Advancing marine invertebrate cell line research: four key knowledge gaps

In Vi o Cellula & De elopmen al Biology - Animal (2025) 61:493–505
Vol.:(0123456789)
h ps://doi.o g/10.1007/s11626-025-01029-y
INVITED REVIEW
Ad ancing ma ine in e eb a e cell line esea ch: ou key knowledge
gaps
Ba uchRinke ich1· Shi leyA.Pomponi2,3
Recei ed: 12 Decembe 2024 / Accep ed: 23 Feb ua y 2025 / Edi o : Cyn hia L. Goodman
© The Au ho (s) 2025
Abs ac
Al hough cell cul u es om ma ine in e eb a es ha e g ea po en ial as aluable ools in a ious scien i ic ields, nea ly all
a emp s o cul u e hese cells in i o ha e consis en ly ailed, and he easons o his emain unclea . The ongoing ailu e o
de elop s able, long- e m cell cul u es om ma ine in e eb a es, despi e a ied species and me hods employed, highligh s
signi ican knowledge gaps in unde s anding hei in i o equi emen s. These gaps impede p og ess, unde sco ing he
complexi y o ma ine in e eb a e cells and he need o inno a i e app oaches o o e come challenges in he ield. When
e iewing ecen li e a u e on he key da a de iciencies and challenges behind he ailu e o de elop ma ine in e eb a e
cell cul u es, we iden i ied and discussed ou majo knowledge gaps: (1) op imizing cul u e media, (2) s a egies o ex end
s emness o isola ed cells, (3) using “omics” o enhance cell cul u e, and (4) selec ing sui able cell ypes o in i o cul u es.
B idging hese gaps is c ucial o ad ancing ma ine in e eb a e cell cul u e sys ems. Ye , gi en he cu en s a e-o - he-
a , add essing hese gaps and ad ancing he discipline necessi a e comp ehensi e, in eg a ed, and species- o cell-speci ic
s a egies, along wi h close collabo a ion among labo a o ies wo king on di e se species.
Keywo ds Ma ine in e eb a es· Cell cul u es· Knowledge gap· S emness· Omics· Media· Cell ypes
In oduc ion
Ma ine in e eb a e cells cul u ed in i o hold immense
po en ial as aluable ools ac oss di e se scien i ic disci-
plines and applied ields, including biological/en i on-
men al s udies (e.g., Rosne e al. 2021, 2024), economical
applica ions, including cul i a ed sea ood (e.g., Musg o e
e al. 2024a; Ro e e al. 2024) and medical esea ch (e.g.,
Da a e al. 2015). The uses o cell cul u es om ma ine
in e eb a es u he o e al e na i es o animal expe imen-
a ion, as well as oppo uni ies o bio echnological inno-
a ion and physiologic/me abolic s udies. In e eb a es,
which comp ise o e 95% o all animal species, ep esen
a signi ican esou ce o such esea ch. Among hem, he
ma ine in e eb a es ha a e known o occu in all habi a s
o he ma ine/oceanic domains, a e es ima ed o accoun o
30–35% o all animal species (O mond e al. 1997; Collie
e al. 2016) and a e no ed o hei dominance in ma ine
ecosys ems and hei as axonomic di e si y. Encompass-
ing o e 20 phyla and ep esen ing 92% o ocean species
(Chen 2021), ma ine in e eb a es p o ide a di e se ange
o cell and issue ypes, which exhibi signi ican a ia ion
bo h be ween and wi hin phyla. These cells u he display
ema kable mo phogene ic po en ial, including mul ipo-
ency, o ipo ency, and e en neoplas ic beha io s (Robe
2010; Rinke ich e al. 2022). This po en ial unde pins hei
high in i o plas ici y, including dynamic changes in s uc-
u es, cell p oli e a ion, egene a ion, and lineage di e en-
ia ion, which can a y conside ably e en among closely
ela ed g oups.
While mammals accoun o only 0.4% o exis ing mul-
icellula axa, hey domina e cell cul u e esea ch, wi h
97% o a ailable cell lines o igina ing om mammalian
species (Bai och 2018), highligh ing a disp opo ion-
a e scien i ic in e es . In he same way, e o s o cul u e
ma ine in e eb a e cells in i o a e no a jus a ecen
* Shi ley A. Pomponi
spomponi@ au.edu
1 Is ael Oceanog aphic & Limnological Resea ch, Na ional
Ins i u e o Oceanog aphy, Tel Shikmona, 3108001Hai a,
Is ael
2 Flo ida A lan ic Uni e si y, Ha bo B anch Oceanog aphic
Ins i u e, Fo Pie ce, FL, USA
3 Wageningen Uni e si y, Biop ocess Enginee ing,
Wageningen, TheNe he lands
/ Published online: 28 Ma ch 2025
RINKEVICH ANDPOMPONI
de elopmen , wi h esea ch in his a ea da ing back nea ly
six decades (Rannou 1968, 1971; Gomo 1971; Vago
1971). The li e a u e e eals ha esea ch e o s ha e
p edominan ly ocused on six ma ine g oups, he Po i -
e a, Cnida ia, C us acea, Mollusca, Echinode ma a, and
U ocho da a (Vago 1971; Bayne 1998; Mo he sill and
Aus in 2000; Po s e al. 2020; Doma -Coulon and Blan-
choud 2022), wi h a ocus on de eloping basic echniques
o cell isola ion and cul u e. Wi h o e 500 publica ions
on aqua ic in e eb a e cell cul u es (Doma -Coulon and
Blanchoud 2022), almos all a emp s o es ablish s able,
long- e m cell cul u es om ma ine in e eb a es ha e
consis en ly ailed, wi h he easons emaining unclea
( e iewed in Vago 1971; Bayne 1998; Rinke ich 1999,
2005, 2011; Mo he sill and Aus in 2000; de Ca al e al.
2007; Yoshino e al. 2013; Cai and Zhang 2014). Wi h
he excep ion o a ecen b eak h ough in de eloping a
cell line om sponges (Hesp e al. 2023), no endu ing o
p oli e a i e cell lines ha e been success ully es ablished
o aqua ic in e eb a es. While hese s udies con ibu ed
ounda ional insigh s, hey equen ly encoun e ed sig-
ni ican challenges, such as sus aining cell iabili y and
achie ing long- e m p oli e a ion. This challenge is u -
he ampli ied by he lack o published eco ds on ailed
me hodologies, as highligh ed by G asela e al. (2012), o
oppo unis ic mic oo ganisms being mis aken o in e -
eb a e cells (Rinke ich 1999). The eluc ance o publish
unsuccess ul expe imen s s ems om hei pe cei ed
unsui abili y o mos scien i ic jou nals ha has hinde ed
p og ess by limi ing insigh s in o he challenges and obs a-
cles in ma ine in e eb a e cell cul u e.
