SHORT COMMUNICATION Open Access
De e mina ion o po e size dis ibu ion a he
cell-hyd ogel in e ace
Aldo Leal-Egaña
1*
, Ul Die ich-B aumann
2
, A ánzazu Díaz-Cuenca
3,4
, Ma cin Nowicki
5
and Augus inus Bade
1
Abs ac
Backg ound: Analyses o he po e size dis ibu ion in 3D ma ices such as he cell-hyd ogel in e ace a e e y
use ul when s udying changes and modi ica ions p oduced as a esul o cellula g ow h and p oli e a ion wi hin
he ma ix, as po e size dis ibu ion plays an impo an ole in he signaling and mic oen i onmen s imuli
impa ed o he cells. Howe e , he majo i y o he me hods o he assessmen o he po osi y in bioma e ials a e
no sui able o gi e quan i a i e in o ma ion abou he ex u al p ope ies o hese nano-in e aces.
Findings: He e, we epo a me hodology o de e mining po e size dis ibu ion a he cell-hyd ogel in e ace, and
he dep h o he ma ix modi ied by cell g ow h by en apped HepG
2
cells in mic ocapsules made o 0.8% and
1.4% w/ algina e. The me hod is based on he es ima ion o he sho es dis ance be ween wo poin s o he
ib il-like ne wo k hyd ogel s uc u es using image analysis o TEM pic u es. Values o po e size dis ibu ion
de e mined using he p esen ed me hod and hose ob ained by ni ogen physiso p ion measu emen s we e
compa ed, showing good ag eemen . A combina ion o hese me hodologies and a s udy o he cell-hyd ogel
in e ace a a ious cell cul u e imes showed ha a e h ee days o cul u e, HepG
2
cells g owing in hyd ogels
composed o 0.8% w/ algina e had mo e coa se o po es a dep hs up o 40 nm inwa ds (a phenomenon mos
no able in he i s 20 nm om he in e ace). This coa sening phenomenon was weakly obse ed in he case o
cells cul u ed in hyd ogels composed o 1.4% w/ algina e.
Conclusions: The me hod pu posed in his pape allows us o ob ain in o ma ion abou he adial de o ma ion o
he hyd ogel ma ix due o cell g ow h, and he consequen modi ica ion o he po e size dis ibu ion pa e n
su ounding he cells, which a e ex emely impo an o a wide spec um o bio echnological, pha maceu ical and
biomedical applica ions.
Backg ound
Algina e is a na u al polysaccha ide, which o ms s able
h ee-dimensional (3D) hyd ogels upon binding di alen
ca ions such as Ca
2+
,S
2+
o Ba
2+
. Due o he high
immune compa ibili y, he use o algina e o en ap cells
has been widely s udied wi h he pu pose o en apping
immo alized and/o ans o med cells which could
eplace mal unc ioning issues o a diseased o gan [1].
Besides, algina e mic ocapsules can be used o es he
ac ion o an icance d ugs on malignan cells embedded
in a 3D en i onmen ( umou -like mic ocapsules) [2].
Owing o he enhanced p oli e a ion capaci y o
immo alized and/o cance cells, he analysis o modi i-
ca ions o he in e ace be ween cell and bioma e ial
wi h cell g ow h is highly desi able. Some me hods o
cha ac e ize he po ous s uc u e o he 3D ne wo ks
ha e been p e iously epo ed, such as me cu y in u-
sion po osime y [3], ni ogen physiso p ion [4], and he
di usion kine ics o ele an solu es [5]. Ne e heless,
hese echniques canno be applied in he p esence o
cells, no do hey gi e in o ma ion abou modi ica ions
p oduced a he cell-bioma e ial in e ace due o cell
p oli e a ion.
Owing o he easibili y o ob aining and analyzing
high esolu ion elec on mic oscope images o c yo ixed
cells embedded in 3D ma ices, i is one o he mos
widely used echniques o analyze ex u al p ope ies o
hyd ogels, o e ing he ad an age o simul aneously
* Co espondence: [email p o ec ed]
1
Depa men o Cell Technology and Applied S em Cell Biology,
Bio echnology and Biomedicine Cen e (BBZ), Uni e si y o Leipzig.
