized

cryobiology is consequently in its infancy.

freezing protocols, and several such

et

al.,

2013a),

conducted.

In

and

contrast,carbonateetal.,documented

exoskeletons;
2014a),

byandmonitoring

Fig. 2. A stony coral (Hydnophora rigida) spawning in Nanwan Bay, Taiwan. Photo
credit=Fuwen Kuo.
55

Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

methods developed to date have beenstress can damage cellular structures anddescribed below. Since cryopreservation

reduce viability (even killing cells; Benson,

can damage cells, tissues, and DNA

2008). It is therefore necessary to reduce(Kopeika et al., 2014), coral cryobiology

chilling sensitivity to minimize thestudies must ensure high viability ofdamage caused by cryopreservation. The

thawed biological material. This topic isdegree of chilling stress increases withalso discussed in this review.

cooling rate (Morris, 1981), and coral
oocytes with high lipid content generally

Cryo-injury

sustain less chilling stress (Lin et al., 2011;

Cryopreservation is a long-term

Tsai & Lin, 2012); this could be due tostorage technique conducted to preserve

coinciding reductions in lipid peroxidation

structurally intact living cells and tissues(Darin-Bennett & White, 1977; Lin et al.,for extended periods of time at very low

2012, 2013b).

temperatures (Tsai & Lin, 2012). Several
factors affect the successcryopreservation,of coralin a loss of water from a cell (Muldrew &limiting of, or even avoiding, cryo-injury,

McGann, 1990), as well as damage to

which

by

cellular structures and macromolecules.

intracellular ice crystal formation. Chilling

However, ice crystal formation can be

typically

notably

intracellularly or extracellularly and result

the

is

most

Ice crystals (Fig. 3) can form

broughtonFig. 3. Extracellular ice formation around a coral oocyte of Junceela fragilis undergoingfreezing. Scale bar=500µm. Photo credit=Chiahsin Lin.
56

Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

State-of-the-art
techniques

prevented by controlling the rate ofcooling (Mazur et al., 1972); rapid coolingfreezingand slow warming generally cause more

Different types of cryopreservation

ice crystal-related injuries than slow

techniques are currently being used forcooling and rapid warming. This finding

coral cryopreservation. The most common

was corroborated by Hagedorn et al.

are slow-freezing (Fig. 4), two-step(2006a) in a study in which coral larvae

freezing, and vitrification. Each techniqueunderwent slow freezing in two steps tohas different features, as well as different

allow the larvae sufficient time topros and cons. Hence, it is necessary todehydrate, thereby preventing ice crystal

understand the details and limitations offormation. Vitrification can also prevent

each when developing protocols. Slow-

ice crystal formation and has been applied

freezing is a process in which cells areto coral oocytes and larvae (Tsai et al.,slowly cooled below their freezing points.

2015b, 2016; Daly et al., 2018; Cirino et

The resulting water efflux rate reducesal., 2019). In addition to chilling stress anddehydration and ice crystallisation impacts.

ice crystal formation, cells may be“Controlled slow-freezing” involves adamaged by the solution itself when theseries of steps in which the sample is pre-cooling rate is too low (Chong, 2015). This

cultured

generally occurs when cells are exposed to

cryoprotected (Benson, 2008). Several

a high concentration of solute for an

studies

excessive period, leading to cellular

programmable freezing (Fig. 5) withdehydration. This weakens the complex

oocytesstructures of proteins and results in loss ofJunceella juncea & J. fragilis; Goh, 2019)lipids and phospholipids (Chong, 2015).

and their Symbiodiniaceae endosymbionts

Weakenedmore(Gerakladium; Zhao, 2017). Short-term,

permeable to electrolytes and are thus

low-temperature preservation and slow-

easily ruptured when cells revert to thefreezing have also been used for theisotonic state (Mazur et al., 1972). The

cryopreservation

solution effect can be reduced by gradually

Echinopora sp. (stony coral; Tsai & Lin,

adding and removing cryoprotectants2012), and 2) Ellisella robusta (gorgonian;(CPAs) to prevent excessive osmotic

Lin et al., 2014; Goh, 2019).

membranesarepressure changes (Adams et al., 2003).

oracclimated

have
of

and

used

gorgonian

of

then

controlled,coralsoocytes(e.g.,

of1)

Two-step freezing is a method inwhich cells are frozen at low temperaturesby immersing samples first in liquid
57Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