The gene al pe sis en lack o success in de eloping cell
cul u es om ma ine in e eb a es, despi e di e se spe-
cies and expe imen al me hodologies, unde sco es c i ical
knowledge gaps in unde s anding he in i o equi emen s
and cellula mechanisms o hese cells. These gaps u he
hinde he de elopmen o no el s a egies o add ess pe -
sis en challenges in he ield, emphasizing he complexi y
o ma ine in e eb a e cellula sys ems and unde sco es he
need o inno a i e app oaches o o e come hese ba ie s.
We ha e e iewed ecen li e a u e on p e alen da a de i-
ciencies and lea ning sho alls unde lying he ailu es in he
de elopmen o cell cul u es om ma ine in e eb a es. This
e iew highligh s ou majo knowledge gaps (Fig.1): (1)
he need o speci ic and op imized imp o emen s in cul u e
media; (2) s a egies o ex end he limi ed in i o s emness
o isola ed cells; (3) le e aging “omics” app oaches o
ad ancing cell cul u e echniques; and (4) es ablishing
clea c i e ia o selec ing he mos sui able cell ypes o
in i o cul u es. Add essing hese gaps, as discussed below,
is essen ial o ad ancing he ield and unlocking he ull
po en ial o ma ine in e eb a e cell cul u e sys ems.
The need o speci ic andop imized
imp o emen s incul u e media (Fig.1)
Cul u e medium is he co ne s one o de eloping any cell
line. Nume ous a emp s ha e been made o de elop cul-
u e media o ma ine in e eb a es, wi h he mos common
s a egy in ol ing he op imiza ion o comme cially a ail-
able (o “o - he-shel ” (OTS)) mammalian o insec cell
cul u e media (see Table1). Fo mammalian cell cul u e,
he demand o mo e consis en , chemically de ined media
led o he de elopmen o media based on nu i ional bio-
chemis y and body luids analyses (F eshney 2000). No
unlike hese i s a emp s o de elop mammalian cell cul-
u e media, some esea che s ha e ied o c ea e de no o
syn hesis o media based on he ex acellula milieu in i o,
such as hemolymph o mollusks o il e ed sea wa e o
c enopho es and cnida ia (Table1). Howe e , hese e o s
ha e no yielded be e esul s han using op imized OTS
media, wi h no signi ican imp o emen in cell su i abili y
o p oli e a ion ( o a e iew, see Balak ishnan e al. 2022).
The selec ion o media o ma ine in e eb a e cell cul u e
is o en based on wha has been used by o he esea che s
ying o cul u e cells om he same species, a he han on
he speci ic nu i ional equi emen s o he o ganisms o cell
ypes. Fo example, he e is s ill limi ed unde s anding o he
in i o nu i ional equi emen s o p oli e a ion and main-
enance o sponge cells (Cai and Zhang2014). Con e sely,
o many molluscan species, he knowledge o osmolali y,
g ow h ac o s, and o he equi emen s is a ailable and could
be applied o he de elopmen o cell lines ( e iewed in Bal-
ak ishnan e al. (2022)).
Mammalian and insec cell cul u e media we e op i-
mized o mee he speci ic nu i ional needs o he cell ype
being cul u ed. They ange om simple composi ion (e.g.,
Eagle’s minimum essen ial medium (EMEM)) o mo e
complex op ions (e.g., Medium 199 (M199), Dulbecco’s
minimum essen ial medium (DMEM), RPMI 1640, Ham’s
(DMEM/F12), and G ace’s insec medium). Leibo i z’s
L-15 medium, o example, was designed wi h a di e en
bu e ing sys em o suppo cells in a CO2- ee a mosphe e.
While mos OTS cul u e media selec ed o ma ine in e e-
b a e cell cul u es end o be mo e complex, wi h some e en
combining wo OTS media (Qa i e al. 2023), i is likely
ha he a ionale behind hei ini ial de elopmen and he
speci ic cell ypes hey we e designed o suppo may no
ha e been ully conside ed du ing he selec ion p ocess (see
Yao and Asayama 2017, o a de ailed e iew o cell cul u e
medium de elopmen ).
Two o he impo an a iables o conside in de eloping
cul u e media a e he use o se um and an ibio ics. While
con amina ion o p ima y cul u es, p ima ily no ed in he
ea ly days ollowing isola ion (Rinke ich 1999; G asela
494
ADVANCING MARINE INVERTEBRATE CELL LINE RESEARCH: KNOWLEDGE GAPS
e al. 2012; Pe s. Obse .), will no be add essed he e, i is
e iden ha p ima y cul u es a e p one o a wide ange o
con aminan s, including p oka yo ic and euka yo ic o gan-
isms (G asela e al. 2012). Se um, such as cal , e al bo ine,
and ho se se um, is o en used in cell cul u es because i
p o ides essen ial g ow h ac o s, p o eins, lipids, ho mones,
and o he compounds ha can s imula e cell di ision. How-
e e , he downside o using a se um is ha he composi ion
is unde ined, leading o a iabili y be ween di e en ba ches.
An ibio ics a e commonly employed o con ol con ami-
nan s, wi h mos esea che s using some combina ions o
penicillin, s ep omycin, gen amicin, and/o ampho e icin
B o a ge bac e ia and ungi. Howe e , hese an ibio -
ics a e ine ec i e agains unicellula euka yo ic pa asi es,
such as one o he mos pe sis en con aminan s, h aus-
ochy ids (Rabinowi z e al. 2006; Qa i e al. 2021), and
do no add ess o he po en ial con aminan s, such as i uses
and mycoplasma. One no able excep ion is Nowo ny e al.
