Tissue and Cell 43 (2011) 115–124
Contents lists available at ScienceDirect
Tissue and
Cell
j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /t i c
e
Adhesive cell cultivation on polymer particle having grafted epoxy polymer chain
Masahiro Yasuda a ,∗,Hiroshi Kunieda a ,Kentaro Ono a ,Hiroyasu Ogino a ,Tomohiro Iwasaki a ,Masaki Hiramoto b ,Wilhelm Robert Glomm c ,Yukio Hirabayashi d ,Shin Aizawa d
a
Department of Chemical Engineering,Osaka Prefecture University,1-1Gakuen-cho,Naka-ku,Sakai,Osaka 599-8531,Japan
b
Department of Metabolic Disorder,Diabetes Research Center,National Center for Global Health and Medicine,1-21-1Toyama,Shinjuku-ku,Tokyo 162-8655,Japan c
Department of Chemical Engineering,Norwegian University of Science and Technology,Sem Sælands vei 4,Trondheim N-7491,Norway d
Department of Functional Morphology,Nihon University School of Medicine,30-1Ootaniguchi Kami-machi,Itabashi-ku,Tokyo 173-8610,Japan
a r t i c l e i n f o Article history:
Received 15July 2010Received in revised form 17December 2010
Accepted 19December 2010
Available online 23 February 2011Keywords:
Graft polymer chain
Poly(glycidyl methacrylate)Suspension polymerization Adhesive cell Co-culture
Extracellular matrix
a b s t r a c t
In this study,we synthesized a new cell immobilization support having poly(glycidyl methacrylate)as a graft polymer chain and used this support for cell cultivation.Base polymer particle was synthesized by suspension polymerization and epoxy polymer chain was extended from particle surface on graft poly-merization.Produced polymer particles had broad particle size distribution ranging from 20to 1000m and the degree of polymerization of grafted polymer chain was ranged from 500to 1000.The effects of various factors,such as grafted polymer chain length and its surface density,composition of base poly-mer network and graft polymer chain,on the cell growth of murine fibroblast cell line (MS-5cell)on polymer particle were studied.This polymer particle could cultivate not only fibroblast cell line but also epidermal cell line (HeLa cell),osteoblast cell line (MC3T3E1cell),and chondrocyte cell line (ch-8cell)on its surface.Growth rate is almost the same as that of cells using poly(styrene)tissue culture dish.To apply this cell cultivation system for examination of cell co-culture,HeLa cell immobilized on 100m of polymer particle was successfully co-cultured with MS-5cell immobilized on 300m of polymer particle for four weeks.
© 2011 Elsevier Ltd. All rights reserved.
1.Introduction
Cell-to-cell adhesions and cell-to-cell junctions play various important roles,such as forming and maintaining of tissues and organs,protection against bacteria penetration,and support for cell differentiation (Hubbell,1995;Sternlicht and Werb,2001;Alberts et al.,2002).Various proteins required for cell adhesion and cell junction have been identified and their roles have been clarified from many studies concerning for molecular biology.However,the mechanisms of signal transduction and cell differentiation induced by cell adhesion and cell junction in vivo have been poorly under-stood.This was because organs and tissues were heterogeneous and signal transduction between cells was composed of many kinds of molecules.Therefore,it was very difficult to identify and isolate activated cell site and to evaluate its functions correctly.Further-more,adhesive cell cultivated by ordinary dish culture spreads two-dimensionally and could not form three-dimensionally struc-ture,such as organs and tissues.Many researchers have desired to reconstruct organs and tissues in vitro (Soria et al.,2000;Poumay and Coquette,2007;Neuss et al.,2008).