Deu sche Pla z 5, 04103, Leipzig, Ge many
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Leal-Egaña e al.Jou nal o Nanobio echnology 2011, 9:24
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© 2011 Leal-Egaña e al; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
ob aining in o ma ion pe aining o bo h he cells and
he ma e ial comp ising he ma ix [6]. Since hyd ogels
a e mos commonly o med by ne wo ks o andomly
in e connec ed polyme s, hey o m complex mic oa ch-
i ec u es o ca i ies wi h a iable shapes and mo pholo-
gies. E en hough well-de ined po e-like s uc u es can
be clea ly obse ed wi h scanning elec on mic oscopy
[7], we need o conside o he app oaches o ex ac ing
accu a e quan i a i e h ee dimensional in o ma ion o
he hyd ogel ma ix om measu emen s made in wo
dimensions.
In his pape we desc ibe a me hodology based on
au oma ed image p ocessing and analysis o ansmis-
sion elec on mic oscopy (TEM) images ob ained om
hyd ogels, and i s applicabili y on de e mining modi ica-
ions o he po e size dis ibu ion a he cell-algina e
in e ace as a esul o cell g ow h.
The me hod was pe o med a e en apping he hepa-
oca cinoma cell line HepG
2
, which ep esen s an exam-
ple o cells wi h enhanced p oli e a i e capaci y.
Findings
Ma e ial and me hods
Elec on mic oscopy images
T ansmission Elec on Mic oscopy (TEM) images we e
ob ained wi h an Elec on Mic oscope (Ca l Zeiss EM
10, Ge many) acco ding o me hods published p e-
iously [8]. B ie ly, he me hod is based on he ixa ion
o algina e mic ocapsules wi h a 2.5% glu a aldehyde
solu ion (Se a, Ge many) dissol ed in a bu e solu ion
composed o 9 g/l NaCl (Ca l Ro h, Ge many), 5.55 g/l
CaCl
2
(Me ck, Ge many) and 10.46 g/l o Mops bu e
(Ca l Ro h, Ge many). A e o e nigh ixa ion (4°C),
algina e mic ocapsules we e sa u a ed wi h 2.0% (w/ )
aga ose (Ca l Ro h, Ge many), and ixed again wi h 2.5%
glu a aldehyde a 4°C o 1 h. Capsules we e insed
h ee imes o 20 min wi h he bu e solu ion. Pos -
ixa ion was pe o med by using 1.0% osmium e oxide
(Me ck, Ge many) a 4°C (2 × 1h), and pos e io
embedded in Du cupan (Sigma-Ald ich, Ge many).
Ul a hin sec ions we e s ained wi h u anyl ace a e and
lead ci a e (Se a, Ge many) [8].
The o al numbe o TEM pic u es ob ained was 72,
assuming a andom dis ibu ion o cells wi hin he algi-
na e capsules.
Tex u al p ope ies o cell- ee algina e mic ocapsules [4]
Measu emen s we e ca ied ou a e d ying he mic o-
capsules in CO
2
beyond he c i ical poin . N
2
adso p-
ion-deso p ion iso he ms we e collec ed using a
Mic ome i ics ASAP2010 gas adso p ion analyze a
77K, a e degassing he samples a 298K o e nigh on a
acuum line. The B unaue -Emme -Telle (BET) speci ic
su ace a ea was e alua ed using adso p ion da a in a
ela i e p essu e ange, 0.05 o 0.2 [9]. Algina e ma ix
po e size dis ibu ion was calcula ed on he basis o he
deso p ion b anches using he Ba e -Joyne -Halenda
me hod (BJH) [10].