Fig. 4. A sequence of images depicting ice crystal formation during controlled, slow
freezing. Photo credit=Chiahsin Lin.
nitrogen vapor or dry ice and then at

nitrogen for rapid freezing and then stored.

cryogenic temperatures for storage. Unlike

Several experiments have been conductedslow-freezing, two-step freezing does notusing

require any specialised or controlled

Symbiodiniaceae (Chong et al., 2016c; Lin

cooling equipment, and generally low

et al., 2019b; Di Genio et al., 2020), coral

CPA concentrations are used (Chong et al.,sperm (Acropora humilis; Viyakarn et al.,2016c). It also allows the sample to2017), and the primary cells of other

undergo osmosis with the CPA and applies

marine invertebrates (Odintsova et al.,a slow cooling rate to minimise the2001). Cooling rates of 40°C/min forformation of intracellular ice crystals (Tsai

Acropora digitifera (Ohki et al., 2014),&

complete

41.7°C/min for A. humilis (Viyakarn et al.,dehydration, cells are immersed in liquid2017), and 20-30°C/min for A. tenuis, A.

Lin,2012).

Following58

two-stepfreezing

withPlatax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

Fig. 5. Programmable cooling freezes samples by gradually cooling them at a controlledrate in a computerized freezer. Photo credit=Chiahsin Lin.
millepora,A.

palmata,and

Fungiaalso been used for P. damicornis tissue

scutaria (Hagedorn et al., 2012, 2017)

balls (Feuillassier et al., 2015).

have been used.

Factors
affectingcryopreservation

Vitrification is a rapid coolingmethod in which cells directly enter asuccessful“glass” state through the use of a

Successful cryopreservation requires

vitrification solution that thwarts ice

a suitable freezing protocol that limits

crystal formation (Chong et al., 2016c). J.

biomolecular damage, thereby resulting injuncea oocytes (Tsai et al., 2015b, 2016),

highSymbiodiniaceae

Cooling rate, CPA composition andcells(Hagedorn&sampleviability

Carter, 2015; Zhao, 2017), and coral larvae

concentration,(Daly et al., 2018) have all been

thawing time can all be optimized tosuccessfully

via

improve cell viability. The cooling rate isvitrification. Cirino et al. (2019) actually

critical for successful cryopreservation, as

managed to cryopreserve larvae housing

it determines whether cells remain inendosymbiotic dinoflagellates by vitrified

equilibrium

cryopreservation;

following nano-goldenvironment or become progressively

laser thawing, the larvae successfully grew,supercooled (in which case there is ansettled, and developed. Vitrification has

increasing possibility of ice formation;

cryopreserved

59

equilibrium

post-thawing.

with

their

time,

and

extracellularPlatax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

Kumar et al., 2003). Cooling rates that are

weight,candiffuseexcessively low or excessively high will

membrane (Tsai et al., 2011), and include

compromise the survival of cells after

glycerol (Gly), ethylene glycol (EG),cryopreservation. Tsai et al. (2010a)

propylene glycol (PG), methanol (MeOH),

recommended a slow cooling rate toand dimethyl sulfoxide (DMSO). They

permit a sufficient degree of cellular

have been used in numerous experiments;

dehydration; however, an excessively slow

for instance, EG was used to vitrify coral

rate may lead to CPA toxicity within

oocytes and larvae (Tsai et al., 2015b;

oocytes in particular and can result in

Cirino et al., 2019), and MeOH was used

extracellular ice formation; the latterto cryopreserve coral oocytes and algae

phenomenon then creates an osmotic(Tsai & Lin, 2012; Chong et al., 2016c).

gradient across the cell membrane thatDMSO has been used to cryopreserve A.

draws water out (Tsai & Lin, 2012). This

humilis sperm (Viyakarn et al., 2017).

consequently limits the amount of cellularNon-permeating CPAs have much higher

water that can crystallize. For coral sperm,

molecular weights and cannot cross cell

oocytes, and larvae, as well as in hospite

membranes; they include sugars (Tsai etand cultured Symbiodiniaceae, the optimalal.,rate of cooling can be determined bypolyvinylpyrrolidone (Pedro et al., 2005;

factoring in the 1) permeability of the cell

Shalueimembrane to water, 2) surface-to-volume

cryopreservation, non-permeating CPAs

ratio of the cell, 3) type and concentration

are typically combined with permeatingof CPA(s), 4) CPA infiltration rate, and

CPAs: MeOH+sucrose for sperm A.

other factors (Tsai et al., 2010b, 2011; Lin

digitifera& Tsai 2012; Viyakarn et al., 2017; Cirino

EG+sucrose for tissue ball P. damicornis

et al., 2019; Di Genio et al., 2020).