∗Corresponding author.Tel.:+81722549299;fax:+81722549911.E-mail address:yasuda@chemeng.osakafu-u.ac.jp (M.Yasuda).Recently,using cell sheet,new tissue reconstruction technol-ogy have been developed and applied for regenerative medicine (Elloumi-Hannachi et al.,2010).In this technology,cell was culti-vated on thin thermo responsive polymer sheet
mainly composing of poly(N -isopropylacrylamide)(PIPAAm)and thermal phase tran-sition successfully divide cell sheet from base thin polymer film (Shimizu et al.,2001,2003;Sasagawa et al.,2010).When adhe-sive cell was cultivated in dish,suspension of cell was prepared by using trypsin to divide cell from dish surface.In this situa-tion,cell to dish conjugation was enzymatically digested and cell to cell conjugation was simultaneously digested.However,cell sheet prepared by cell sheet technology had not only extracellu-lar matrix between cell and adhered base polymer sheet but also cell-to-cell junctions between cells (Kushida et al.,1999).Using cell sheet technology,the regeneration of myocardium,cornea,skin,periodontium,blood vessel,and so on,have been applied for clin-ical applications (Elloumi-Hannachi et al.,2010)and good clinical results were obtained for long term tissue preservation (Ide et al.,2006).
Cell sheet was good technology for reconstruction of homoge-neous tissue.However,to build complex organization composed of heterogeneous cells,such as hematopoietic microenvironment in bone marrow and pancreas tissue for product of insulin,three-dimensional cell support and growth system in which het-erogeneous cells were preserved without growth rate selection
0040-8166/$–see front matter © 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.tice.2010.12.007
116M.Yasuda et al./Tissue and Cell43 (2011) 115–124
must be developed.For large scale cultivation of adhesive cell in fluidized bed,fixed bed,and rotating wall vessel reactor,polymer particle(microcarrier)whose size was micron order was effective and they can immobilize various cells having various growth rates (Bryan,2000;Pörtner et al.,2005).Many researchers have stud-ied the effects of microcarrier surface properties such as chemical nature,charge density,roughness,wettability,rigidity and so on, on cell attachment and growth(Kato et al.,2003and references therein).Among these properties,the amino group content has been considered a critical factor for cell attachment and growth (Kato et al.,2003).A kind of short linear oligopeptides(Arg-Gly-Asp,RGD sequence)had an important function for cell adhesion (Mizuno and Glowacki,1996;Wang et al.,2002;Reeve et al., 2003;Zheng et al.,2004;Kurihara and Nagamune,2005;Bigi et al., 2005).Coating of extracellular matrix,such as collagen,laminin, andfibronectin,was effective to cultivate adhesive cell on arti-ficial material.However,suitable extracellular matrix required was dependent on the difference in the cell species.If extracel-lular matrix required for cell growth and cell immobilization and secreted from cell is in situ immobilized on a support,we can estab-lish new cell immobilization system.This was the starting point of our research.
In our previous study,we developed new graft-polymerization method using a polymerizable azo initiator,2,2 -azobis[N-(2-propenyl)-2-methylpropionamide](APMPA)having both two vinyl groups and o
ne azo group(Yasuda et al.,2010a).This method can be applied for suspension polymerization and we can easily syn-thesize polymer particle having grafted functional polymer chain. When glycidyl methacrylate was used for graft co-polymerization, epoxy grafted polymer chain which can rapidly react with amino group of protein was introduced into particle surface.We assumed that a thin layer formed by the reaction between proteins orien-tated from cell secretion or dissolving in serum and epoxy group of grafted polymer chain enhanced cell adhesion and growth on both particle surface and particle gap.Furthermore,oxygen and nourish-ment transport through this thin layer would increase the number of cell grown on both particle surface and particle gap and enable long term cultivation.
Therefore,the objective of this work was development of new three-dimensional cell cultivation method using this graft polymer particle composed of poly(glycidyl methacrylate)and optimization of various factors for cell adhesion and growth.For this purpose,the effects of graft polymer chain length,its surface density,composi-tion of base polymer network and graft polymer chain on the cell growth of murinefibroblast cell line(MS-5cell)on polymer parti-cle werefirstly studied.Then,to apply this cultivation method for other kind of adhesive cell and co-culture,epidermal cell line and osteoblast cell line was cultivated in the presence of grafted poly-mer particle.MS-5cell cultivated for5days using grafted polymer particle was added to HeLa cell whose doubling time was5-fold faster than that of MS-5cell and cultivated for long term.
