Sho Communica ion
C yogenic, bu no hypo he mic, p ese a ion dis up s he ex acellula
ma ix o cell shee s
Sa a F ei as-Ribei o
a,b
, And eia F. Ca alho
a,b,c
, Daniel B. Rod igues
a,b
, Luís Ma ins
a,b
,
Rica do A. Pi es
a,b
, Ve a M. Mendes
d
, B uno Manadas
d,e
, Ma iana Ja nalo
,g
, Rica do Ho a
,g
,
Rui L. Reis
a,b
, Rog´
e io P. Pi aco
a,b,*
a
3B’s Resea ch G oup, I3Bs – Resea ch Ins i u e on Bioma e ials, Biodeg adables and Biomime ics, Uni e si y o Minho, Headqua e s o he Eu opean Ins i u e o
Excellence on Tissue Enginee ing and Regene a i e Medicine, A ePa k, Pa que de Ciˆ
encia e Tecnologia, Rua A e 1, Edi ício 1 (Sede), 4805-694 Ba co, Guima ˜
aes,
Po ugal
b
ICVS/3B’s–PT Go e nmen Associa e Labo a o y, B aga/Guima ˜
aes, Po ugal
c
Ins i u o de Ciˆ
encias Biom´
edicas Abel Salaza (ICBAS), Uni e sidade Do Po o, Po o, Po ugal
d
CNC-Cen e o Neu oscience and Cell Biology, Uni e si y o Coimb a, Coimb a, Po ugal
e
Ins i u e o In e disciplina y Resea ch, Uni e si y o Coimb a, Coimb a, Po ugal
Depa men o Plas ic and Recons uc i e Su ge y, and Bu n Uni y, Cen o Hospi ala de S˜
ao Jo˜
ao, Po o, Po ugal
g
Facul y o Medicine - Uni e si y o Po o, Po ugal
ARTICLE INFO
Keywo ds:
Cell shee s
Ex acellula ma ix
Biop ese a ion
Hypo he mic p ese a ion
C yogenic p ese a ion
ABSTRACT
Cell shee (CS)-based app oaches hold signi ican po en ial o issue egene a ion, elying on he ex acellula
ma ix (ECM) o success. Like in na i e issues, he ECM p o ides s uc u al and biochemical suppo o cellula
homeos asis and unc ion. E ec i e p ese a ion s a egies ha main ain ECM in eg i y a e c i ical o enhance
he he apeu ic po en ial o CS-based app oaches. While c yogenic and hypo he mic p ese a ion me hods o e
po en ial solu ions, hei impac on CS ECM s uc u e is no ully unde s ood. The e o e, a comp ehensi e
analysis o he ECM o hASCs CS ollowing c yogenic and hypo he mic p ese a ion o 3 and 7 days, was
conduc ed. Al hough p o eomic analysis indica ed ha c yop ese a ion had no signi ican e ec on he o e all
composi ion o he ECM, i induced signi ican ECM s uc u al al e a ions, pa icula ly dis up ing collagen o -
ganiza ion, which was no obse ed ollowing hypo he mic p ese a ion. These s uc u al changes we e
accompanied by al e a ions in mechanical p ope ies, including a educ ion in elas ic modulus. In con as ,
hypo he mic p ese a ion main ained ECM in eg i y and mechanical p ope ies simila o he con ol. The
no able ECM s uc u al changes ollowing c yogenic p ese a ion can po en ially impac cellula beha io ,
including adhesion, p oli e a ion, and di e en ia ion, he eby a ec ing he e icacy o CS he apies in i o. This
sugges s ha hypo he mia may o e a p omising al e na i e o c yop ese a ion o p ese ing CS in eg i y and
unc ionali y.
1. In oduc ion
The ex acellula ma ix (ECM) is a i al componen o e e y issue.
I consis s o a la ge a ie y o mac omolecules whose composi ion and
speci ic s uc u es a y om issue o issue. The main cons i uen s o
he ECM a e p o eoglycans and ib ous p o eins, o which collagens,
elas ins, ib onec ins and laminins a e he mos abundan [1]. These
componen s associa e o o m he complex ne wo k ha cons i u es he
physico-chemical mic oen i onmen whe e cells li e, p o iding hem
s uc u al suppo as well as en i onmen al signals, which may lead o
* Co esponding au ho . 3B’s Resea ch G oup, I3Bs – Resea ch Ins i u e on Bioma e ials, Biodeg adables and Biomime ics, Uni e si y o Minho, Headqua e s o
he Eu opean Ins i u e o Excellence on Tissue Enginee ing and Regene a i e Medicine, A ePa k, Pa que de Ciˆ
encia e Tecnologia, Rua A e 1, Edi ício 1 (Sede), 4805-
694 Ba co, Guima ˜
aes, Po ugal.
E-mail add esses: [email p o ec ed] (S. F ei as-Ribei o), [email p o ec ed] (A.F. Ca alho), [email p o ec ed]
(D.B. Rod igues), [email p o ec ed] (L. Ma ins), [email p o ec ed] (R.A. Pi es), [email p o ec ed] (V.M. Mendes), [email p o ec ed]
(B. Manadas), [email p o ec ed] (M. Ja nalo), [email p o ec ed] (R. Ho a), [email p o ec ed] (R.L. Reis), [email p o ec ed]
(R.P. Pi aco).
Con en s lis s a ailable a ScienceDi ec
Bioac i e Ma e ials
jou nal homepage: www.keaipublishing.com/en/jou nals/bioac i e-ma e ials
h ps://doi.o g/10.1016/j.bioac ma .2024.12.019
Recei ed 7 Augus 2024; Recei ed in e ised o m 11 No embe 2024; Accep ed 17 Decembe 2024
Bioac i e Ma e ials 46 (2025) 301–310
A ailable online 25 Decembe 2024
2452-199X/. 2024 The Au ho s. Publishing se ices by Else ie B.V. on behal o KeAi Communica ions Co. L d. This is an open access a icle unde he CC BY
license (
h p://c ea i ecommons.o g/licenses/by/4.0/ ).
p oli e a ion, di e en ia ion, o dea h [1].