(2021), who added plasmocin as a p ophylac ic measu e in
cnida ian cell cul u es (Table1). Jus as cul u e media a e
op imized by es ing a ious combina ions o componen s
and assessing cul u es o iabili y and p oli e a ion, a simi-
la app oach should be applied o de e mine he mos e ec-
i e an ibio ic (o combina ion he eo ) o con ol he spe-
ci ic ype(s) o con amina ion while p ese ing he iabili y
and p oli e a i e capaci y o he cul u ed cells (e.g., G asela
e al. 2012). Fo applica ions such as he in i o p oduc ion
o ma ine-de i ed pha maceu icals, i will e en ually be nec-
essa y o de elop an ibio ic- ee cell lines. The impo ance
o main aining s ic asep ic echnique, pa icula ly du ing
cell isola ion, along wi h egula media changes, canno be
o e emphasized (G asela e al. 2012).
Table1 lis s some media used o e he las decade o
cell cul u e o sponges, c enopho es, cnida ia, c us aceans,
Figu e1. Fou key knowledge gaps iden i ied as ba ie s o he success ul de elopmen o ma ine in e eb a e cell cul u es.
495
RINKEVICH ANDPOMPONI
Table 1. Cell cul u e media, cell ypes, and cul u e applica ions o ma ine in e eb a e cell cul u e since 2014. No e ha all OTS media ha e been adjus ed o osmola i y, and mos media con-
ained an ibio ics
O ganisms Cul u e media Cell ypes Applica ion Re e ence
Po i e a: nume ous species o Dem-
ospongiae
M-199, M1, OpM1 A chaeocy e-en iched ac ions in
ea ly s udies; mixed cells in las
decade
In i o p oduc ion o ma ine na u al
p oduc s
Mun oe e al. (2019), Conkling e al.
(2019), Hesp e al. (2023)
C enopho a: Mnemiopsis leidyi Fil e ed seawa e , c enopho e mes-
oglea se um
Ec ode mal cells, smoo h muscle
cells, diges i e cells, spe m cells
Cellula di e en ia ion and physiol-
ogy; cellula basis o egene a ion
Vandepas e al. (2017)
Cnida ia: Anemonia i idis G ace’s modi ied insec medium Endode mal and gas ode mal cells
om egene a ing en acles, cnido-
cy es, dino lagella e symbion s
E ec o he mal s ess Ven u a e al. (2018)
Cnida ia: Nema os ella ec ensis and
Pocillopo a damico nis Leibo i z’s L-15 medium Cnidocy es, nema osomes, pu a i e
gas ode mal and epide mal cells
Model de elopmen : unc ional cell
biology
Nowo ny e al. (2021)
Cnida ia: 5 species o oc oco als Au ocla ed il e ed seawa e Agg ega es o glandula cells, g an-
ulocy es, acuolocy es, cilia ed
cells, se e al non-cha ac e ized
cell ypes
Models o s udy biomine aliza ion Hue e-S au e e al. (2015)
C us acea: Indian mud c ab Scylla
se a a Leibo i z’s L-15 medium, M 199,
G ace’s insec medium, MEM,
DMEM, TC-100 insec medium,
IPL-41 insec medium, RPMI
P ima y muscle cells, hemocy es E ec s o hea y me als; diseases Si akuma e al. (2019)
Mollusca: 23 species o bi al es,
gas opods, cephalopods
Eagle’s MEM, Medium 199, Leibo-
i z’s L-15 medium
S em-like cells in man le; hemo-
cy es; epi helial cells o gills and
diges i e gland; diges i e gland
cells; neoplas ic cells
P ima ily sho - e m expe imen s
ocused on speci ic applica ions
Re iewed by Balak ishnan e al.
(2022)
Mollusca: C assos ea i ginica Leibo i z’s L-15 medium o Op i-
MEM
De i ed om explan s o hea , gill,
man le, adduc o muscle, gonad,
diges i e gland
Iden i ica ion o no el cell mo phol-
ogies o s udies o eco oxicology,
i ology, immunology, disease
Po s e al. (2020)
Echinode ma a: An edon medi e -
anea Leibo i z’s L-15 medium Undi e en ia ed (amoebocy es,
coelomocy es) and plu ipo en
cells (phagocy es, g anulocy es,
dedi e en ia ed cells)
Cell pheno ypes esponsible o a m
egene a ion
Di Benede o e al.(2014)
Echinode ma a: se e al species o
holo hu ians
Leibo i z’s L-15 medium Cells om egene a ing gu udi-
men s: sphe ical-, o al-, spindle-
shaped cells
De elopmen al and egene a i e
biology
Bello e al. (2015)
Echinode ma a: Apos ichopus
japonicus Leibo i z’s L-15 medium In es inal cells: ound and spindle-
shaped
Wang e al. (2020)
Tunica a: Bo yllus schlosse i 5 a ia ions o “ unica e g ow h
medium” (TGM): ASW,
DMEM/F-12, RPMI 1640
blood cells (se e al ypes) In i o p oduc ion o biopha ma-
ceu icals; s em cells; de elopmen-
al biology
Qa i e al. (2023)
496
ADVANCING MARINE INVERTEBRATE CELL LINE RESEARCH: KNOWLEDGE GAPS
mollusks, echinode ms, and unica es. I also highligh s he
cell ypes isola ed o cul u e, and he applica ions explo ed
in hese s udies. The mos common medium selec ed o e
he pas decade is Leibo i z’s L-15 medium, likely due o i s
design o incuba ing cells in a CO2- ee a mosphe e. How-
e e , ecen success in cul u ing sponge cells in a ia ions
o Medium 199 (Conkling e al. 2019; Hesp e al. 2023)
sugges s ha , a leas o sponges, a medium speci ically
designed o a CO2- ee a mosphe e may no be equi ed.
I is wo h no ing ha cells om species wi h pho osyn-
he ic symbion s, such as some cnida ia, can be co-cul u ed
wi h hei symbion s and main ained wi hou he use o
o he nu ien s in he cul u e medium (Hue e-S au e e al.
2015). The below e iew o cul u e medium knowledge gaps
ocuses on wo g oups, sponges and unica es, o which
di e en app oaches o medium de elopmen ha e been
explo ed, yielding some o he mos p omising esul s.
Sponges Cell cul u e medium is a complex mix u e o nu i-
en s and g ow h ac o s in which he con ibu ion o each
componen is c ucial o he long- e m main enance and
g ow h o each speci ic cell ype (P ice 2017). To op imize
cul u e media o sponge cells, esea che s ha e u ilized a -
ious me hods, including he Placke –Bu man design wi h
esponse su ace me hodology and uni o m design (Zhao e
al. 2005), one-dimensional sea ch app oaches (Willoughby
and Pomponi 2000), and gene ic algo i hms (Mun oe e al.