Cell cul u e
HepG
2
cells (ob ained om he depa men al cell bank
o he S em Cell Biology labo a o y, Uni e si y o Leip-
zig, Ge many) we e cul i a ed in DMEM (Bioch om,
Ge many) supplemen ed wi h 15% / oe al bo ine
se um (GIBCO, Sco land), 100 ng/ml sodium py u a e
(Sigma-Ald ich, Ge many) and 50μg/ml Gen amycin
(PAA labo a o ies, Aus ia).
Cell encapsula ion
HepG
2
cells we e immobilized in 0.8% and 1.4% w/
algina e-CaCl
2
mic ocapsules o 500μm diame e
acco ding o me hods desc ibed p e iously [4,8]. A com-
me cially a ailable encapsula ion sys em (Inno ech, IE-
50R) wi h a 250μm nozzle was used. This sys em p o-
duces capsules wi h a diame e o up o 500μm. In all
cases, he ini ial numbe o immobilized HepG
2
pe mL
algina e was 1.5·10
6
(app oxima ely 100 cells pe cap-
sule). The iabili y o he immobilized cells be o e he
p ocess o encapsula ion was de e mined by he T ipan
Blue exclusion me hod (Sigma-Ald ich, UK), whe e he
iabili y o HepG
2
eached 95%.
De e mina ion o cell and/o agg ega es sizes
Analysis o cells and/o agg ega es adii was ca ied ou
by using he p og am Axio ision (Ca l Zeiss, Ge many)
a e images cap u e o cells and/o agg ega es wi h an
Axio e HRC came a (Ca l Zeiss, Ge many) moun ed
on an in e ed mic oscope (Zeiss Axio e 200). Ana-
lyses o size dis ibu ion we e ca ied ou wi h a mini-
mum numbe o 200 capsules, which we e placed in a 4
well pla e con aining 500 μL media, 0.05% / concen-
a ion o Calcein A/M (In i ogen, USA) and 0.25% /
o E hidium homodime I (In i ogen, USA).
Image Analysis
Au oma ed analysis o ansmission elec on mic oscopy
(TEM) was accomplished using he ollowing p o ocol:
i s ly, ela i ely high image-inhe en con as basically
makes au oma ic image segmen a ion (algina e s. ca -
i ies) s aigh o wa d by applying bina iza ion using a
simple h esholding, howe e , p ep ocessing is equi ed
in o de o compensa e o local con as luc ua ions,
so ha image inhomogenei y co ec ion using a high-
pass il e [11] was applied. Image noise was emo ed
doing a p ese ing edge-smoo hing using o al a ia ion
il e ing [12]. Addi ionally, cohe ence-enhancing shock
il e ing [13] was done o u he in ensi y all di ec ed
algina e s uc u es. P e-p ocessed TEM images we e
hen pa i ioned in o algina e and ca i y segmen s using
bina iza ion. The minimum accep ed lumen a ea was se
o app ox. 275nm². Measu emen s o hese bina y
images we e pe o med using an unsigned Euclidean
image dis ance ans o ma ion [14] p o iding o all
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backg ound pixels a espec i esho es dis ance o he
su ounding algina e, he eby ob aining alues o ela i e
adii o hese ca i ies. The numbe o imes he same
alue was epea ed is he ea e dubbed he equency.
All dis ance ans o ma ion-based measu emen s we e
accomplished along he skele on be ween wo opposi e
algina e ib ils. Disc e e alues o adii o he algina e
ca i ies a e named in his pape as ela i e po e adius
( p ). Fo images ob ained a e cell en apmen , we ca -
ied ou he p o ocol desc ibed abo e, ollowed by co -
ela ing measu emen s o ela i e po e adii o he
pe pendicula dis ance om he in e ace cell-bioma e -
ial, assuming a maximum dis ance o 400 nm. This was
ca ied ou by delinea ing he cell con ou o gene a e a
mask, which was used as a s a ing poin o measu e-
men s, again accomplished based on a compu a ional
e ec i e Euclidean dis ance ans o m. In o de o
ob ain a dis ibu ion o alues in pe cen , p be ween
10 and 70 nm we e g ouped in a disc e e clus e . All
image p ocessing was accomplished using he compu e
algeb a sys em MATHEMATICA
®
(Wol am Resea ch,
Inc., U bana-Champaign, Illinois, USA) including he
Digi al Image package w i en by Jens-Pee Kuska.