Conside ing i s impo ance o so many undamen al cellula p o-
cesses, he ECM is a c ucial pa o e e y issue enginee ing and egen-
e a i e medicine s a egy. Cell Shee (CS) Enginee ing-based s a egies,
in pa icula , ely hea ily on cell-sec e ed ECM, which is he main
cons i uen o CS cons uc s. F om single-cell shee s o mul ilaye ed
cons uc s, using one o mo e cell ypes, nume ous s udies ha e shown
he po en ial o hese ECM- ich cons uc s o he egene a ion o a wide
ange o issues [2] such as co nea [3], myoca dium [4], a icula
ca ilage [5], bone [6] and skin [7]. Howe e , he widesp ead clinical
applica ion o hese cons uc s will depend on de eloping success ul
me hodologies ha p ese e CS p ope ies, pa icula ly he ECM, om
he ab ica ion si e o he bedside. C yogenic p ese a ion and hypo-
he mic p ese a ion eme ge as po en ial candida es o ha pu pose.
Slow eezing is he mos adi ional o m o c yogenic p ese a ion
and has mainly been used o p ese e single-cell suspensions [8–11].
C yop o ec an di usion and uni o m cooling occu easily in cell sus-
pensions, p o ec ing cells om ice o ma ion. Due o hei hickness, his
is mo e di icul o achie e in complex sys ems such as issues and o -
gans. ECM damage ollowing c yop ese a ion has been obse ed in
se e al issues, mainly in collagen and elas in o hea al es [12] and
loss o laye ed s uc u e [13] in co neal epi helial CSs.
Hypo he mic empe a u es ha e long been used in se e al medical
applica ions [14,15], namely o gan p ese a ion du ing anspo a ion
om dono o ecipien [16]. These empe a u es slow down
ene gy-dependen p ocesses like p o ein syn hesis, anspo sys ems,
and cell cycle p og ession. This way, cells can be suspended du ing a
sho pe iod o ime wi h a simpli ied me hod, a oiding cellula and
s uc u al damage om ice o ma ion and changes in solu e concen a-
ion caused by ex eme empe a u e shi s du ing eezing. Howe e ,
p olonged exposu e o hypo he mic empe a u es can ha e some
damaging e ec s, especially a he cellula le el [17,18].
In his wo k, a di ec compa ison o he e ec s o c yogenic s hy-
po he mic p ese a ion on he ECM o CSs was pe o med by looking a
pu a i e changes on ECM composi ion and s uc u e ollowing bo h
p ese a ion me hodologies. Fo each p ese a ion me hod, he gold
s anda d p ese a ion solu ion was used. Human adipose s omal cells
(hASCs) CSs we e ei he s o ed a −196 ◦C using e al bo ine se um
(FBS) wi h 10 % ( / ) dime hyl sul oxide (DMSO), o a 4 ◦C using he
p ese a i e hypo he mosol (HTS). A e 3 and 7 days o p ese a ion,
ECM p o ein abundance, s uc u al in eg i y and mechanical p ope ies
we e assessed.
2. Ma e ials and me hods
2.1. Isola ion o human adipose s omal cells
Human subcu aneous adipose issue was ob ained om su gical
p ocedu es pe o med a Cen o Hospi ala Uni e si ´
a io S˜
ao Jo˜
ao, a e
pa ien ’s w i en in o med consen , and in he scope o a collabo a ion
p o ocol app o ed by he e hical commi ees o bo h ins i u ions o his
wo k (Comiss˜
ao de ´
E ica do Cen o Hospi ala Uni e si ´
a io S˜
ao Jo˜
ao/
Uni e si y o Minho: 217/19; CEICVS 008/2019). hASCs we e ob ained
as p e iously desc ibed [19]. B ie ly, a e being cu in o small pieces,
adipose issue was diges ed wi h a solu ion o 0.05 % (w/ ) collagenase
ype II (Sigma Ald ich, USA) o 45 min a 37 ◦C unde agi a ion and
hen cen i uged o ob ain s omal ascula ac ion (SVF). The ob ained
SVF was incuba ed wi h ed blood lysis bu e , cen i uged and he su-
pe na an esuspended in Minimum Essen ial Medium
alpha-modi ica ion (
α
-MEM) (Li e Technologies, Uni ed Kingdom) sup-
plemen ed wi h 10 % ( / ) FBS and 1 % ( / ) an ibio ic/an imyco ic.
The nuclea ed cells we e coun ed using a solu ion o 3 % ( / ) ace ic
acid (VWR, Uni ed Kingdom) and 0.05 % ( / ) me hylene blue (Sigma
Ald ich, USA) in wa e , pla ed in cul u e lasks (Falcon, Uni ed
Kingdom) in he same medium as be o e, expanded in cul u e wi h
equen medium changes and passaged a e eaching 80 % con luence.
Cells we e used up o passage 4.
2.2. Cell shee s p oduc ion and de achmen
A he beginning o each expe imen al se up, cells we e de ached,
cen i uged and pla ed in 6 well issue cul u e polys y ene (TCPS) pla es
(Falcon, Uni ed Kingdom) a a densi y o 31,500 cells/cm
2
. hASCs we e
hen g own o 5 days wi h
α
-MEM supplemen ed wi h 50
μ
g/mL
asco bic acid (Wako, USA) o p oduce CS. CS we e hen de ached by
mechanical manipula ion om he pla es. B ie ly, he medium was
eplaced by Dulbecco’s phospha e bu e saline (DPBS) (The mo Scien-
i ic, USA), and o ceps we e used o mechanical peeling. A e , CS we e
ei he subjec ed o he di e en p ese a ion s udies (hypo he mic and
c yogenic) o cha ac e ized immedia ely, o ming in his case he
"be o e p ese a ion" (BP) g oup.