(Feuillassier et al., 2014a).

2018),
etfreelyacrosslipids,
al.,

(Ohki

et

2013).

al.,andDuring2014)

andCPAs protect cells from freezing andEquilibration time refers to the period

initially cause cells to shrink, later

during which cells are mixed with CPAs

swelling to a new equilibrium volume as

before undergoing cryopreservation. This

the CPAs disperse throughout the cell

enables osmosis to occur without affecting(Adams et al., 2003). There are two types

cell viability. The equilibrium timeof CPAs: 1) membrane-permeating and 2)

normally depends on the type of cells andnon-membrane-permeating; each plays aCPAs used and can be adjusted to enhancedifferent role during cryopreservation.

osmosis. For example, the equilibrium

Permeating CPAs are low moleculartime should be shorter when high60

Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

permeability CPAs are used (Chong,viability must be assessed. Mitochondria2015); only 2 min of equilibration timeplay a vital role in cell energy metabolism

were required to prepare coral oocytes andby providing ATP. However, the use oflarvae for vitrification (Tsai et al., 2015b,

CPAs during freezing disrupts coral2016) and cryopreservation (Cirino et al.,mitochondrial distribution and membrane2019).

potential,

For

A.

humilis

sperm

and

which

then

lowersATP

Symbiodiniaceae, times of 15 (Viyakarn etproduction (Tsai et al., 2014a). In a low-al., 2017) and >30 min (Di Genio et al.,temperature study of Echinopora sp.,2020) were required, respectively.

mitochondria DNA copy numbers wereThawing is the last stage of theused as a proxy of coral oocyte health after

cryopreservation process and represents

treatment

the period during which cells are revived.

concentrations (Tsai et al., 2015a). Vital

Variousbestains can also be used to assess viability

employed: room temperature thawing

since only intact membranes will bar their(Bougrier & Rabenomanana, 1986), water

entry (Mazur, 1970). Both fluoresceinbath thawing (Tsai et al., 2016), and laser

diacetate (FDA) and propidium iodide (PI)warming (Cirino et al., 2019). However,

have been used in coral cryopreservation

the thawing time must be precise andresearch (Tsai et al., 2010b; Lin et al.,accurate. For instance, rapid thawing can2011); the former requires cellular esterase

improve the survival of coral samples

activity and an intact membrane whereas(Tsai et al., 2016) since it restricts the re-

PI is a more traditional vital stain (Jones &growth of ice crystals. However, rapid

Senft, 1985; Tsai et al., 2008). FDA+PI

thawing can also be lethal to some frozen

were used in conjunction in gorgonian

samples because of the resulting osmotic

coral oocytes, though the data were

stress. Symbiodiniaceae (Chong et al.,spurious given the unique membrane2016c), J. juncea and J. fragillis (Lin et al.,characteristics of these soft corals (Tsai et2011), and Echinopora sp. (Tsai & Lin,

al., 2011).

thawing

methodscanwithdifferent

CPA2012) cells have been thawed in water

Optimizing
cryopreservation
protocols for different cell/tissue
types

baths. Cirino et al. (2019) utilized gold
nanorods to absorb and then emit laserderived energy to rapidly and uniformly
rewarm samples.

The cryopreservation of coral sperm,

To determine whether cells havesurvived

cryopreservation,

post-thaw
61

oocytes,larvae,tissueballs,

embryos,
andfragments,

Symbiodiniaceae

Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004(including cultured samples) requires

can be used for seeding shrinking

different freezing techniques and CPAs

populations; the gametes also 1) provide

since differing cell/tissue types respondeasy and inexpensive transport of genetic

uniquely to dehydration, CPAs, chilling,material among living populations, 2)

and ice crystals. We now discuss theextend generation intervals, and 3) serve as

current

a source material for research (Ballou,

protocols

in use forcoral

cryopreservation.

1992; Wildt et al., 1997; Holt & Pickard,
1999).