2019). These s udies led o imp o emen s in me abolic and
es e ase ac i i y, DNA and p o ein con en , and, in some
cases, inc eases in cell numbe s. Ye , he de eloped media
a e gene ally species-speci ic, as he op imized medium o -
mula ions did no yield he same esul s when applied o
di e en sponge species (Zhao e al. 2005; Conkling e al.
2019). This species-speci ici y emains a common challenge
in sponge cell cul u e.
Designing and op imizing cul u e media equi e me hods
ha can handle nume ous componen s wi h complex in e ac-
ions. Gene ic algo i hms (GAs) ha e been used o op imize
pa ame e s like insec cell g ow h (Ma eijn e al. 2003),
mic oalgal cells (Camacho-Rod íguez e al. 2015; López-
Rosales e al. 2015), sponge cell me abolism (Mun oe e al.
2019), and yeas p oduc ion (E schmann e al. 2004). GAs
op imize mul iple pa ame e s (medium componen s) wi hou
needing de ailed in o ma ion on compound concen a ions
o cellula me abolism (Ma eijn e al. 2003). They simula e
na u al selec ion by andomly gene a ing a “popula ion” o
expe imen al condi ions, allowing he bes pe o me s o
“ma e” and e ol e h ough c osso e and mu a ions un il
op imal esul s a e achie ed (Rangana h e al. 1999; Weus-
e -Bo z 2000; Ma eijn e al. 2003).
The GA o e s se e al ad an ages o e o he medium
op imiza ion me hods o sponge cell cul u e. I allows o
he selec ion o componen concen a ion om a ange o
alues, wi hou needing p io knowledge o in e ac ion o
cy o oxici y, enabling he e alua ion o mo e componen s
in less ime (Ma eijn e al. 2003; E schmann e al. 2004).
Typically, a GA can each nea -op imum esul s in unde 10
gene a ions (Weus e -Bo z 2000), es ing hund eds o e en
housands o medium composi ions. Fo example, op imiz-
ing he amino acid composi ion o Dysidea e he ia sponge
cells ook jus ou gene a ions, leading o he c ea ion o
he i s op imized a ia ion o Medium 199, M1 (Mun oe e
al. 2019). Using he same GA wi h M1 medium as he base
esul ed in a new medium a ia ion, OpM1, wi h op imized
concen a ions o i amins, ace elemen s, lipids, g ow h
ac o s, and se um (Hesp e al. 2023). Bo h M1 and OpM1
imp o ed me abolic ac i i y and cell p oli e a ion in p i-
ma y cell cul u es compa ed o ea lie media. Sup isingly,
al hough M1 did no esul in cell p oli e a ion in he spe-
cies o which i was de eloped, a es o he medium on 12
andomly selec ed species om di e se o de s esul ed in
signi ican cell p oli e a ion o nine species (Conkling e al.
2019). When cells o he deep-wa e sponge Geodia ba e i
we e cul u ed wi h OpM1, hey exhibi ed a apid g ow h
a e and nea ly 100 popula ion doublings (Hesp e al. 2023).
I onically, he wo species used in he Pomponi lab o dec-
ades (Dysidea e he ia, Axinella co uga a) did no p oli e -
a e in ei he o hese media, emphasizing he impo ance o
no limi ing medium op imiza ion o he esea che ’s “model
species.”
Tunica es A he o he end o he phylogene ic spec um
a e he unica es, wi h he species Bo yllus schlosse i being
a longs anding ocus o ma ine in e eb a e cell cul u e
esea ch o decades. S udies on his species ha e explo ed
he ini ia ion o p ima y cell cul u es om emb yos, epi he-
lial cells, and ci cula ing blood cells, assessing he e ec s
o g ow h ac o s on p ima y cul u es, es ablishing a de ined
medium o blood cells, and e alua ing s emness signa u es
in epi helial monolaye s (Rinke ich and Rabinowi z 1993,
1994, 1997, 2000; Rabinowi z and Rinke ich 2004, 2005,
2011; Rabinowi z e al. 2009; Qa i e al. 2023). Despi e
he occu ence o se e al ypes o s em cells in B. schlosse i
blood, epi helium, and emb yos (Rinke ich and Rabinow-
i z 1993; Rabinowi z and Rinke ich 2004, 2011; Rabinow-
i z e al. 2009; Qa i e al. 2022), no cell lines ha e been
es ablished.
Qa i e al. (2023) ecen ly conduc ed a comp ehensi e
analysis o B. schlosse i blood cell p oli e a ion in i e di -
e en medium o mula ions, wi h “ unica e g ow h medium”
(TGM) as he base. This TGM base con ains L-glu amine,
HEPES bu e , sodium py u a e, and e al bo ine se um.
Fi e a ia ions o he TGM base we e p epa ed wi h o wi h-
ou OTS media (DMEM/F-12 (Ham) o RPMI 1640), wi h
o wi hou a i icial seawa e (ASW), and each wi h di e en
combina ions o an ibio ics, as ollows: TGM1-DMEM/F-12
497

RINKEVICH ANDPOMPONI
(Ham), penicillin, s ep omycin; TGM2-RPMI 1640, peni-
cillin, s ep omycin; TGM3-ASW, penicillin, s ep omycin,
ampho e icin B, gen amicin; TGM4-DMEM/F-12 (Ham),
ASW, gen amicin, penicillin, s ep omycin; and TGM5-
DMEM/F-12 (Ham), ASW, penicillin, s ep omycin (Qa i
e al. 2023). Cul u es we e moni o ed using con ocal mic os-
copy, wi h each cell ype dis inguished by i s au o luo es-
cence emission in ensi y ac oss he blue, g een, ed, and
a - ed channels. P oli e a ion was measu ed h ough immu-
no luo escence de ec ion o p oli e a ing cell nuclea an igen
(PCNA+). Wi hin he i s week, an inc ease in cell p oli -
e a ion was obse ed among dis inc blood cell ypes. The
dis ibu ion o cell ypes a ied ac oss he i e media and
changed o e ime. PCNA+ ac i i y also a ied among he
media and changed o e ime, wi h a ious blood cell ypes
p oli e a ing a di e en imes. Wi hin 1mon h, medium-
speci ic p ima y cul u es we e de eloped. This is an impo -
an inding, p o iding compelling e idence o he po en ial
o de elop cell- ype speci ic cul u es o unica es and o he
ma ine in e eb a es.