Simila o he measu emen s o ela i e po e’s adii,
a e ea men o he images wi h he p ocedu es
desc ibed p e iously, measu emen s o diame e s o he
algina e ib ils we e ca ied ou by measu ing he dis-
ance ans o m masked ou along a ib il skele on. The
p ecision o ou me hod depends o he image esolu-
ion, whe e in case o he pic u es used in his pape
(ob ained wi h an ampli ica ion o 20000X), 1 pixel
ep esen s 2.34 nm².
Resul s and Discussion
Figu e 1 shows 2D images o he ma ix nano-a chi ec-
u e o he algina e hyd ogel mic ocapsules. The hyd o-
gel ma ix is o med by a ne wo k o ib il-like
s uc u es which can be iden i ied and disc imina ed
om he su ounding ca i ies by compu a ional p o-
g ams. These ca i ies a e named in his pape as ela i e
Figu e 1 Illus a ion o he me hod o de e mine po e size dis ibu ion de eloped in his wo k. Image A depic s a hyd ogel as i is
ypically obse ed using ansmission elec on mic oscopy. Image B shows he esul s o he image segmen a ion a e bina iza ion. Image C
shows he esul o a Euclidean dis ance ans o ma ion. Image D gi es an o e lay o he po e egion image skele on ( ed lines) wi h he o iginal
image. Image skele ons a e one-pixel wide cen e axes. They a e de ined ia he se o inne po e pixels. The se is de ined ia local dis ance
maxima wi h espec o algina e segmen s. Scale ba co esponds o 250 nm.
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po es ( p). In his wo k we measu ed he sho es dis-
ance be ween wo opposi e poin s o hese ib il-like
s uc u es, gene a ing a simula ed skele on, which
allowed us o es ima e he dimensions o he p. Hal o
his dis ance, named in his pape as he ela i e po e
adius ( p ), was used as he c i e ia o de ining he
sizes o hese ca i ies. In addi ion, he equency in he
de e mina ion o he same alues o p s was analyzed,
wi h he pu pose o s udying he po e size dis ibu ion.
This analysis allows us o compa e di e en concen a-
ions o hyd ogels, and he po e size dis ibu ion mea-
su ed wi h o he s anda dized me hods.
To analyze he eliabili y o ou image analysis, he
alues o he po e size dis ibu ion o cell- ee mic ocap-
sules we e compa ed wi h hose ob ained by ni ogen
physiso p ion on d ied mic ocapsules. Al hough his
echnique is widely used o measu e su ace a eas in
powde s and po ous ne wo ks, i can also p o ide use ul
in o ma ion abou po e size in he mesopo ous ange.
The iso he ms ob ained a e p esen ed in Figu e 2, and i
is possible o obse e a simila beha iou o hose o
ype IV and hys e esis ype H
3
acco ding o he IUPAC
classi ica ion [15], ypical o mesopo ous solids wi h
s ong adso ben -adso ba e in e ac ions, indica ing he
p esence o la ge mesopo es wi h a size dis ibu ion ha
con inues in o he mac opo e domain (po es > 50 nm).
Type H
3
loops a e usually gi en by adso ben s con ain-
ing sli -shaped po es in good ag eemen wi h he
obse ed ne wo k ca i ies. The adso p ion a low ela-
i e p essu e allowed us o e alua e he speci ic su ace
a ea o he samples by he BET me hod, assuming a
monolaye o N
2
molecules co e ing 0.162 nm
2
. Speci ic
su ace a eas o 245 and 532 m
2
.g
-1
ha e been ob ained
o capsules made o 0.8% and 1.4% w/ algina e espec-
i ely, in a ep oducible and well-co ela ed measu e-
men wi h he inc ease in biopolyme ma e ial pe
capsule o simila dimensions (app oxima ely 500 μmin
diame e ).