Progressincryopreservation

sperm

Sperm

cryopreservationisespecially important now that it is harderfor corals to survive in situ due to climate

The cryopreservation of sperm playschange and other anthropogenic impacts

an important role in preserving paternal(discussed in more below).

genetic diversity and has benefited theMost coral sperm cryopreservationaquaculture industry since a source ofstudies have used DMSO as the major

viable sperm ensures that the life cycle can

CPA (Hagedorn et al., 2012; Tsai et al.,be completed ex situ (Martinez-Paramo et

2014b; Viyakarn et al., 2017; Tab. 1).

al., 2016). Banking frozen invertebrate

Although most studies have shown thatsemen also has important implications forsperm frozen

hybridization,

breeding,with DMSO have higher viability thangynogenesis, domestication, and stock

other CPAs (e.g., EG & PG; Hagedorn etconservation, and cryopreserved gametesal., 2006b, 2012, 2017), DMSO is highly

selective

Tab. 1. Cryopreservation of coral sperm. CPA=cryoprotectant. NOEC=”No observed effect”
concentration.
Species

Experimental or
cryopreservation methodTwo-step freezing

Cryoprotectant

Reference

2 M DMSO

Viyakarn et al. (2017)

Acropora tenuis
Acropora millepora
Junceella juncea
Junceella fragillis
Acropora digitifera

Two-step freezing

10% DMSO

Hagedorn et al. (2017)

CPA effectTsai et al. (2014b)

Two-step freezing

NOEC up to 3 M for MeOH or
DMSO
0.9 M sucrose with 20% MeOH

Acropora palmata
F. scutaria
Fungia scutaria

Two-step freezing

10% DMSO

Hagedorn et al. (2012)

Slow freezing

10% DMSO

Hagedorn et al. (2006b)

Acropora humilis

62

Ohki et al. (2014)

Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

toxic to coral sperm and can lead to 1) thecoral oocytes have substantial chilling

immediate

tails

tolerance at 0 and 5°C, but not at -5°C; for(Hagedorn et al., 2006b) and 2) post-thaw

this reason, we recommend vitrification

osmotic stress; we recommend, then, that

(Tsai et al., 2015a, 2016) since it limits

researchers consider alternatives. Ohki etchilling injury. That being said, oocyteal. (2014) successfully used 0.9 M sucrose

susceptibility to cryopreservation varies

with 20% methanol with A. digitifera

dramatically across taxa. For instance, J.

sperm. This high viability may befragillis has higher tolerance towards

attributed to the fact that sugars are

CPAs compared to other gorgonians,

compatible osmolytes that protect cells

while Echinopora robusta demonstrates

against osmotic shock during extracellularhigher tolerance to chilling compared towater depletion caused by ice formation.

other hermatypic corals (Tsai et al., 2016).

They also preserve the structural and

The latter observation has been attributed

functional integrity of membranes at low

to high lipid content (Lin et al., 2012);

temperatures (Crowe et al., 1990).

elevatedswelling

of

their

concentrationsofphosphatidylethanolamines and fatty acids

Oocyte cryopreservation

are linked to high membrane fluidity,

Coral oocyte cryopreservation (Tab.

whichprotects

cells

from

cold-

2) was first developed in 2010b by Tsai ettemperature stress (Lin et al., 2012, 2013b).

al. though remains a challenge due to theirHigh membrane fluidity reduces thehigh sensitivity to CPAs and chilling, as

amountwell as their low membrane permeabilities

cryopreservation, which thereby limits(Lin et al., 2011, 2012, 2014; Tsai et al.,

CPA toxicity issues (Lin et al., 2014). The

2010b, 2016). Furthermore, the high lipid

most

content of oocytes results in low surface

cryopreservation of coral oocytes to date isarea-to-volume

are

MeOH, which protects cells by permeating

associated with freezing and thawing

through the cell membrane and reducing

difficulties (Lin et al., 2013b). The limited

the formation of ice crystals (Tsai et al.,time available to collect coral gametes also

2014a). However, MeOH is a poor

imposes a limitation. Broadcast spawning

vitrificant; EG is instead the CPA ofcorals generally release gametes (Fig. 2)

choice when cryopreserving coral oocytes

only during the spring or summer fullvia vitrification (Tsai et al., 2015a, 2016).

ratios,whichmoon periods, and for < 1 h on just 2-3
nights. According to Tsai & Lin (2012),
63

of

CPAs

effective

neededCPA

forduringthePlatax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

Tab. 2. Cryopreservation of coral oocytes. CPA=cryoprotectant. Mt=mitochondrial.
Species