While he de elopmen o cul u e media ypically p i-
o i izes cell p oli e a ion (expansion), i may be necessa y
o cul u e di e en ia ed cells. This would equi e swi ch-
ing om a medium ha suppo s expansion o one ha p o-
mo es di e en ia ion ( o e iew, see Yada e al. 2020). The
esul s o Qa i e al. (2023) p o ide a p omising example o
he po en ial o use di e en media o cul u e a ious ma ine
in e eb a e cell ypes, each wi h dis inc p oli e a i e capac-
i ies and unc ions.
S a egies oex end helimi ed in i o
s emness o isola ed cells (Fig.1)
S emness e e s o he cha ac e is ics and p ope ies ha
de ine s em cells, pa icula ly in he con ex o hei abil-
i y o sel - enew. P ese ing he “s emness” o s em cells,
and emaining undi e en ia ed, is essen ial o success ul
applica ion in a wide ange o scien i ic and medical ields
(Hu ley 2015), as in he cell cul u e ield. In mammalian
sys ems, he in i o cul u e o s em cells has p og essed
apidly, d i en by inno a ions like niche-based me hods (Pal
and Das 2017), 3D sphe oid and o ganoid cul u es (Yen e
al. 2023), bio eac o s, 3D sca olds (Yi e al. 2018), and
coa ing cul u e dishes wi h a achmen -inducing componen s
(e.g., gela in, Ma igel, collagen; McKee and Chaudh y
2017). These me hods add ess many limi a ions o adi-
ional 2D cul u es, o e ing signi ican po en ial o enhance
s em cell he apeu ic applica ions in egene a i e medicine
and beyond.
The applica ion o s emness echnologies o ma ine in e -
eb a e cell cul u es is s ill in i s ea ly s ages, e ealing a sig-
ni ican knowledge gap. Unlike he signi ican ad ancemen s
in e eb a e cell cul u es and cell lines, decades o esea ch
ha e ye o p oduce a pe manen cell line om ma ine in e -
eb a es, wi h only one ecen success ul app oach in ol ing
sponge cells (Hesp e al. 2023). Unde in i o condi ions,
isola ed cells om many ma ine in e eb a e axa cease di i-
sion and en e quiescence wi hin 24–72h. To add ess his
esea ch challenge, i is essen ial o modi y he uni e sal qui-
escen s a e, enabling ce ain cells o acqui e plu ipo ency,
allowing o inde ini e di ision and achie ing immo ali y
(Rinke ich 1999, 2005, 2011; Anoop e al. 2021; Suda shan
e al. 2023). Po en ial s em cell ac i i ies can u he be elu-
cida ed using assays ha in ol e egene a ion p ocesses in
ma ine in e eb a es (Rinke ich e al. 2010; Le anoni e al.
2024; Musg o e e al. 2024b). Ha nessing s em cells om
ma ine in e eb a es o ex ended in i o s emness o e s a
p omising app oach o o e coming cu en obs acles, hough
i emains a challenging ield. Se e al au ho s (de Ca al e
al. 2007; Sun e al. 2007; Odin so a 2009; Rinke ich 2011;
Doma -Coulon and Blanchoud 2022; Mohaje e al. 2024)
ha e sugges ed ha ex ended in i o s emness could be
achie ed using (a) adul s em cells om ma ine o ganisms o
(b) me hodologies de eloped o mammalian-induced plu i-
po en s em (iPS) cells o o umo igenesis (e.g., Anoop e
al. 2021; Suda shan e al. 2023; no discussed he e).
Ma ine in e eb a es, including sponges, cnida ians,
la wo ms, c us aceans, mollusks, echinode ms, and ascid-
ians, possess subs an ial pools o adul s em cells ha a e
i al o main enance, egene a ion, and asexual ep oduc-
ion (Balla in e al. 2018, 2022; Rinke ich e al. 2022).
Ye ma ine in e eb a e s em cells (MISC) exhibi a wide
ange o occu ences and phylum-speci ic cha ac e is ic
mo phologies and beha io s, wi h he ypical well-cha ac-
e ized sponge a chaeocy es and choanocy es, hyd ozoan
I-cells, pla yhelmin h and acoel neoblas s, and unica e
hemoblas s (Doma -Coulon and Blanchoud 2022;E esko-
sky e al. 2022, 2024; Rinke ich e al. 2022). Rep esen ing
up o 40% o an o ganism’s cells, hey play c ucial oles
in p ocesses such as whole-body egene a ion, do mancy,
agame ic asexual ep oduc ion, and inde e mina e g ow h,
u he ecognized as alid uni s o selec ion (Rinke ich e
al. 2009, 2022; Vanni e al. 2022b). MISC a ise a a ious
li e s ages, displaying bo h di e en ia ed and undi e en i-
a ed pheno ypes and o en exhibi ing amoeboid mo emen
(Doma -Coulon and Blanchoud 2022; Rinke ich e al.
2022). Typically, plu i- o o ipo en , MISC may exp ess
ge m-cell ma ke s, bu hey usually lack ge m-line seques-
a ion and do no eside in dis inc niches (Doma -Coulon
and Blanchoud 2022; Ma inez e al. 2022; Rinke ich e al.
2022; Vanni e al. 2022b).
As o he abo e, he iden i ica ion, isola ion, and cha -
ac e iza ion o s em cells in aqua ic in e eb a es emain
majo echnical challenges, o en equi ing species-spe-
ci ic app oaches and he use o alida ed s em cell ma ke s
498
ADVANCING MARINE INVERTEBRATE CELL LINE RESEARCH: KNOWLEDGE GAPS
(Doma -Coulon and Blanchoud 2022; Rinke ich e al.
2022). Fo ins ance, in he b anching co al S ylopho a pis-
illa a, s em cells we e no iden i ied in he cell a las
de i ed om bo h la al and adul issues, despi e using
enzyma ic o mechanical dissocia ion me hods and known
ma ke s (Le y e al. 2021). Fu he mo e, esea ch e o s
ha e es ablished p o ocols o isola ing iden i ied s em-like
cells in only a ew species (e.g., Hayashi e al. 2006; Sun
e al. 2007; Hemm ich e al. 2012; Reyes-Be mudez e al.