2.3. Hypo he mic p ese a ion
A e CS de achmen , DPBS was eplaced wi h hypo he mosol (Bio-
Li e Solu ions, USA) and CS we e placed a 4 ◦C. A e a s o age pe iod o
3 and 7 days, CS we e washed wi h DPBS and cha ac e ized immedi-
a ely, o ming he HP g oup.
2.4. C yogenic p ese a ion
Following de achmen , DPBS was emo ed, and a poly( inylidene
di luo ide) (PVDF) memb ane (Millipo e Co po a ion, U.S.A.) was
placed o e he CS o allow hei manipula ion. CSs we e hen placed in
a Te lon con aine wi h a solu ion o FBS wi h 10 % ( / ) DMSO, ozen
a −20 ◦C o 2h and −80 ◦C o e nigh . Finally, he con aine s we e
s o ed a −190 ◦C in a d y apou phase c yo ank (Biosys em A c ic 24
s a ebou ne c yogenics, USA). A e 3 and 7 days o s o age, CSs we e
washed wi h DPBS and cha ac e ized immedia ely, o ming he CP
g oup.
2.5. ECM ex ac ion
CSs we e decellula ized o assess ECM composi ion. CSs we e
ans e ed o a mic ocen i uge ube and incuba ed wi h a solu ion o
20 mM NH
4
OH and 1 % ( / ) T i on x-100 in wa e (all om Sigma
Ald ich, USA) o 3 min a 37 ◦C. A e a cen i uga ion s ep, he
emaining pelle was incuba ed wi h 200U DNase (The mo Scien i ic,
USA) and cOmple e p o ease and phospha ase inhibi o s mini able s
(Me ck, Ge many) acco ding o manu ac u e ins uc ions. Samples
we e cen i uged, he supe na an disca ded, and he pelle solubilized
in SDS bu e (7 % (w/ ) 0.5 M T is.HCl/0.4 % SDS pH 6.8 bu e ), 30 %
(w/ ) glyce ol, 10 % (w/ ) SDS, 0,93 % (w/ ) DTT and 0.012 % (w/ )
b omophenol blue, in wa e ). Finally, samples we e incuba ed o 20
min a 40 ◦C, 20 min a 65 ◦C and 10 min a 90 ◦C. To al p o ein was
quan i ied using a mic o Pie ce 660 nm P o ein Assay Reagen wi h he
Ionic De e gen Compa ibili y Reagen (all om The mo Scien i ic
Pie ce, USA), acco ding o he manu ac u e ins uc ions.
2.6. P o eomics
Sho GeLC-SWATH-MS (Sequen ial Window Acquisi ion o all
Theo e ical Mass Spec a/Da a Independen Acquisi ion - DIA) was
pe o med as p e iously desc ibed [20]. B ie ly, 30
μ
g o each sample
was subjec ed o in-gel diges ion a e a pa ial SDS-PAGE un using
p ecas gel (4–20 % Mini-P o ean® TGX™ Gel, Bio-Rad). Mass spec-
ome y da a we e acqui ed in wo di e en acquisi ion modes:
Da a-Dependen Acquisi ion (DDA) o he pooled samples and DIA o
each indi idual sample. P o ein iden i ica ion and lib a y cons uc ion
we e pe o med using P o einPilo ™ ( 5.0.1, Sciex), and he ela i e
quan i ica ion was pe o med using SWATH™ p ocessing plug-in o
PeakView™ ( 2.2, Sciex).
S. F ei as-Ribei o e al.
Bioac i e Ma e ials 46 (2025) 301–310
302
2.7. His ological analysis
A e o malin ixa ion wi h 10 % ( / ) bu e ed o malin solu ion,
CSs we e embedded in his ogel (The mo Scien i ic, USA) o p o ec hem
du ing he pa a in embedding p ocess. CSs we e hen p ocessed in a
MICRON STP120-2 spin issue p ocesso (MICRON, Ge many),
embedded in pa a in (The mo Scien i ic, USA), and se ially sec ioned
in o 4
μ
m- hick sec ions.
2.8. Immunohis ochemis y
CS sec ions we e depa a inized and ehyd a ed in a MICROM
HMS740 au oma ic s aine (MICROM, Ge many). An igen e ie al was
pe o med by ea ing he sec ions wi h ci a e bu e a 95 ◦C o 4 min,
ollowed by aliza in ed ea men o 10 min o quench backg ound
luo escence. Sec ions we e insed wi h PBS, non-speci ic binding
blocked wi h a 3 % (w/ ) BSA solu ion and hen incuba ed o e nigh a
4 ◦C wi h p ima y an ibodies abbi an i-human laminin (1:30) (Abcam,
Uni ed Kingdom), abbi an i-human ib onec in (1:50) (Abcam, Uni ed
Kingdom) and abbi an i-human ype I collagen (1:50) (Abcam, Uni ed
Kingdom). A e epea ed washes in PBS, samples we e incuba ed wi h
Alexa Fluo 488 donkey an i- abbi seconda y an ibody (1:500) (Mo-
lecula p obes, USA) o 45 min a oom empe a u e. Cells we e washed
in PBS and cell nuclei we e coun e s ained wi h DAPI. Sec ions we e
analyzed wi h an AxioImage Z1m mic oscope (Zeiss, Ge many), and
images we e acqui ed and p ocessed wi h Zeiss Zen so wa e e sion 2.6
(Zeiss, Ge many).
2.9. Cell shee hickness quan i ica ion
Immunohis ochemis y images we e p ocessed using ZEN 3.8 so -
wa e (Zeiss, Ge many) using he leng h ool (Fig. S1). To do so, images o
ype I collagen, he mos abundan ECM p o ein, we e used. CS hickness
was measu ed in 3 di e en loca ions ac oss 3 andomly selec ed ields
o samples om 4 di e en popula ions, wi h 34 measu emen s pe
condi ion.
2.10. Masson ich ome s aining
CS sec ions we e s ained wi h Masson’s T ich ome (MT) ki (Bio-
Op ica, I aly) acco ding o he manu ac u e ’s ins uc ions. B ie ly,
sec ions we e depa a inized wi h xylene, ehyd a ed in g aded e hanol
se ies and s ained wi h ou di e en s ains. Weige ’s i on hema oxylin
is used o nuclei, pic ic acid is used o e y h ocy es, a mix u e o acid
dyes is used o cy oplasm, and aniline blue is used o connec i e issue.