Experimentalor

CPA(s)

Reference

Physiological assessment---

Lin et al. (2019c)

Physiological assessment---

Lin et al. (2018)

Ultrastructural observations---

Tsai et al. (2016)

Vitrification

3.5 M PG+1.5 M EG+

Tsai et al. (2016)

cryopreservation method
Echinopora gemmacea
Oxypora lacera
E. gemmacea
O. lacera
O. lacera
E. gemmacea
Montipora incrassata
M. hispida
Galaxea fascicularis
Merulina ampliata
Junceella fragilis
Ellisella robusta

2 M MeOH

Junceella juncea

Vitrification

3.5 M PG+1.5 M EG+

Tsai et al. (2015a)

2 M MeOH
E. gemmacea

Mt DNA content

0.5 M MeOH

Tsai et al. (2015b)

J. juncea

Ultrastructural observations---

Tsai et al. (2014c)

Lipid phase transition---

Lin et al. (2014)

E. gemmacea

Mt DNA content

1 M MeOH

Tsai et al. (2014a)

Platygyra daedalea

Lipid content & composition---

Lin et al. (2013b)

Lipid content & composition---

Lin et al. (2012)

E. gemmacea

Chilling

0.5 M MeOH

Tsai & Lin (2012)

J. juncea

Chilling

1 M MeOH

Lin et al. (2011)

CPA effects

MeOH<EG< PG<DMSO

Tsai et al. (2011)

CPA effects

MeOH<DMSO<PG=EG

Tsai et al. (2010b)

J. fragillis
J. juncea
J. fragillis

E. gemmacea
Echinophyllia aspera
O. lacera
Astreopora expansa
J. juncea
J. fragillis

J. fragillis
J. juncea
J. fragillis
E. gemmacea

Coral larvae and adult tissue
cryopreservation

provide a biological platform for crossing
different genotypes to potentially increase

Corals can reproduce both sexually
and asexually.

Therefore,

resilience(Hagedorn

et

al.,

2012).

preserving

Recently, both asymbiotic (Daly et al.,sexually derived cells such as oocytes and2018) and symbiotic (Cirino et al., 2019)sperm can preserve biodiversity and evencoral larvae have been successfully
64Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

cryopreserved by vitrification and nano-coral embryos are non-existent, and adultlaser warming. In the latter study, 55% ofcoral cryopreservation is in its infancy

thawed Seriatopora caliendrum larvae(Tab. 3.). This is due to the complex, dual-

survived treatment, and 9% were able tocompartmental

settle. It is hope that more endosymbiotic

gastrodermal cells, as well as their atypicalcoral species will be cryopreserved in thelipid composition, both of which increase

near future, though both vitrification andcryopreservation difficulty (Feuillassier etlaseral., 2014a). Furthermore, the calcium

warming

conditions

must

be

optimized for each new species test.

natureofcoral

carbonate exoskeleton of adult corals

Compared with coral sperm andthwarts

oocytes, studies on the cryopreservation of

cryopreservation

success(Feuillassier et al., 2014b). DMSO, MeOH,

Tab. 3. Cryopreservation of adult and larval corals. CPA=cryoprotectant.
SpeciesMaterial

Experimental or
cryopreservation
methodCPA(s)

Reference

Seriatopora caliendrum

LarvaeVitrification &laser warming

2M EG+1M PG

Cirino et al. (2019)

Fungia scutaria

LarvaeVitrification &laser warming

10% PG+5%DMSO+1 M
trehalose

Daly et al. (2018)

Pocillopora damicornis

Tissue ball

Vitrification

1.5 M EG+1.5 M
Gly+1.5 M DMSO

Feuillassier et al.
(2015)

P. damicornis

Apex

CPA effects

EG>MeOH>DMSO>Gly

Feuillassier et al.
(2014b)P. damicornis

Tissue ball

CPA effects≤ 4 M EG, MeOH, Gly,or DMSO

Feuillassier et al.
(2014a)P. damicornis

Adult fragment

Chilling≤ 1.5 M DMSO

Hagedorn et al.
(2013)

F. scutaria

Larva

Analysis ofinternal
osmolality---

Hagedorn et al.
(2010b)P. damicornis

Larvae

CPA effects, ice
nucleation
temperature, &permeability

PG

Hagedorn et al.
(2006a)F. scutaria

65Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

and Gly have all been used as CPAs ingenera successfully cryopreserved to date.

