Determination of Pore Size Distribution at the Cell-Hydrogel Interface

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 h p://www.jnanobio echnology.com/con en /9/1/24 © 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 Leal-Egaña e al.Jou nal o Nanobio echnology 2011, 9:24 h p://www.jnanobio echnology.com/con en /9/1/24 Page 2 o 7 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. Leal-Egaña e al.Jou nal o Nanobio echnology 2011, 9:24 h p://www.jnanobio echnology.com/con en /9/1/24 Page 3 o 7 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 Leal-Egaña e al.Jou nal o Nanobio echnology 2011, 9:24 h p://www.jnanobio echnology.com/con en /9/1/24 Page 4 o 7 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. Leal-Egaña e al.Jou nal o Nanobio echnology 2011, 9:24 h p://www.jnanobio echnology.com/con en /9/1/24 Page 5 o 7 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 1. Sg oi A, Mai G, Mo el P, Bae schige RM, Gonelle-Gispe C, Se e-Beinie V, 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. 2. Yu L, Chen M, Cheung K: D ople -based mic o luidic sys em o mul icellula umo sphe oid o ma ion and an icance d ug es ing. Lab Chip 2010, 10:2424-2432. 3. Vay K, Schele S, F iess W: New insigh s in o he po e s uc u e o poly(d, l-lac ide-co-glycolide) mic osphe es. In J Pha m 2010, 402:20-6. 4. Leal-Egaña A, Díaz-Cuenca A, Bade A: De e mina ion o he decay a e cons an o hepa ocy es immobilized in algina e mic ocapsules. J Mic oencapsul 2010, 27:86-93. 5. Gau ie A, Ca pen ie B, Du esne M, Vu Dinh Q, Paullie P, Legallais C: Impac o algina e ype and bead diame e on mass ans e s and he me abolic ac i i ies o encapsula ed C•A cells in bioa i icial li e applica ions. Eu opean Cells and Ma e ials 2011, 21:94-106. 6. Guilhe me M, Faja do A, Moia T, Kuni a M, Gonçal es M, Rubi a A, Tambou gi E: Po ous nanocomposi e hyd ogel o inyled mon mo illoni e- 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 h p://www.jnanobio echnology.com/con en /9/1/24 Page 6 o 7 7. Seck T, Melchels F, Feijen J, G ijpma D: Designed biodeg adable hyd ogel s uc u es p epa ed by s e eoli hog aphy using poly(e hylene glycol)/ poly(d,l-lac ide)-based esins. J Con ol Release 2010, 148:34-41. 8. Leal-Egaña A, Hein ich JM, Smi h M, Nowicki M, Bade A: A simple non- des uc i e me hod o he ixa ion and immunos aining o cul u ed cells encapsula ed in algina e. Bio echnol Appl Biochem 2006, 44:143-150. 9. Rouque ol F, Rouque ol J, Sing K: Adso p ion by powde s and po ous solids: p inciples, me hodology and applica ions. London, Academic P ess; 1999. 10. Sing K, Williams R: Empi ical P ocedu es o he Analysis o Physisop ion Iso he ms. Adso p ion Science and Technology 2005, 23:839-853. 11. Gonzalez R, Woods R: Digi al Image P ocessing New Je sey: Pea son Educa ion; 2008. 12. Chan T, Oshe S, Shen J: The digi al TV il e and nonlinea denoising. In T Image P ocess. Volume 10. IEEE; 2001:231-241. 13. Weicke J: Cohe ence-enhancing shock il e s. LNCS 2003, 2781:1-8. 14. Sai o T, To iwaki J: New algo i hms o n-dimensional Euclidean dis ance ans o ma ion. Pa e n Recogn 1994, 27:1551-1565. 15. Sing K, Williams R: Physiso p ion hys e esis loops and he cha ac e iza ion o nanopo ous ma e ials. Adso p Sci Technol 2004, 22:773-782. 16. Ralla K, Sohling U, Rieche s D, Kaspe C, Ru F, Schepe T: Adso p ion and sepa a ion o p o eins by a smec i ic clay mine al. Biop ocess Biosys Eng 2010, 33:847-861. 17. Ge e z J, Gillies G, To qua o S: Simula ing umo g ow h in con ined he e ogeneous en i onmen s. Phys Biol 2008, 5:036010. 18. Cheng G, Tse J, Jain R, Munn L: Mic o-en i onmen al mechanical s ess con ols umo sphe oid size and mo phology by supp essing p oli e a ion and inducing apop osis in cance cells. PLoS one 2009, 4: e4632. 19. Hu mache D: Bioma e ials o e cance esea ch he hi d dimension. Na Ma e 2010, 9:90-93. 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. Submi you nex manusc ip o BioMed Cen al and ake ull ad an age o : • Con enien online submission • Tho ough pee e iew • No space cons ain s o colo igu e cha ges • Immedia e publica ion on accep ance • Inclusion in PubMed, CAS, Scopus and Google Schola • Resea ch which is eely a ailable o edis ibu ion Submi you manusc ip a www.biomedcen al.com/submi Leal-Egaña e al.Jou nal o Nanobio echnology 2011, 9:24 h p://www.jnanobio echnology.com/con en /9/1/24 Page 7 o 7