A e wa ds, sec ions we e dehyd a ed and moun ed wi h esinous
moun ing medium En ellan® (Me ck, Ge many). His ological sec ions
we e analyzed unde a Leica DM750 mic oscope (Leica, Ge many).
2.11. Fas g een s aining
CS sec ions we e s ained wi h as g een FCF (Sigma-Ald ich, USA),
as p e iously desc ibed [21]. B ie ly, sec ions we e depa a inized wi h
xylene, ehyd a ed in g aded e hanol se ies, and nuclei coun e s ained
wi h DAPI a 1:1000 o 10 min a RT. Fo as g een FCF s aining,
samples we e dehyd a ed using a se ies o me hanol-in-wa e solu ions
(30 %, 50 %, 70 %, and 100 % me hanol; 1 min each). Samples we e
hen incuba ed in 50
μ
g/mL o Fas G een FCF (Sigma-Ald ich, USA) in
me hanol o 30 min a RT. A e wa ds, he sec ions we e insed in pu e
me hanol o emo e unbound s aining and moun ed in DEPEX (SERVA,
Ge many). His ological sec ions we e analyzed unde a Zeiss LSM980
Ai yScan 2 (Zeiss, Ge many) con ocal mic oscope wi h ZEN 3.8 so wa e
(Zeiss, Ge many).
2.12. A omic o ce mic oscopy
CSs we e immobilized on a Pe i dish wi h he help o an ace a e
shee on op, wi h a cen al hole o 6 mm. All measu emen s we e ca ied
ou in PBS a 37 ◦C, which is compa ible wi h cell su i al. The nano-
mechanical p ope ies o CSs we e analyzed wi h a NanoWiza d 3 (JPK
Ins umen s, Ge many), using a conical ip (hal cone angle 15◦) unde
o ce mapping mode. Be o e analysis, he p obe was calib a ed unde
PBS using he con ac -based me hod. Fo each sample 8 ×8 maps we e
eco ded using squa e acquisi ion ames o 10 ×10
μ
m
2
. A leas h ee
o ce maps we e collec ed a di e en sample posi ions. The Young
modulus o each analyzed sample posi ion was calcula ed om he
co esponding o ce cu e by i ing he He z model using JPK SPM
Da a P ocessing so wa e (JPK Ins umen s) and a Poisson a io ixed a
0.5.
2.13. S a is ical analysis and p o eomics da a p ocessing
All da a e e s o 4 independen dono s (n =4). S a is ical analyses
we e pe o med using G aphPad P ism 9 so wa e (G aphPad So wa e
Inc.). P o eomics da a we e p ocessed using OmicsPlayg ound pla o m
[22]. Log CPM alues o all p o eins we e hen used o UMAP, hea -
map, unc ional anno a ion, and di e en ially exp essed p o ein anal-
ysis. Hea maps display he op 50 p o eins wi h he highes s anda d
de ia ion. Di e en ially exp essed p o eins we e analyzed using
no end.limma wi h an FDR o 0.05. P o ein anno a ion was pe o med
using he COMPARTMENTS da a se .
3. Resul s
3.1. Hypo he mia main ains p o ein abundance pa e n
To assess he e ec s o p ese a ion on he ECM, p o eomic analysis
was ca ied ou bo h be o e (BP) and a e p ese a ion (Fig. 1A). Fi s ly,
he composi ion o he ob ained p o eome a e decellula iza ion was
cha ac e ized. To iden i y ea u es en iched in CSs p o eome, di e ences
in p o ein abundance we e analyzed ela i e o he baseline (BP) o
ei he hypo he mic p ese a ion (HP) o c yogenic p ese a ion (CP),
a e 3 (HP3 o CP3) and 7 days (HP7 o CP7) (Fig. 1B). F om he es ed
condi ions, hypo he mic p ese a ion o 3 days main ained a p o ein
abundance pa e n close o ha obse ed be o e p ese a ion, sug-
ges ing minimal al e a ions. Howe e , p olonged p ese a ion in hy-
po he mic condi ions led o a no able shi in he obse ed pa e n. A
signi ican shi in p o ein abundance was also obse ed o c yo-
p ese ed CSs, ega dless o p ese a ion ime. Unsupe ised hie a -
chical clus e analysis o he op 50 exp essed p o eins con i med he
simila i y in p o ein abundance pa e ns be ween BP and HP3, manly
gi en by p o eins g ouped in he o ange clus e (Fig. 1C). To iden i y
en iched ea u es, p o eins in he di e en clus e s unde wen anno a-
ion using a compa men da a se (Fig. 1D). En iched ea u es indica e
co ela ion wi h se e al cellula compa men s, especially plasma
memb ane and adhesion molecules in all clus e s. The o ange clus e
exhibi s he highes co ela ions and highligh s he simila i ies be ween
BP and HP3, as well as he di e ences o he o he g oups.