cryopreservation experiments of adult

It will be critical to cryopreserve all suchcoral fragments (Hagedorn et al., 2012)

types because some, such as Durusdinium,

and tissue balls (Feuillassier et al., 2014a),are known to have higher thermal

withtolerance (Stat et al., 2009).

thenon-permeating

EG+sucrose tested with adult

CPAs
coral

Different freezing methods have beenbranches (Feuillassier et al., 2014b) andused in Symbiodiniaceae cryopreservation

tissue balls (Feuillassier et al., 2015).

studies. For instance, two-step freezing

Although alcohol+sugar CPA cocktails

with EG and PGs as the CPAs was used formay reduce the toxicity of the alcohol andDurusdinium and Gerakladium (Lin et al.,limit cellular damage (Renard, 1991), such

2019b; Chong et al., 2016c), though

was not the case with P. damicornis tissues

vitrification with DMSO as the CPA was(Feuillassier et al., 2014a), in which

superior for Cladocopium (Hagedorn &immersion in 0.2 M sucrose+0.75 M

Carter, 2015). However, because different

DMSO+0.75 M MeOH+0.75 M EG did

CPAs were used in studies with different

not confer any survival benefit afterendosymbiont types, it is currently notcryopreservation.

possiblewhethertoknowwhetherdifferential cryopreservation success is

Symbiodinaceae cryopreservation
Coralsowe

their

due to the type of Symbiodiniaceae, theevolutionary

CPA used, or the freezing method. With

success to their mutualistic relationshiprespectwithfamilyvitrification and two-step freezing have

Symbiodiniaceae; these endosymbionts

both been executed successfully (Zhao,

supply nutrients and energy to their host

2017; Lin et al., 2019b). In the former, allcorals and are consequently obligatory forfreezable water is removed from the cells

all reef-building corals (Whitehead &after sugar addition, and the highly

Douglas, 2003; Russell, 2013). Given theconcentrated

concerns with climate change-induced

compartment vitrifies as described above.

bleaching raised above, Symbiodiniaceae

Duringcryopreservation will play a critical role inSymbiodiniaceae cells are permeabilized

the future restoration of coral populations

with CPAs before being vapor-frozen in(Chong et al., 2016a). Unfortunately, thisLN2 (Santiago-Vázquez et al., 2007;

field is in its nascent stages (Tab. 4), with

Chong et al., 2016c; Lin et al., 2019b;

only several of the nine Symbiodiniaceae

Thongpoo et al., 2019). In both processes,

dinoflagellates

of

the

66

to

the

freezing

internal
two-stepmethod,aqueousfreezing,

Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

Tab. 4. Cryopreservation of Symbiodiniaceae. Mt=mitochondrial. CPA=cryoprotectant.
Species/hostExperimental or
cryopreservation
methodTwo-step freezing

CPA(s)

Reference

Various CPAs at different
concentrations

Di Genio et al. (2020)

Luminometry &flow cytometry
Mt DNA content
Two-step freezing
Gene expression
Gene expression
Vitrification &controlled slowcoolingTwo-step freezing
Vitrification

MeOH=DMSO<EG<PG<
Gly
2M PG
2M PG+2M EG
1 M MeOH+0.4 M sucrose
1 M MeOH+0.4 M sucrose
1 M Gly

Lin et al. (2019a)

1 M MeOH+0.4 M sucrose
10% DMSO+5 %MeOH+0.5 M trehalose

Chong et al. (2016c)
Hagedorn & Carter (2015)

Cladocopium/F. scutaria, /P. damicornis,/P. compressa

Chilling---

Hagedorn et al. (2010a)

Breviolum/Pseudopterogorgia elisabethae

Two-step freezing

20% ethanol or 20% MeOH

Santiago-Vazquez et al. (2007)

Symbiodinium (cultured)
Cladocopium (cultured)
Fugacium (cultured)
Gerakladium/Junceella fragilis
Durusdinium (cultured)
Durusdinium (cultured)
Gerakladium/J. fragilis
Gerakladium/J. fragilis
Gerakladium/J. fragilis

Gerakladium/J. fragilis
Cladocopium/Fungia scutaria /Pocillopora
damicornis /Porites compressa