Table 1 shows he compa ison o he esul s ob ained
in mic ocapsules made o 0.8% and 1.4% w/ algina e,
using ou image analysis and he N
2
-adso p ion-deso-
p ion. The good ag eemen be ween he esul s o bo h
me hods is clea , wi h e o s lowe han 5.0%. The
esul s in Table 1 indica e ha algina e hyd ogels ha e a
wide dis ibu ion o ela i e po es, wi h dimensions up
o oughly 70nm. Quan i ies o po es smalle han 10
nm co espond o app oxima ely 50% in he case o algi-
na e 0.8% w/ , and app oxima ely 60% in he case o
algina e 1.4% w/ mic ocapsules, indica ing ha bo h
ma ices seem o be e y simila in e ms o po e size
dis ibu ion. Beside he de e mina ion o he dimension
o he ca i ies o ming he algina e ma ix, ou me ho-
dology allowed us o de e mine he algina e ib il-like
s uc u e wid h, which is highe in he case o algina e
1.4% han in he capsules made o 0.8% w/ (Table 2).
I is impo an o no e ha al hough he hyd ogel
ma ix allows easy di usion o se e al nu ien s wi h
small molecula weigh (e.g. glucose, oxygen), he p e-
sence o a high popula ion o po es smalle o 10 nm
could es ic he di usion o some p o eins, such as
albumin and/o hemoglobin (S okes adius o 3.1-3.5
nm and 2.4 nm espec i ely) [16].
I is impo an o ema k ha he sensi i i y o ou
me hod elies on he mic og aph image esolu ion.
Thus, he use o image analysis becomes a powe ul
s a egy o he analysis o meso- and nano- po ous
Figu e 2 N
2
adso p ion (black illed symbols) - deso p ion
(un illed symbols) a 77K iso he ms o supe c i ical CO
2
d ied
capsules made o 0.8% ( iangles) and 1.4% w/ (ci cles).
Table 1 Compa ison o alues o ela i e po e adius ( p ) de e mined by N
2
adso p ion-deso p ion and image
analyses in cell- ee mic ocapsules made o 0.8% and 1.4% w/ algina e
Range p (nm) 0.8% w/ Algina e 1.4% w/ Algina e
N
2
-adso p ion (%) Image analysis (%) N
2
-adso p ion (%) Image analysis (%)
p ≤10 48.9 ± 1.3 49.4 ± 2.3 59.8 ± 1.5 62.8 ± 1.8
10 < p < 20 15.4 ± 1.1 16.7 ± 2.3 16.1 ± 1.2 19.9 ± 1.1
20 < p < 25 13.3 ± 1.0 11.1 ± 2.2 8.9 ± 1.0 9.4 ± 1.6
25 < p < 40 11.3 ± 0.8 9.3 ± 1.9 8.8 ± 0.7 4.7 ± 0.4
40 < p < 70 9.0 ± 0.5 11.0 ± 0.7 5.3 ± 0.5 2.9 ± 0.3
70 < p 2.2 ± 0.3 3.0 ± 0.3 1.1 ± 0.2 1.0 ± 0.2
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ma e ials, p esen ing clea ad an ages o o he s a egies
o cha ac e iza ion o ex u al p ope ies o hyd ogels.
A e cha ac e iza ion o cell- ee algina e hyd ogel,
HepG
2
cells we e en apped in mic ocapsules made o
0.8% and 1.4% w/ algina e, and cul u ed o 6 days,
analyzing agg ega ion and p oli e a ion as inc eases in
he size o single cells and agg ega es. Since algina e
lacks domains o p o eases, en apped cells canno
mig a e in o he ma ix, gene a ing sphe ical agg ega es
a e p oli e a ion, which can be analyzed by measu ing
hei diame e s [17]. As Figu e 3 shows, cells en apped
in 0.8% w/ mic ocapsules inc eased hei size much
mo e han hose immobilized in 1.4% w/ .