2.Materials and methods
2.1.Materials
2,2 -Azobis(isobutyronitrile)(AIBN),2,2 -azobis[N-(2-pro-penyl)-2-methylpropionamide](APMPA),1,4-butanediol digly-cidyl ether,1,4-dioxane,2-(diethylamino)ethyl methacrylate (DEAEMA),diethylaminoethyl methacrylate,ethanol,glycidyl methacrylate(GMA),hydrochloric acid,2-hydroxyethyl methacry-late(HMA),methacrylic acid(MA),methanol,methyl methacrylate (MMA),pentaerythritol triacrylate(PETA)and trypan blue were purchased from Wako Pure Chemical Co.(Osaka,Japan).Cresol red,hydroquinone,phosphoric acid,poly(vinyl pyrrolidone)K-90(molecular mass is about360,000)(PVP),sodium hydroxide, toluene were purchased from Nacalai Tesque(Kyoto,Japan). Coomassie brilliant blue G-250,and pentaerythritol triacrylate were purchased from Sigma–Aldrich Co.(St.Louis,MO,USA). Bovine serum albumin(BSA)(molecular mass is about66,000)was purchased from Invitrogen(Carlsbad,CA,USA).All of the reagents were used without purification.
2.2.Preparation of polymer particle having graft polymer chain
We used polymer particle having graft polymer chain as cell sup-port.In1000ml of three-neckflask,2.0g
of AIBN,3.0g of APMPA, 38.0g of MMA,38.0g of PETA,and560ml of water containing1wt% PVP were added.The reaction mixture stirred at70◦C with300rpm for3h and then at80◦C with300rpm for2h.Produced polymer particle having azo group derived from APMPA was washed with distilled water and methanol for three times by repeating suspen-sion andfiltration of the particle.Next,in1000ml three-neckflask, 10g of polymer particle having azo group,0–25g of GMA,0–25g of comonomer,and250g of toluene were added.The reaction mix-ture was stirred at100◦C with150rpm for8h(standard condition). Produced particle having grafted polymer chain was washed as described above.Here after,we call polymer particle having graft polymer chain as epoxy grafted polymer particle.
For the preparation of poly(propylene)(PP)plate having graft epoxy chain,10mm×50mm of PP plate was put on glass laboratory dish and was irradiated with125kGy(25kGy h−1×5h)of␥-ray.In N2atmosphere,PP plate,5g of MA,20g of GMA,8g of sodium dodecyl sulfate,and400g of Milli-Q water was added to a100ml offlask and the reaction mixture was incubated at60◦C for8h. After reaction,plate was rinsed with methanol and water.
Polymer particle and PP plate was dipped in ethanol and stored at4◦C.Prior to particle addition to cell,ethanol was removed by decantation or centrifugation.Polymer particle and PP plate were washed in several changes of Milli-Q water.
2.3.Analysis of polymerization and produced particle
Monomer conversions of polymerization were measured by gravimetric method(Yasuda et al.,2001).Each particle diameter distribution was measured using a laser particle diameter analyzer MICROTRAC FRA(Microtrac Inc.,Montgomery,PA,USA).Amount of epoxy group of polymer particle was measured by hydrochloric acid-dioxane method(Weiss and Frederic,1970).Particle surface was analyzed as tapping method using an atomic force microscope (SPM-9500J3;Shimadzu,Kyoto,Japan).Adsorb rate of BSA to poly-mer particle was measured by Brad-ford method(Bradford,1976). BSA size in buffer solution was measured using a Zetasizer Nano ZS(Malvern).The particle pore and its distribution were measured by mercury porosimetry(Pascal140and240,Thermo Scientific, Waltham,MA,USA).About0.5g of sample was charged to the glass sample electrode tube(dilatometer CD-3PL,Thermo Scientific)and degassed by Pascal140for30–60min and mercury was introduced into the pore.Pascal140can measure micrometer level of pore (>3m).Macro-pore and meso-pore ranged from3m to7nm was measured by Pascal240after the measurement of Pascal140 in which particle large gap wasfilled with mercury.The dilatome-terfilled with mercury using Pascal140was placed in the oil cell and air was eliminated byfilling oil.Pascal240added pressure on glass sample electrode tube tofill mercury against meso-pore
until 200MPa.Both results were combined by programs installed by con-trol PC.To check epoxy group introduced into PP plate,IR spectra of PP plate was measured by FT-IR spectrophotometer(FT-IR-410, JASCO,Tokyo,Japan)attached with ATR measurement unit(ZnSe prism).