3.2. Di e en ially exp essed p o eins
P o ein abundance pa e ns we e u he cha ac e ized by co ela-
ion and di e en ial exp ession analysis. Fi s ly, co ela ion analysis
aimed a elucida ing he ela ionships be ween p ese a ion condi ions
and p o ein exp ession pa e ns (Fig. 2A). Speci ically, when compa ing
CP3 and CP7, a s ong posi i e co ela ion ( =0.7429) was obse ed,
sugges ing a consis en pa e n o p o ein abundance ac oss c yogenic
p ese a ion condi ions. HP7 and HP3 exhibi ed a mode a e posi i e
co ela ion ( =0.4132), indica ing some deg ee o simila i y in p o ein
abundance p o iles be ween he wo hypo he mic p ese a ion ime
S. F ei as-Ribei o e al.
Bioac i e Ma e ials 46 (2025) 301–310
303
pe iods. When compa ing di e en p ese a ion me hods, a posi i e
co ela ion was also obse ed, bu i s impac a ied wi h inc easing
p ese a ion imes. Speci ically, when compa ing HP3 wi h CP3, a weak
posi i e co ela ion was e iden ( =0.2739). In ac , i was he lowes
among all compa isons. In e es ingly, he co ela ion be ween di e en
p ese a ion me hods inc eased wi h longe p ese a ion imes ( =
0.5276), sugges ing a endency o simila p o ein exp ession p o iles
wi h inc easing p ese a ion imes ega dless o he p ese a ion
me hods. A e examining he in luence o p ese a ion condi ions on
p o ein abundance pa e ns, an analysis ocused on di e en ial p o ein
con en was pe o med (Table S1). This analysis aimed o iden i y p o-
eins ha exhibi s a is ically signi ican di e ences in abundance be-
ween he expe imen al condi ions in es iga ed, using he le els be o e
p ese a ion - BP - as benchma k (Fig. 2B). The use o c yogenic p es-
e a ion did no yield any signi ican di e en ial abundances compa ed
o he BP condi ion. Con e sely, HP3 p esen ed only wo p o eins wi h
di e en ial abundances compa ed o BP. This numbe inc eased o ou
a e 7 days o hypo he mic p ese a ion. A Venn diag am was used o
isually ep esen he o e lap o di e en ially exp essed p o eins
(Fig. 2C). Speci ically, wo p o eins we e obse ed o be di e en ially
exp essed in bo h HP3 and HP7 samples, wi h an addi ional wo p o eins
di e en ially exp essed in HP7. Among hese p o eins, only one was
ela ed wi h ECM, namely, EGF-con aining ibulin-like ex acellula
ma ix p o ein 2 (EFEMP2). EFEMP2 is pa o he co e ma isome, a
subse o he ma isome, which is he collec ion o all p o eins ha
cons i u e and in e ac wi h he ECM, acco ding o he de ini ion by
Naba e al. [23]. This p o ein was hen analyzed in mo e de ail in he
nex sec ion.
Fig. 1. Hypo he mia main ains p o ein abundance pa e n. A) Schema ic ep esen a ion o CSs ECM ex ac ion and subsequen p o eomic analysis. B) UMAP
plo s o each condi ion colo ed by ela i e log-exp ession. Red indica es a p o ein is o e exp essed in a speci ic g oup, blue ha i is down egula ed compa ed o he
a e age alues o all samples. Dis ance me ic is co a iance. C) Hea map displaying unsupe ised hie a chical clus e ing o op 50 p o eins. Membe s o di e en
clus e s (indica ed by ba s on he igh o he hea map) a e lis ed on he u he igh o he hea map in colo ed a eas. D) Hea map o he unc ional anno a ion o
each clus e we e ed indica es highe co ela ion and blue lowe co ela ion. Anno a ion was pe o med using compa men s da a se .
S. F ei as-Ribei o e al.
Bioac i e Ma e ials 46 (2025) 301–310
304
3.3. Ma isome p o ein abundance is p ese ed o all es ed p ese a ion
me hods
To un eil po en ial e ec s o he p ese a ion p o ocols on ECM-
ela ed p o eins, he CSs ma isome was explo ed. O 1034 iden i ied
p o eins, 86 we e ecognized as ECM componen s based on he human
ma isome da ase [24]. A o al o 46 co e ma isome p o eins we e
iden i ied, comp ising 6 p o eoglycans, 26 glycop o eins, and 14 colla-
gens, along wi h 40 ECM-associa ed p o eins, including 4 sec e ed ac-
o s, 11 ECM-a ilia ed p o eins, and 25 ECM egula o s (Fig. 3A)
(Table S2). Among he de ec ed collagens we e a ious ib illa ypes,
such as ypes I, III, V, and XI, as well as non- ib illa ypes, such as ypes
IV, VI, VIII, XII, and XVI. Addi ionally, key cons i uen s o he co e
ma isome, such as ib onec in and laminin, we e iden i ied wi hin he
glycop o eins classi ica ion. To in es iga e changes o he ma isome
p o ein signa u e be o e and a e p ese a ion, an unsupe ised hie -
a chical clus e analysis was pe o med on he op 50 p o eins ound in
he ECM (Fig. 3B). The analysis e ealed a close ma isome p o ein
pa e n o baseline (BP) and HP3, while emaining condi ions we e
clus e ed a he away, indica ing mo e changes o ma isome p o ein
abundance o hese la e g oups (Fig. 3B). Gi en he s uc u al
impo ance o co e ma isome p o eins, an ini ial analysis o hei
abundances o each condi ion e ealed no s a is ically signi ican di -
e ences be ween he condi ions (Fig. 3C). This was also he case when
subdi iding co e ma isome p o eins acco ding o p o ein ypes.
Immunohis ochemis y analysis (Fig. 3D) co obo a ed hese indings,
showing no appa en changes in p o ein exp ession o key ECM con-
s i uen s collagen ype I, ib onec in, and laminin be o e and a e
Fig. 2. Di e en ially exp essed p o eins. A) Pai wise sca e plo s o di e en ial exp ession p o iles o con as s in analysis. Simila p o iles show high co ela ion
wi h poin s close o he diagonal. B) Volcano plo o di e en ially exp essed p o eins o each con as (FDR =0.05). C) Venn diag am wi h he dis ibu ion o he
di e en ly exp essed p o eins.