2021). Ano he p omising app oach in ol es gene ically
modi ied ma ine in e eb a es, whe e ansgenic epo e s
o s emness p ope ies can be enginee ed (e.g., in Hyd a,
Juliano e al. 2014). Howe e , apping in o he di e se ange
o speci ic echniques de eloped o o he axa (including
di e en species wi hin he same phylum) holds g ea po en-
ial o b oade gene aliza ion and applica ion.
Few publica ions o da e ha e speci ically ocused on
de eloping cell cul u es om ma ine in e eb a es using
pu i ied o en iched s em cells (e.g., Zhang e al. 2003;
Sun e al. 2007; Reyes-Be mudez e al. 2021). Mohaje e
al. (2024) ecen ly compiled di e se insigh s in o MISC
de ec ion by e iewing s udies ha ocus on iden i ying
adul s em cells in a ious ma ine in e eb a e o ganisms,
wi hou ci ing pape s ha applied MISC in cell cul u es.
Ye , in se s o 4-day p ima y sponge cell cul u es, pu i ied
a chaeocy es (adul s em cells) om Hymeniacidon pe le e
showed a signi ican 2.5- old inc ease in o al cell numbe ,
showcasing hei po en ial o de eloping sponge cell cul-
u es o p oduce aluable sponge-de i ed pha maceu icals
(Sun e al. 2007). Following he same a ionale, Zhang e al.
(2003) u ilized sponge p immo ph cul u es de i ed om an
a chaeocy e-dominan cell popula ion ha we e en iched ia
a Ficoll g adien , a he han he ypical mixed-cell popula-
ion me hod. This app oach esul ed in signi ican inc eases
in DNA syn hesis, cell p oli e a ion (up o h ee old), cell
g ow h (up o ou old), and, in long- e m cul u es, enhanced
me abolic ac i i y o he p immo phs. Reyes-Be mudez e
al. (2021) p esen ed gene exp ession p o iles o cul u ed
co al (Ac opo a digi i e a) cells, highligh ing he egula-
o y gene ne wo ks in ol ed in plu ipo ency and di e en ia-
ion. In cul u es de i ed om he co al’s ip agmen s ( he
colony’s as es -g owing issues), he in i o ansc ip ion
p o ile esembled ha o ea ly la ae, wi h o e exp ession
o o hologs o p eme azoan and Hyd a s em cell ma ke s,
along wi h ansc ip s associa ed wi h cell di ision, mig a-
ion, and di e en ia ion.
iPS cells a e a ype o s em cells gene a ed by ep og am-
ming soma ic (adul ) cells o a plu ipo en s a e. The mos
commonly used genes o ep og amming soma ic cells in o
iPS cells (known as he Yamanaka ac o s) a e Oc 4, Sox2,
Kl 4, and c-Myc, all ound in ma ine in e eb a es (Rink-
e ich e al. 2022; Vanni e al. 2022a). Ye , he e is a g ea e
knowledge gap ega ding he use o iPS cells o ma ine
in e eb a e cell cul u es. In e eb a e sys ems, he gen-
e a ion o iPS cells om soma ic cells mimics on ogene ic
p ocesses, bu may no pa allel na u al in i o mechanisms
(Cha e jee e al. 2015). Ye , he iPS echnology, whe e
soma ic cells ha e been modi ied o acqui e an emb yonic
s em-cell-like capaci y, holds g ea p omise o de eloping
ma ine in e eb a e cell cul u es, as i can o e come he
majo challenge o cellula quiescence obse ed in i o
a e isola ing adul cells (Rinke ich 2011). Wi h his in
mind, mo e esea ch is equi ed o e alua e he po en ial o
es ablished mammalian echniques in he ma ine in e e-
b a e cells a ena, ying o employ endogenous homologs o
Yamanaka ac o s. Clea ly, mo e e o s in o ma ine in e -
eb a e plu ipo ency may add o his subjec as addi ional
Yamanaka-like ac o s could be unco e ed. Resea ch on
iPS cell cul u e condi ions in he mammalian sys ems has
p ima ily ocused on wo key a eas: enhancing ep og am-
ming e iciency and quali y, and de eloping human iPS cell
cul u e sys ems o clinical applica ions (Mochiduki and
Oki a 2012). Following he abo e, iPS cells de i ed om
ma ine in e eb a es could hen emain in a p imi i e s a e
and eadily p oli e a e in o a ious cell lineages, le e aging
hei inna e capaci y o sel - enewal and di e en ia ion o
acili a e he c ea ion o immo al cell lines. To da e, no
s udies ha e applied iPS echnology o de elop cell cul u es
om ma ine in e eb a es. In addi ion o he use o s em
cells and he iPS cells me hodology o ex ending he limi ed
in i o s emness, ecen publica ions (Anoop e al. 2021;
Suda shan e al. 2023) u he a emp ed he app oaches o
cell hyb idiza ion and ec opic exp ession o mu a ed genes.
Le e aging “omics” app oaches
o ad ancing cell cul u e echnique (Fig.1)
In he las wo decades, apid ad ancemen s in mamma-
lian cell cul u e and cell enginee ing ha e highligh ed he
impo ance o using “omics” echniques o be e unde s and
cellula mechanisms and pa hways, including apop osis,
cell p oli e a ion, cell s imula ion o quenching, and he
e ec s o physicochemical en i onmen s (Rinke ich 2005;
Kuys e mans e al. 2007; Čupe lo ić-Cul e al. 2010; Zhang
e al. 2013; Fa ell e al. 2014; Liu e al. 2019; O’B ien
and Hu 2020), as well as o applied app oaches such as
cance esea ch (Be g e al. 2017) and d ug de elopmen
(Bu iani e al. 2012). The “omics” echniques ha a e in e-
g a ed wi h cell cul u e de elopmen enable he de ec ion o
comp ehensi e changes in exp ession a he ansc ip omic
(mRNA), p o eomic (p o ein), and me abolomic (me abolic)
le els, among o he s. These usages imp o e unde s anding
cell cul u e sys ems, as well as he s a uses (in exp ession
le els) depic ed be ween di e en labo a o ies wo ldwide
on he same cell line o among successi e passages in an
499
RINKEVICH ANDPOMPONI
es ablished cell line, such as HeLa cells (Liu e al. 2019).