Thongpoo et al. (2019)
Lin et al. (2019b)
Chong et al. (2017)
Chong et al. (2017)
Zhao, (2017)

dehydration followed by extracellular(Altman et al., 1993). Additionally, it canfreezing prevent intracellular ice crystal

be hard to distinguish SYTOX® fromformation.

chlorophyll fluorescence (Lebaron et al.,

In addition to the ATP assays

1998; Zetsche & Meysman, 2012). A

discussed above, numerous methods havepreferable approach may be the CellTiter-

been described for evaluating the viability

Blue assay, in which the metabolic activityof Symbiodiniaceae. The aforementionedof cells is assessed based on their ability tovital staining dyes penetrate cells withconvert the redox dye resazurin into thedamaged membranes though can be labor-

fluorescentintensive

Santiago-Vázquez

unlessflow

cytometryisemployed.

Furthermore,

they

can

overestimate

the

of

viablenumberend

product
etal.

viability of Breviolum.
Pulse amplitude-modulated (PAM)

because dead cells do not always havefluorometry,which

rupturedmaximumdark-adapted

permeable(2007)

successfully used this approach assess theSymbiodiniaceae (Lin et al., 2019a)andresorufin.

membranes
67

measures
yield

the
of

Platax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

photosystem II (Fv/Fm), has been used as

too marginalized for coral survival, theira proxy for photosynthetic efficiency ingenetic material can be cryopreserved untilcorals, and Hagedorn et al. (2015) useda point in time in which coral propagation

Fv/Fm values as a proxy for health ofis once again viable in situ (Mayfield et al.,cryopreserved

Since2019). Different genotypes could even be

cryopreservation experiments can generatecrossed to increase genetic diversity anda large number of samples to be analyzed

potentially yield more environmentallyby any such viability assay, the ideal

resilientapproach may well be one that can be

specimens could be reared in captivity to

conducted quickly. In the end, cell survival

reduce aquarium trade impacts on in situ

and growth are the easiest-to-interpret, andpopulations. Currently, coral cryobanks

likely most relevant, indicators of thecan be found at Taiwan’s NMMBA

viability of cryopreserved and thawed cells.

(Mayfield et al., 2019; Di Genio et al.,Cladocopium.

populations.

Thawedcoral

2020; Fig. 6), the Hawaii Institute ofCoral cryo-banking

Marine Biology (USA), and the Taronga

Gene banks preserve genetic material

Western Plains Zoo (Australia; Hagedornfrom different species in different forms

and Spindler, 2014).

and for varying purposes, including

Conclusions

scientific studies, breeding efforts, andconservation

endeavors.

One

bank,

The

generalcryopreservation

Genome 10K, aims to sequence at least

protocol involves 1) the initial freezing of

one species from each genus of vertebrates

the samples, followed by 2) long-termin the animal kingdom for researchstorage, and finally 3) thawing for

purposes (Koepfli et al., 2015). Cryobanks

subsequent culture or fertilization in theare one form of gene bank, and many exist

case of gametes. Future efforts should seek

across the globe. They are particularlytoimportantandcomplex tissue types, in which case morebiopreservation (both discussed above),advanced diagnostic approaches may be

and the Frozen Ark Project (Clarke, 2009)

needed to assess health post-thaw (e.g.,aims to preserve samples for both researchultrastructural observations and molecularand preservation. National Museum of

biotechnology). Not only will these data

Marine Biology and Aquarium (NMMBA)

allow us to understand the physiologicalhas been involved with the Frozen Ark

implications of extreme low-temperature

project since 2015. If the oceans become

exposure on coral gametes, embryos,

foraquaculture

68

cryopreserve

increasingly

morePlatax 17: 53-76, 2020
doi: 10.29926/platax.202012_17.0004

Fig. 6. Cryopreserved coral cells, larvae, and Symbiodiniaceae are deposited incryobanks for long-term storage. Researchers from Taiwan’s National Museumof Marine Biology and Aquarium an are actively collecting and cryopreserving
coral cells to be used for research (e.g., genetic modification & gene mapping
projects) and as a backup source of genetic material for future reef restoration
endeavours. Photo credit=Chiahsin Lin.
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mammaliancell
viability
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Cryopreservation of spermatozoa of thelarvae, and, but they will be critical tooptimizing protocols that give thawed
samples the best chance at survival in thenew, and ideally improved, milieu in
which they will ultimately reside.

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