S. F ei as-Ribei o e al.
Bioac i e Ma e ials 46 (2025) 301–310
305
Fig. 3. Cell shee s ma isome p o iling. A) To al numbe o p o eins, and numbe o iden i ied ma isome p o eins (le ). Rep esen a ion o each ma isome
ca ego y, and espec i e numbe o ECM p o eins iden i ied ( igh ). B) Hea map displaying unsupe ised hie a chical clus e ing o op 50 p o eins o ECM. Membe s
o di e en clus e s (indica ed by ba s on he igh o he hea map) a e lis ed on he u he igh o he hea map in colo ed a eas. C) Dis ibu ion plo s o condi ion in
he di e en ca ego ies o he ma isome as indica ed. Resul s we e analyzed using K uskal-Wallis es wi h Dunn’s mul iple compa ison pos - es . D) Rep esen a i e
immunocy ochemis y images o exp ession o majo ECM p o eins ype I collagen, ib onec in and laminin (all in g een). Cell nuclei was coun e s ained wi h DAPI
(blue) Immunocy ochemis y was pe o med a e emo ing cul u es om p ese a ion. Scale ba : 50
μ
m. E) Quan i ica ion o CS hickness be o e and a e p es-
e a ion. Da a a e p esen ed as iolin plo illus a ing he ke nel densi y dis ibu ion equency o CS hickness and analyzed using one-way ANOVA wi h Tukey
mul iple compa ison pos - es (*p <0.0332, **p <0.0021 and ****p <0.0001).
S. F ei as-Ribei o e al.
Bioac i e Ma e ials 46 (2025) 301–310
306
p ese a ion, ega dless o he p ese a ion me hod applied, which was
u he suppo ed by do blo analysis (Fig. S2). Howe e , a educ ion in
CS hickness was obse ed pos -p ese a ion, sugges ing di ec damage
o ECM s uc u e due o he p ese a ion p ocesses a he han changes
o p o ein abundances (Fig. 3E).
3.4. C yop ese a ion al e s ma ix o ganiza ion and s uc u e
A e obse ing changes in hickness ollowing p ese a ion, o he
s uc u al and mechanical changes o CS we e in es iga ed. His ological
analysis wi h MT s aining e ealed a p onounced dis up ion o ECM
uni o mi y ollowing c yogenic p ese a ion, especially o p olonged
p ese a ion pe iods, aligning wi h he obse ed educ ion in CS hick-
ness (Fig. 4A). Some deg ee o ECM damage was also obse ed o he
HP7 condi ion al hough no as se e e as o he c yogenic condi ions. As
sugges ed by he o he da a, HP3 was he condi ion ha be e main-
ained ECM s uc u e in compa ison wi h he baseline BP condi ion. A
ine analysis o collagen s uc u e made using con ocal mic oscopy
a e as g een s aining, e ealed agmen a ion and dis up ion in
collagen uni o mi y, co obo a ing he mac oscopic s uc u al al e -
a ions obse ed abo e (Fig. 4B). Howe e , when examining s uc u al
in eg i y a he nanoscale, no di e ences we e obse ed be ween con-
di ions, sugges ing ha he p ima y s uc u al damage ollowing p es-
e a ion occu s a a mac oscopic a he han nanoscale le el (Fig. S3).
No ably, CSs p ese ed unde hypo he mic condi ions main ained he
o ganiza ion o collagenous s uc u es simila o he BP s a e, which was
u he mo e e lec ed in hei mechanical p ope ies measu ed by Bio-
AFM (Fig. 4C). Young’s modulus was assessed h ough nanoinden a ion,
whe e a p obe inden s he sample su ace o gene a e a o ce-
displacemen cu e. Th ee o ce maps we e collec ed om dis inc e-
gions on each cell shee , wi h each map con aining mul iple measu e-
men s (inden a ions) ac oss a de ined a ea. Each poin on he esul ing
g aph ep esen s a single measu emen , illus a ing s i ness dis ibu ion
wi hin he cell shee . The obse ed a ia ion in Young’s modulus e-
lec ed he he e ogeneous s i ness o cell shee componen s: so e
cellula egions yield lowe modulus alues, while s i e , aligned
collagen ib ils p oduce highe alues. Ac oss all p ese a ion condi-
ions, CS s i ness dec eased compa ed o he BP condi ion. Howe e ,
bo h he HP3 and HP7 condi ions p esen ed a ke nel densi y dis ibu ion
equency simila o BP condi ion, indica ing a be e p ese a ion o he
s i e componen s o CSs, in ma ked con as wi h CSs p ese ed unde
c yogenic empe a u es (Fig. 4D).
4. Discussion
The ECM plays a c ucial ole in CS echnology, p o iding he s uc-
u al amewo k necessa y o cell a achmen , p oli e a ion, and di -
e en ia ion [1]. As he p ima y componen o CSs, he ECM dic a es
hei mechanical p ope ies and de e mines hei abili y o o m unc-
ional issue-like cons uc s. Gi en he delica e na u e o CSs and hei
eliance on he ECM o s uc u al suppo and signaling cues, app o-
p ia e p ese a ion me hods a e essen ial o s eamline hei clinical
implemen a ion. C yogenic p ese a ion, conside ed he gold s anda d,
and hypo he mic p ese a ion a e wo possible p ese a ion app oaches
[16,25]. Ye , no di ec compa ison be ween hese me hods has been
made o his pu pose. The e o e, in his s udy, we aimed o comp e-
hensi ely cha ac e ize he composi ional, s uc u al, and mechanical
impac o c yogenic and hypo he mic p ese a ion on CSs ECM.
The chosen p ese a ion ime ame was selec ed because i in-
co po a es he pe iods du ing which maximal damage occu s o bo h
p ese a ion s a egies. I , on he one hand, hypo he mic p ese a ion is
inhe en ly ime-sensi i e, as cellula me abolism is only pa ially sup-
p essed and biochemical ac i i y con inues, on he o he , i is widely
known ha in c yop ese a ion damage p ima ily occu s du ing he
eeze- haw p ocess [26]. Fu he mo e, examining he ECM composi ion
o CSs be o e and a e p ese a ion is essen ial o assessing he
p ese a ion’s e ec s.and educing sample complexi y is i al o be e
isualize he p o eins o in e es [27,28]. The applied decellula iza ion
p ocess emo ed a subs an ial po ion o he cellula con en and
allowed he en ichmen o he sample in ECM p o eins. O hese, he
ma isome e e s o he collec ion o p o eins ha o m o a e associa ed
wi h he ECM. This includes bo h he s uc u al ECM p o eins such as
collagens, ib onec ins, and elas ins, known as he co e ma isome, and
he b oade se o ma isome-associa ed p o eins, comp ising enzymes,
g ow h ac o s, cy okines, and o he molecules ha in e ac wi h o
egula e he ECM [23,29]. While he iden i ied ECM p o eins encom-
passed bo h co e ma isome and ma isome-associa ed ca ego ies, his
analysis p ima ily ocused on co e ma isome p o eins, d i en by he key
ole hese s uc u al p o eins play in main aining CS in eg i y, and hus
hei s abili y and unc ionali y. Among hese s uc u al p o eins, col-
lagens a e he mos abundan , ep esen ing o e 30 % o he ECM con-
en [30]. The he ein iden i ied ib illa collagens play a c ucial ole in
de ining he s uc u al a chi ec u e and mechanical p ope ies o CSs
[31], while p o eoglycans add s eng h and s uc u e o he ECM.