The applica ion o mul i-omics echniques u he imp o es
he p og ession in cell cul u e op imiza ion and biop ocess
designs (Fa ell e al. 2014). As an example, p o eomics can
e eal he o e all ex en o pep ide exp essions wi hin a spe-
ci ic cell cul u e condi ion. When combined wi h ansc ip-
omic and me abolomic app oaches, p o eomics p o ides
aluable insigh s in o how cells unde in i o condi ions
espond o he a ious epe oi e o condi ions. Me abo-
lomics, which ep esen s he global quan i a i e assessmen
o me aboli es unde in i o condi ions, p o ides c ucial
da a o sys em-le el analysis and modeling o biological
p ocesses when conduc ed alongside o he “omics” meas-
u emen s (Čupe lo ić-Cul e al. 2010). Wi h ad ancemen s
in cu en and eme ging echnologies, “omics” esea ch may
hus e ol e o add ess mo e complex sys emic ques ions and
se e as a aluable ool in he de elopmen o cell cul u es
om ma ine in e eb a es.
We iden i y a signi ican knowledge gap in he appli-
ca ion o “omics” app oaches o de eloping cell cul u es
om ma ine in e eb a es. Recen ad ancemen s in nex -
gene a ion sequencing echniques, pa icula ly in single-
cell ansc ip omics, a e allowing esea che s o cha ac e -
ize s em-like cells ac oss a g owing numbe o axa (e.g.,
Musse e al. 2021; Rinke ich e al. 2022), ma king an
essen ial i s s ep owa d hei isola ion and in i o cul u e.
Fu he , insigh s in o in i o issue homeos asis, cell p oli -
e a ion dynamics, and soma ic s em cell niches (Ma inez
e al. 2022) can aid in selec ing speci ic issue spo s wi h
high p oli e a i e po en ial. Ye , despi e declining cos s o
sequencing and “omics” echnologies and inc easing e o s
o cha ac e ize di e en ia ed and s em cells in ma ine in e -
eb a es, sugges ing “omics” da a will soon be a ailable o
mos axa (Doma -Coulon and Blanchoud 2022), he e
emains a sho age o s udies demons a ing he b oad acces-
sibili y o such da a ac oss nea ly all ma ine in e eb a e
axa. Fu he , while he e is a b oad ange o “omics” s udies
ocusing on ma ine in e eb a es a he whole-o ganism le el
(e.g., Imbs e al. 2021; Romano e al. 2022; Kül z e al.
2024), e y li le has been di ec ed owa ds he use o omics
me hodologies o he de elopmen o p ima y cell cul u es
om ma ine in e eb a es (bu see Kawamu a e al. 2021),
wi h limi ed scien i ic app oaches.
One such app oach is he wo k o Tsuchiya e al. (2023)
ha conduc ed ansc ip omic analysis o p ima y lymphoid
cells in ku uma sh imp (Ma supenaeus japonicus) o unde -
s and gene exp ession changes unde hei cul u e condi-
ions. RNA sequencing a ou cul u e ime poin s (days 1,
3, 4, 6) iden i ied h ee gene exp ession pa e ns: (1) down-
egula ed on days 3–6, (2) up egula ed on days 3–4, and
(3) up egula ed on day 6. No ably, he sh imp VEGF3 and
i s ecep o showed signi ican down egula ion, con i med
by qPCR. These indings highligh ed he need o imely
VEGF signaling supplemen a ion in o cul u ed cell medium
o main ain s able, long- e m sh imp lymphoid cell cul u es.
P omising in e media e app oaches ha may open he oad
o cell cul u e s udies in ol e he indi ec use o “omics”
echniques o in i o s udies o ma ine in e eb a es. This
is pa icula ly aluable o de eloping in i o eco oxicol-
ogy models, such as assessing a he ansc ip omic le el he
e ec s o hea y me als on bi al e hemocy es (de Boissel
e al. 2017) o a he p o eomics le el, he oxici y o nano-
pa icles on bi al e issues (De Felice and Pa olini 2020).
The same implies o app oaches sea ching o he in eg a-
ion be ween immune cells and s em cells in ma ine in e -
eb a es (Balla in e al. 2021) o elucida ing p o eomic ou -
comes om pu i ied cell ypes o c ay ish hemopoie ic issue
(Söde häll and Junkunlo 2019). The applica ion o “omics”
echnologies o ma ine in e eb a e cell cul u es is s ill in i s
ea ly s ages, highligh ing a signi ican knowledge gap.
Es ablishing clea c i e ia o selec ing
hemos sui able cell ypes o in i o
cul u es (Fig.1)
As no ed ea lie , he selec ion o species plays a c ucial
ole in he success o es ablishing a cell line, pa icula ly
conside ing he species-speci ici y o he cul u e media
de eloped o ma ine in e eb a e cell cul u e (Zhao e al.
2005; Conkling e al. 2019). I he goal o he esea ch is o
de elop p ima y cul u es o cell lines o a speci ic applica-
ion ( a he han de eloping a cell line pe se), he choice o
species (and cell ypes) could po en ially impede he suc-
cess ul es ablishmen o long- e m cul u es. A con inued
lack o success in cell p oli e a ion using he esea che ’s
“model species” may wa an in es iga ion o cells om
o he ela ed species (Conkling e al. 2019).
Selec ion o cell ypes o ma ine in e eb a e in i o
cul u es may be based on he pu a i e s emness o he cells,
he in ended use o he cul u es, and/o cell lineage (Fig.1).
“O he cell ypes” (Fig.1) includes cells o which hei
emb yonic o igin is no known and o which hei mo -
phologies change in cul u es. Fo example, sponges do no
ha e ue ge m laye s (E esko sky and Dondua 2006) and
sponge cells a e able o ansdi e en ia e (Adamska 2018),
which makes cha ac e iza ion o cell lineage challenging.
Indeed, cell lines may no be necessa y o sho - e m s ud-
ies ha can add ess hypo heses using p ima y cell cul u es,
and he op imiza ion o cul u e a iables will depend on
he hypo heses being es ed. Fo example, And ade e al.
(1999) used a chaeocy e-en iched ac ions o cells om
he sponge Teichaxinella mo chella (= Axinella co uga a),
cul u ed o 48h in Medium 199, o iden i y he biosyn he ic
o igin o s e ensine, a sponge alkaloid, using adiolabeled
amino acid p ecu so s. Balak ishnan e al. (2022) p o ide
500
ADVANCING MARINE INVERTEBRATE CELL LINE RESEARCH: KNOWLEDGE GAPS
a comp ehensi e e iew o applied esea ch in oxicology,
pa hology, and neu ophysiology using p ima y molluscan
cell cul u es.