Measu emen s o p sizes and equency we e ca ied
ou on days 0, 3 and 6, in a simila manne o he de e -
mina ions pe o med in cell- ee hyd ogels. These alues
we e co ela ed wi h a hi d pa ame e measu ed pe -
pendicula ly inwa ds om he algina e ma ix o he
cell. This analysis allows us o quan i y he ex ension
(dep h) o which he cells can modi y he ma e ial
ma ix in e ms o po e size dis ibu ion.
Ou esul s show signi ican modi ica ions in he pa -
e n o po e size dis ibu ion, mos ly obse ed in case o
cells en apped in hyd ogels made o 0.8% algina e,
whe e an inc ease in he p esence o po es smalle han
10 nm was clea ly obse ed (Figu e 4). Fu he mo e,
hese modi ica ions we e obse able up o dep hs o 40
nm om he in e ace, wi h he highe coa sening
de ec ed wi hin he i s 20 nm om he in e ace. By
con as , only sligh de o ma ions we e obse ed in he
expe imen s pe o med wi h hyd ogels made o 1.4% w/
algina e (app oxima ely 40 nm om he in e ace),
whe e coa sening o po es seems o be much slowe and
mo e homogeneous han in he so e capsules.
The highe esis ance o he mo e highly concen a ed
hyd ogel o mechanical de o ma ion can be explained
by inc eases in bo h he pe cen age o po es smalle
han 10 nm, and he hickness o he algina e ib il-like
s uc u es, due o inc eased c osslinking o algina e
polyme .
Acco ding o ecen publica ions, immobilized cells
wi hin algina e hyd ogels a e submi ed o comp ession
o ces which lead single cells o gene a e cellula mic o-
sphe oids [18]. Thus, analyses o adial de o ma ion o
he algina e ma ix due o cell g ow h and he conse-
quen modi ica ion o he po e size dis ibu ion pa e n
can gi e us e y impo an in o ma ion abou modula-
ion o a es o molecula di usion o nu ien s/was e
p oduc s, in o ma ion which is no only ex emely use ul
o biomedical applica ions [1], bu also o s udying he
de elopmen o p ima y umou s in umo -like mic o-
capsules [2,19], as men ioned p e iously.
I is impo an o men ion ha me hods o cell ixa-
ion can sligh ly diminish cell size, and he e o e a sho
dis ance be ween cells and he ma e ial in e ace can be
obse ed in se e al cases. Ne e heless, as shown in Fig-
u es 1 and 4, his does no a ec he po e size dis ibu-
ion and he ex u al p ope ies o he ma ix ma e ial,
ensu ing he eliabili y o ou me hod. As a inal ema k,
i is impo an o no e ha al hough ou me hodology
has been no es ed wi h o he polyme s, because i is
based on image analysis o TEM pic u es, s udies o
modi ica ions o he cell-hyd ogel in e ace may be pos-
sible in di e en ypes o hyd ogels which main ain hei
ex u al p ope ies a e ixa ion.
Table 2 Compa ison o alues o ib il-like adii ( l )
de e mined by image analyses in algina e mic ocapsules
made o 0.8% and 1.4% w/ algina e
Range (nm) 0.8% w/ Algina e(%) 1.4% w/ Algina e(%)
l ≤2.34 48.7 ± 2.8 30.3 ± 2.4
2.34 < l < 4.68 48.9 ± 3.2 64.0 ± 3.1
4.68 < l < 7.02 2.3 ± 0.3 5.5 ± 0.5
7.02 < l < 9.36 0.1 ± 0.01 0.3 ± 0.1
9.36 < l 0.0 ± 0.0 0.0 ± 0.0
Figu e 3 Sizes o HepG
2
cell popula ion (indi idual li ing cells
and agg ega es) wi hin mic ocapsules made o 0.8% (A) and
1.4% w/ (B) algina e, du ing days 0 (yellow iangles), 3 ( illed
ci cles), and 6 (open ci cles) o cul u e.