Among he obse ed glycop o eins, ib onec ins and laminins a e c i -
ical o ECM assembly and cellula adhesion by in e ac ing wi h in eg-
ins, g ow h ac o s, cy okines, and o he ECM molecules [32]. Ou
analysis also e ealed smalle amilies o ECM p o eins wi h speci ic
oles in s uc u al o ganiza ion. This is he case o ibulin-2 and i s
in ol emen in he o ma ion o la ge p o eoglycan ne wo ks, and
ib illins, ha p o ide issue ex ensibili y and linking o elas ic ibe s
[33]. A e iden i ying hese key ECM p o eins, he in luence o p ese -
a ion me hods on hei ela i e con en was in es iga ed. Looking a
ela i e p o ein abundance using a di e en ially exp essed p o ein
analysis e ealed no signi ican di e ences be ween condi ions o ECM
p o eins, excep o EFMP2, up egula ed a e 7 days unde hypo he mic
empe a u es. While EFEMP2 is desc ibed as in e ac ing wi h o he ECM
componen s and cell su ace p o eins, i is no hough o play a p e-
ponde an ole in ECM s uc u e and, he e o e, has no been u he
explo ed. When examining he exp ession pa e ns o he mos abundan
ECM p o eins using immunohis ochemis y, he esul s co obo a ed he
indings om he di e en ial exp ession analysis. Wi h ECM p o ein
con en emaining ela i ely unchanged, al e a ions in ECM s uc u e
ollowing p ese a ion we e analyzed. Unlike c yop ese a ion, hypo-
he mic empe a u es o e a milde al e na i e ha a oids cellula and
s uc u al damage caused by ice nuclea ion and osmo ic s ess om
c yop o ec an s [34,35]. Al hough nume ous hypo he mic p ese a ion
solu ions exis , mos a e ailo ed o whole o gan p ese a ion. Speci -
ically de eloped o TE p oduc s, HTS is capable o main aining ionic
and osmo ic balance, inhibi ing acidosis, and p e en ing cell swelling
[36,37]. Addi ionally, i s sca enging capaci y e ec i ely educes he
p oduc ion o eac i e oxygen species (ROS), p e en ing he ac i a ion
o a ious cellula s ess pa hways connec ed o oxida i e s ess, which
in u n can lead o cell dea h [38]. These a ibu es collec i ely play a
undamen al ole in p ese ing cellula iabili y and ECM in eg i y
unde hypo he mic condi ions. This was con i med in he p esen s udy,
wi h CSs p ese ed wi h HTS be e main aining hei s uc u al in eg-
i y in compa ison wi h c yop ese ed CSs. Howe e , a educ ion in CSs
hickness was no ed o longe p ese a ion imes. While he p ecise
ac o s d i ing his educ ion a e elusi e, simila phenomena ha e
al eady been desc ibed in he li e a u e o o he cold p ese a ion
scena ios [39,40]. Indeed, he esul s he ein disclosed o he c yogenic
p ese a ion g oup showed a educ ion in CS hickness simila o ha
obse ed o ex ended hypo he mic p ese a ion. Unlike in acellula
ice, ex acellula ice o ma ion is ypically conside ed benign o
single-cell c yogenic p ese a ion. Howe e , i s e ec s on densely
packed cell-ECM s uc u es can be de imen al. The in ica e o ganiza-
ion o ECM ibe s and hei in e ac ions wi hin he issue a chi ec u e
make hem pa icula ly suscep ible o damage du ing c yop ese a ion
[41]. As ice c ys als g ow in he ex acellula space du ing c yop ese -
a ion, hey al e he s uc u e o ECM, especially a ec ing collagen and
elas in o ganiza ion [42]. This dis up ion o he ECM s uc u e is
S. F ei as-Ribei o e al.
Bioac i e Ma e ials 46 (2025) 301–310
307
Fig. 4. C yop ese a ion al e s ma ix o ganiza ion and s uc u e. A) Rep esen a i e Masson T ich ome s aining images. Collagen is s ained in blue and cells and
o he componen s in ed. S aining was pe o med a e emo ing cul u es om p ese a ion. Scale ba : 50
μ
m ( op) and 20
μ
m (bo om) B) Rep esen a i e as g een
s aining images. Collagen s uc u es depic ed in whi e and cell nuclei coun e s ained wi h DAPI (blue) Scale ba : 5
μ
m C) Schema ic ep esen a ion o Bio AFM
me hodology. D) ECM mechanical p ope ies gi en by Bio AFM analysis. young’s modulus (Pa) alues we e ob ained be o e and a e emo ing cul u es om
p ese a ion. Da a a e p esen ed as iolin plo illus a ing he ke nel densi y dis ibu ion equency o young’s modulus and analyzed using one-way K uskal–Wallis
es wi h Dunn’s mul iple compa ison pos - es (**p <0.0021 and ****p <0.0001).