M.Yasuda et al./Tissue and Cell43 (2011) 115–124117
2.4.Materials for cell cultivation
Dipotassium hydrogen phosphate,25wt%glutaraldehyde solution,36wt%paraformaldehyde,saccharose,and sodium dihy-drogen phosphate were purchased from Wako Pure Chemical Co.Antibiotic–antimycotic(100×)solution,Dulbecco’s modified Eagle’s medium(D-MEM),fetal bovine serum(FBS),and5wt% trypsin aqueous solution containing0.2wt%EDTA·4Na were pur-chased from Sigma–Aldrich Co.
2.5.Cells and cell passage
Human epidermal cell line(HeLa cell),human chondrocyte cell (Ch-8cell),murinefibroblast cell line(MS-5cell),and murine osteoblast cell line(MC3T3E1cell)were offered from school of medicine of Nihon University.These cells were cultivated with D-MEM containing10vol%FBS and1vol%of antibiotic–antim
ycotic (100×)solution(cell culture media).In CO2incubator(5%CO2) (APC-30D,ASTEC Co.,Ltd.,Shimemachi,Japan),each cell was cul-tured using polystyrene dish(3020-100,IWAKI Glass Co.,Ltd., Tokyo,Japan)at37◦C.Phosphate buffered saline(PBS(−))was com-posed of4.0g of sodium chloride,0.10g of potassium chloride, 0.10g of dipotassium hydrogen phosphate,and0.10g of sodium dihydrogen phosphate in500ml of Milli-Q water and adjusted pH using1M of NaOH aqueous solution.When80–90%of bottom surface of polystyrene dish was covered with cell,culture super-natant was removed with aspiration and adhered cell surface was washed with5ml of PBS(−)for3times by pipetting.One milliliter of0.5wt%trypsin aqueous solution containing0.2wt%EDTA·4Na (EDTA–trypsin solution)was then added to the polystyrene dish. For3–5min incubation at37◦C,floated and suspended cell in the EDTA–trypsin solution was collected in15ml of PP centrifuge tube. Five milliliter of PBS(−)was added to the polystyrene dish to col-lect remaining cell and cell was collected into15ml of PP centrifuge tube by pipetting.PP centrifuge tube was centrifuged at4◦C with 1500rpm for5min and cell pellet was suspended with5ml of PBS(−).Cell dispersion was centrifuged again and the centrifu-gation and suspension procedure were repeated three times(cell washing).Finally cell was suspended with D-MEM containing10% (v/v)FBS and1/4–1/8of cell suspension of cell was added to a new polystyrene dish.2.6.Cultivation of mammalian cell in the presence of polymer particle
Ch-8cell,HeLa cell,MC3T3E1cell,and MS-5cell cultivated on polystyrene dish were removed from polystyrene dish by EDTA–trypsin solution and cell washing was repeated for three times.Cell suspension and polymer particle suspension with D-MEM containing10%(v/v)FBS were pored into15ml of PP tube and the mixture was softly mixed by rotating PP tube.Cell and particle suspension was stand at37◦C for60min to24h in CO2 incubator.And then the suspension was added to cell adhesive polystyrene dish(3020-100)or normal polystyrene dish(SH-90-15;ASAHI GLASS Co.,Ltd.)and incubated in CO2incubator.The number of cell on polymer particle was counted using Burker-turk counting chamber.Cell viability was checked using trypan blue staining.Cell morphology on polymer surface was checked hematoxylin-eosin or Alcian blue stain.Cell photograph was taken by using microscope(CKX41,Olympus Co.Ltd.,Tokyo,Japan).