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Acknowledgemen s
The au ho s hank D . John Ha dy and Eileen Lin z o p oo - eading and
cons uc i e c i icism du ing he p epa a ion o his manusc ip . We
g a e ully acknowledge he inancial suppo p o ided by he Spanish
Go e nmen , Depa men o Science and Inno a ion, MICINN (Plan Nacional
BIO2009-13903-C02-02). Aldo Leal-Egaña is g a e ul o he inancial suppo
by a g an om he Ge man Academic Exchange Se ice (Deu sche
Akademische Aus auschdiens ). Ul -Die ich B aumann is g a e ul o he
long and ui ul coope a ion wi h D . Jens-Pee Kuska who died in 2009 a
he young age o 45.
Au ho de ails
1
Depa men o Cell Technology and Applied S em Cell Biology,
Bio echnology and Biomedicine Cen e (BBZ), Uni e si y o Leipzig.
Deu sche Pla z 5, 04103, Leipzig, Ge many.
2
Ins i u e o Medical In o ma ics,
S a is ics, and Epidemiology (IMISE), Uni e si y o Leipzig, Hä els aße 16-18,
04107 Leipzig, Ge many.
3
Ma e ials Science Ins i u e o Se ille (Spanish
Na ional Resea ch Council (CSIC) - Uni e si y o Se ille), Cen o de
In es igaciones Cien í icas Isla de la Ca uja, A da. Ame ico Vespucio no. 49,
41092 Se illa, Spain.
4
Ne wo king Resea ch Cen e on Bioenginee ing,
Bioma e ials and Nanomedicine (CIBER-BBN), Spain.
5
Ins i u e o Ana omy,
Medicine Facul y, Uni e si y o Leipzig, Liebigs asse 13, 04103 Leipzig,
Ge many.
Au ho s’con ibu ions
ALE concei ed and designed he me hod, pe o med he expe imen s and
in e p e ed he da a. UDB pe o med he image analysis and concei ed he
me hod. ADC pe o med he ex u al analysis and in e p e ed he da a. MN
ob ained he elec on mic oscopy images. ALE, ADC, and UDB p epa ed he
manusc ip . AB and ADC c i ically e ised he in ellec ual con en o he
manusc ip and ga e he inal app o al o he e sion o be published. All
Au ho s ead and app o ed he inal manusc ip .
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Recei ed: 20 Feb ua y 2011 Accep ed: 27 May 2011
Published: 27 May 2011
Re e ences
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Buhle LH: T ansplan a ion o encapsula ed hepa ocy es du ing acu e
li e ailu e imp o es su i al wi hou s imula ing na i e li e
egene a ion. Cell T ansplan 2011.
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cons an o hepa ocy es immobilized in algina e mic ocapsules.
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Impac o algina e ype and bead diame e on mass ans e s and he
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c osslinked mal odex in-co-dime hylac ylamide as a highly s able polyme
ca ie o con olled elease sys ems. Eu Polyme J 2010, 46:1465-1474.
Figu e 4 Analysis o ansmission elec on mic oscopy images o he cell bioma e ial in e ace on mic ocapsules made o 0.8% (A, B,
and C) and 1.4% w/ (D, E and F), a day 0 (A and D), 3 (B and E), and 6 (C and F). A ows show zones whe e clea cons ic ion o he
algina e in e ace due o cell p oli e a ion is obse ed. Scale ba ep esen s 10 nm.
Leal-Egaña e al.Jou nal o Nanobio echnology 2011, 9:24
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doi:10.1186/1477-3155-9-24
Ci e his a icle as: Leal-Egaña e al.: De e mina ion o po e size
dis ibu ion a he cell-hyd ogel in e ace. Jou nal o Nanobio echnology
2011 9:24.
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and ake ull ad an age o :
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Leal-Egaña e al.Jou nal o Nanobio echnology 2011, 9:24
h p://www.jnanobio echnology.com/con en /9/1/24
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