S. F ei as-Ribei o e al.
Bioac i e Ma e ials 46 (2025) 301–310
308
signi ican , as i has been iden i ied as he cause o he apid de e io-
a ion obse ed in some implan ed hea al es a e c yop ese a ion
[12,43,44]. Despi e being less densely packed han mos issues, CSs
exhibi simila s uc u al ea u es. P e ious s udies ha e e ealed ha
c yop ese a ion can ha e no able e ec s on he o iginal laye ed
s uc u e o CSs. Speci ically, esea ch on co neal epi helial CSs has
documen ed e idence o ECM s uc u al damage ollowing c yop ese -
a ion [45]. Simila ly, ou esul s demons a ed ECM s uc u al al e -
a ions a e exposu e o c yogenic empe a u es. These changes in CS
s uc u e signi ican ly impac ed mechanical p ope ies. which a y
based on ibe o ien a ion and he linkage and in e ac ion o di e en
ibe ypes [46]. Changes in alignmen and in e connec edness o
collagen ib ils wi h o he ECM componen s signi ican ly in luence
mechanical p ope ies. Exposu e o c yogenic empe a u es dec eased
CS s i ness, which co ela ed well wi h he ex ensi e damage o he
collagen ne wo k shown by he agmen a ion and discon inui y o ECM
and collagen ib ils obse ed a e MT s aining and speci ic collagen
s aining. Ne e heless, al hough ha ing pe o med measu emen s a
a ious loca ions on he CS, i is essen ial o acknowledge ha inden-
a ion echniques may no ully ep esen he dynamics o he en i e
issue. Finally, he obse ed e ec s may a y depending on bo h he cell
ype and he numbe o CS laye s. The na u e o CS cons uc s depends
on ac o s like cell p oli e a ion a es and he quan i y and composi ion
o ECM p oduced by he cells, and hus shee s de i ed om di e en cell
ypes, and especially hose o ganized in mul ilaye ed s uc u es, may
espond di e en ly o p ese a ion me hods. Mul ilaye ed cons uc s, in
pa icula , ep esen mo e densely packed cell-ECM a angemen s
whe e he in e ac ions among ECM ibe s a e mo e in ica e, ende ing
hem, on he one hand, po en ially mo e suscep ible o
c yop ese a ion-induced damage, bu , on he o he , mo e obus and
he e o e po en ially mo e esilien o such insul . This is some hing ha
should be speci ically es ed in u u e wo ks, al hough we belie e he
o e all conclusions o he p esen wo k a e alid o CS cons uc s wi h
low complexi y and hickness.
5. Conclusions
This s udy p o ides comp ehensi e insigh s in o he e ec s o c yo-
genic and hypo he mic p ese a ion on CS ECM composi ion as well as
on i s s uc u e and mechanical p ope ies. Ou indings demons a e
ha exposu e o c yogenic empe a u es esul ed in signi ican al e -
a ions in ECM s uc u e and, in pa icula , dis up ions o collagen o -
ganiza ion. Con e sely, hypo he mic p ese a ion had a less
p onounced e ec on ECM s uc u e, wi h CSs p ese ing, o a la ge
ex en , hei o iginal s uc u al in eg i y and mechanical p ope ies. The
obse ed al e a ions in ECM s uc u e ha e impo an implica ions o
he clinical implemen a ion o CS-based he apies. While no signi ican
al e a ion in e ms o ECM p o ein abundance was ound, he no able
ECM s uc u al changes ollowing c yogenic p ese a ion can po en-
ially impac CSs physiologic unc ion, he eby a ec ing hei e icacy
CS when used clinically as he apies. This sugges s ha hypo he mia
may be he bes al e na i e o p ese ing CS in eg i y and unc ionali y
om he ab ica ion si e o he bedside.
CRediT au ho ship con ibu ion s a emen
Sa a F ei as-Ribei o: W i ing – e iew & edi ing, W i ing – o iginal
d a , Me hodology, In es iga ion, Fo mal analysis, Da a cu a ion,
Concep ualiza ion. And eia F. Ca alho: In es iga ion, Fo mal anal-
ysis. Daniel B. Rod igues: In es iga ion, Fo mal analysis. Luís Ma -
ins: Fo mal analysis. Rica do A. Pi es: In es iga ion, Fo mal analysis.
Ve a M. Mendes: Valida ion, In es iga ion, Fo mal analysis. B uno
Manadas: Valida ion, Fo mal analysis. Ma iana Ja nalo: Resou ces.
Rica do Ho a: Resou ces. Rui L. Reis: Supe ision, Resou ces.
Rog´
e io P. Pi aco: W i ing – e iew & edi ing, W i ing – o iginal d a ,
Valida ion, Supe ision, Me hodology, Funding acquisi ion, Da a
cu a ion, Concep ualiza ion.
E hics app o al and consen o pa icipa e
Human subcu aneous adipose issue was ob ained om su gical
p ocedu es pe o med a Cen o Hospi ala Uni e si ´
a io S˜
ao Jo˜
ao, a e
pa ien ’s w i en in o med consen , and in he scope o a collabo a ion
p o ocol app o ed by he e hical commi ees o bo h ins i u ions o his
wo k (Comiss˜
ao de ´
E ica do Cen o Hospi ala Uni e si ´
a io S˜
ao Jo˜
ao/
Uni e si y o Minho: 217/19; CEICVS 008/2019).
Decla a ion o compe ing in e es
Rui L. Reis is an associa e edi o o Bioac i e Ma e ials and was no
in ol ed in he edi o ial e iew o he decision o publish his a icle.
The emaining au ho s decla e ha he e a e no compe ing in e es s.
Acknowledgme
This esea ch was unded by EU Ho izon 2020 esea ch and inno-
a ion p og am unde he Eu opean Resea ch Council (ERC) g an
CapBed (805411), he Po uguese Science and Technology Founda ion
(FCT) UIDB/04539/2020, UIDP/04539/2020, by The Na ional Mass
Spec ome y Ne wo k (POCI-01–0145-FEDER-402–022125
Re . ROTEIRO/0028/2013). SFR acknowledges doc o al ellowship PD/
BD/135252/2017 and RAP acknowledges indi idual g an CEECIND/
05623/2022.
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.bioac ma .2024.12.019.
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