3.Results and discussion
3.1.Characterization of polymer particle
Forflasks and dishes used for adhesive cell culture,hydroxyl group and carboxyl group were introduced by corona discharge treatment(Nunclon Delta®treatment)and plasma treatment(Lee et al.,2000).Since the introduction of hydroxyl group and car-boxyl group was effective for adhesive cell cultivation,we de
cided that these hydrophilic functional groups were introduced by graft polymerization using APMPA.Table1shows a composition and condition of suspension polymerization and graft polymerization. To distinguish each support,we defined support number and listed in this table.Support numbers from1to13were epoxy grafted polymer particle.Support number14was polymer particle whose size was almost the same as those of support numbers3and their epoxy group was only incorporated with base polymer network.To control the amount of epoxy group incorporated in graft polymer chain,GMA amount in reaction mixture of graft polymerization was varied from0g to25g(supports1–6and support15).To vary the surface density of graft polymer chain,APMPA amount on suspen-sion polymerization was varied from0.75g to3g(supports7–8and support3).APMPA amount was corresponded the number of initia-
Table1
Composition and condition of suspension polymerization and graft polymerization.
Support number Composition of suspension polymerization Composition and condition of graft polymerization MMA[g]GMA[g]APMPA[g]PETA[g]PVP[g]GMA[g]HMA[g]MA[g]DEAEMA[g]Time*[h]
138–338 5.64250––8 238–338 5.6422.5 2.5––8 338–338 5.64205––8 438–338 5.6412.512.5––8 5
38–338 5.64520––8 638–338 5.64 2.522.5––8 738–0.7538 5.64205––8 838– 1.538 5.64205––8 938–338 5.64205––4
10**38–338 5.64205––2
reaction between pvp and amino1138–338 5.6420–5–8
1238–638 5.6420–5–8
1338–11.438 5.6420–5–8
14–38–38 5.64N.D.
1538–338 5.64025––8
1638–338 5.640–25–8
1738–338 5.640––258
18Poly(propylene)plate20–58
*Reaction time of graft polymerization.
**Support number10was the same as3.
N.D.:not done.
118M.Yasuda et al./Tissue and Cell43 (2011) 115–124
Table2
Average size of polymer particle and its standard deviation of epoxy grafted polymer particle.
Support no.Average size[m]Standard deviation[m]
1318.6152.5
2300.7106.9
3261.793.2
4330.0153.3
5242.091.2
6229.174.1
7446.0193.2
8231.879.8
9322.089.3
10357.7142.9
11353.6160.6
12348.9181.8
13255.871.9
14219.531.2
15266.5141.3
tion point on graft polymerization and it was proportionally related with the number of graft polymer cha
in.When HMA was used for comonomer on graft polymerization,the conversion of graft polymerization decreased at a high APMPA amount on suspension polymerization.Therefore,MA was used for comonomer on graft polymerization(support number was11,12,and13,respectively). The reaction time of graft polymerization was shortened by half or quarter hours to shorten graft polymer chain length(support number9and10).Supports15,16,and17had graft hydrophilic polymer chain only composed by HMA,MA,or DEAEMA.Support 18was poly(propylene)plate treated with␥-ray irradiation and graft polymer composed of GMA and MA was introduced on its surface.
In the synthesis of epoxy grafted polymer particle,the con-version on suspension polymerization was almost91–98%and average particle sizes were shown in Table2.The size distribu-tion of epoxy grafted polymer particle was wide.This was because droplet breakup and coalescence occurred randomly and droplet sizes varied according to a normal probability distribution on sus-pension polymerization(Yasuda et al.,2010b).The density of every epoxy grafted polymer particles was about1.2–1.3g cm−3.There-fore,polymer particle quickly precipitated in the aqueous culture medium containing cells.On the graft polymerization,conversion of monomer was ranging from8to37%.The degree of polymer-ization for grafted polymer chain was estimated as the remaining monomer amount in the reaction mixture on the graft polymeriza-tion.We assumed that the initiator efficiency of APMPA f
ragment incorporated base polymer network was0.3(Yasuda et al.,2010a) and the initiator efficiency was constant within every compo-sitions of reaction mixture as shown in Table1.The estimated degree of polymerization for graft polymer chain was about rang-ing from100(support10)to800(support1).We could control degree of polymerization of graft polymer chain with reaction time. Degrees of polymerization of support3,9,and10were411,202, and100,respectively.The amount of epoxy group was propor-tional with the amount of GMA in the reaction mixture of graft polymerization.The amount of epoxy group incorporated in graft polymer chain was ranging from0to1.63×10−3mol(g-particle)−1 as shown in Table3.When APMPA amount on suspension poly-merization was varied from0.75g to3g,the numbers of graft epoxy chain of support3,7and8which were estimated by ini-tiator efficiency and APMPA amount,were8.07×1018(number of graft epoxy chain)(g-particle)−1,4.03×1018(number of graft epoxy chain)(g-particle)−1,and2.14×1018(number of graft epoxy chain)(g-particle)−1,respectively.