A c i ical ac o o conside is he sou ce o he cells
(emb yos, la ae, ju eniles, o adul s) which will depend
on he ype o cells equi ed o de elop a cell line o add ess
a speci ic hypo hesis. Fo example, i he goal is o isola e
s em cells, emb yos o la ae a e he ob ious choice. How-
e e , ob aining emb yos o la ae om some species may
be challenging i hei ep oduc i e cycles a e unknown o
hey do no elease la ae when aken om hei na u al
en i onmen .
The de elopmen o ma ine in e eb a e cell cul u es has
adi ionally ocused on using issues, o gans, o pa o he
en i e o ganism as he sou ce o cells. Fo sponges, as an
example, agmen s o he whole animal a e dissocia ed in o
single cells, some o which may e e in o a s em-like s a e
and/o ansdi e en ia e (Adamska 2018). In he cnida ians,
howe e , when agmen s o he en i e specimen a e dissoci-
a ed in o single cells, hese cells emain in suspensions wi h-
ou a isible e-di e en ia ion, usually s aying in e minally
di e en ia ed s a e (Doma -Coulon and Blanchoud 2022).
In con as , when he ec ode mal issue is speci ically iso-
la ed, hen ec ode mal monolaye s a e speci ically de eloped
(Rabinowi z e al. 2016). Table1 p o ides a lis o he mos
commonly used ma ine in e eb a e cell ypes o e he pas
decade o cell cul u es de elopmen . Me hods o isola ion
and sepa a ion a e no he ocus o his e iew; ye , hey a e
desc ibed in he pape s ci ed in Table1.
The e may be aluable insigh s o gain om ecen e o s
o de elop cul i a ed mea s in gene al, and cul i a ed c us-
acean mea , in pa icula ( e iewed by Musg o e e al.
2024a). Since no c us acean cell lines cu en ly exis , he
ocus has been on isola ing and cul u ing adul muscle s em
cells om lobs e ail muscles, wi h some success (Jang e
al. 2022). Howe e , since his app oach in ol es sac i icing
he sou ce o ganism, al e na i e me hods a e being explo ed
o es ablish an immo alized cell lines wi h bo h p oli e a-
i e and myogenic po en ial om non-le hal sou ces (Mus-
g o e e al. 2024a). Among he mos p omising sou ces a e
hema opoie ic s em cells, which a e ound in many ma ine
in e eb a es (Rinke ich e al. 2022) and ha in c us a-
ceans show some po en ial o di e en ia ing in o muscle
cells (Musg o e e al. 2024a).
This leads back o a discussion o s emness and “omics.”
To b eak h ough he bo leneck ha has hinde ed he de el-
opmen o ma ine in e eb a e cell lines may equi e mo e
esea ch o isola e and iden i y pu a i e s em cells o he
a ge ed species, he use o immuno luo escen an ibodies
o cha ac e ize bo h undi e en ia ed and di e en ia ed cells
o de e mine hei p oli e a i e capaci y (Jang e al. 2022;
Qa i e al. 2023; Musg o e e al. 2024a), and he use
o “omics” echnologies o e ine he selec ion. Con inuing
o use he same ial-and-e o app oach wi hou a be e
unde s anding o he p oli e a i e and unc ional capabili-
ies o he cells we a e a emp ing o cul u e will only lead o
inc emen al imp o emen s. A b eak h ough may come wi h
he use o mo e sophis ica ed ools.
Discussion
I is well documen ed ha isola ed cells om a wide ange o
ma ine in e eb a es cease di ision in i o wi hin 24–72h
a e isola ion, en e ing s a es o cellula quiescence (Rink-
e ich 2011). This happens despi e he ac ha many ma ine
in e eb a es ac oss a b oad ange o axa ( om sponges,
cnida ians, and la wo ms o u ocho da es) possess plu i-
po en and e en o ipo en adul s em cells h oughou hei
li es. These cells a e u ilized no only o main aining he
animal’s body bu also o biological p ocesses like asex-
ual ep oduc ion, budding, and whole-body egene a ion
(Rinke ich e al. 2022). This widesp ead quiescen s a e
mus be al e ed so ha a leas some o he quiescen cells
egain plu ipo ency, enabling hem o di ide and achie e
immo ali y.
In he p esen o e iew, we iden i ied and examined ou
key knowledge gaps in he de elopmen o cell cul u es om
ma ine in e eb a es, including he op imiza ion o cul u e
media, he de elopmen o s a egies o p olong he s emness
o isola ed cells, le e aging “omics” echnologies o imp o e
cell cul u e, and he selec ion o app op ia e cell ypes o
in i o cul i a ion (Fig.1). Gi en he cu en s a e-o - he-
a in his ield, add essing hese gaps and pe haps e en
in eg a ing app oaches o add ess he gaps a e c i ical o
ad ancing ma ine in e eb a e cell cul u e sys ems.
The li e a u e e eals ha he biological easons o
he ailu e o es ablish long-las ing and p oli e a ing cell
cul u es om ma ine in e eb a es emain unclea , high-
ligh ing he need o deepe insigh s. These insigh s may
in ol e de eloping specialized app oaches a he han
elying on uni e sal echniques, such as designing ailo ed
cul u e media, employing speci ic cell dissocia ion p o o-
cols, selec ing di e en cells, issues, o gans, o o ganisms
o he s udy, and u ilizing issue agmen s o adhe en /
non-adhe en cells. The li e a u e u he depic s ha i
is p e e able o cus omize hese s a egies o a pa icula
model issue, o gan, o species, a he han adop ing a one-
size- i s-all app oach (Vago 1971; Rannou 1971; Bayne
1998; Rinke ich 1999, 2005; Mo he sill and Aus in 2000;
Conkling e al. 2019; Po s e al. 2020; Balak ishnan
e al. 2022; Doma -Coulon and Blanchoud 2022). This
con as s wi h he assump ion ha all cells ac oss di e en
axa wi hin he kingdom Animalia sha e simila nu ien
equi emen s, a e egula ed by iden ical de elopmen al and
physiological-biochemical pa hways, and a e in luenced
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