When we took pre-adhesion tests of HeLa cell and MS-5cell using various supports,support3is superior of all.Therefore,sup-Table3
Amount of epoxy group of epoxy grafted polymer particle.
Support no.Amount of epoxy group[×10−3mol/g-particle]
1 1.63
2 1.08
3 1.03
40.704
50.482
60.276
140
port3was selected as a model polymer particle.We studied particle surface roughness before and after graft polymerization using an atomic force microscope(AFM).Fig.1a and b shows AFM topogra-phies of polymer particles after suspension polymerization prior to the graft polymerization(pre support3)and epoxy grafted polymer particle(support3),respectively.As shown in Fig.1b,the rough-ness of support3was ranging from0to31.37nm while polymer particle not having graft chain was ranging from0to14.87nm. Since the surface of dry polymer particle was measured by AFM, about15nm of poly
mer layer consisting of grafted polymer chain covered with the surface of polymer particle at the least.We
esti-
Fig.1.(a)Atomic force microscope of topography of polymer particle having azo group before graft polymerization(pre support3).A particle was put on the sample stage and particle surface was analyzed as tapping method.(b)Atomic force micro-scope of topography of epoxy grafted polymer particle after graft polymerization (support3).
M.Yasuda et al./Tissue and Cell43 (2011) 115–124119
Table4
Pore area distribution of support3.
Pore diameter[nm]Incremental pore area Cumulative pore area From To[m2(g-particle)−1][m2(g-particle)−1]
101.151.50.1950.195
47.530.20.5010.696
29.320.3 1.035 1.732
19.915.8 1.088 2.819
15.512.0 1.595 4.414
11.99.1 1.569 5.984
9.07.6 1.3617.345
mated maximum graft length by using0.25nm of monomer unit length(poly(ethylene)value)and411of polymerization degree of graft polymer chain.Maximum thickness of graft polymer layer was obtained as about100nm.In aqueous solution,thickness of graft layer exists in between15nm and100nm.According to the measurement of surface area distributions by mercury porosime-try,the pore area ranging from7nm to100nm of support3was 7.35m2(g-particle)−1and pore area distribution is summarized in Table4.The outer surface area estimated by the size(261.7m)was 1.91×10−2m2(g-particle)−1.This result indicated that the major-ity of the surface of this support was surface roughness as shown in Fig.1a and the pore surface inside particle.Therefore,as shown in Fig.1b,graft polymer chainfilling up surface roughness and pil-ing up surface was resulted in the large amount of functional group which can immobilize extracellular matrix.
3.2.BSA adsorption on polymer particle
When laminin(this is one of extracellular matrix)was added to polymer particle,the amount of immobilized laminin was quite low and we could not obtain reproducible data for absorption amount by Bradford method.This was because ECM is macromolecule,its occupied area is big,and wide molecular mass distribution makes its adsorption behavior heterogeneous.Therefore,to evaluate both adsorption amount and adsorption rate of protein to polymer par-ticle,well-characterized and homogeneous bovine serum albumin (BSA)was selected as model protein and0.2mg(ml)−1of BSA added to the suspension of polymer particle(0.1g of dry polymer particle) and time course of free BSA existed in the supernatant was mea-sured.Fig.2shows the time course of BSA adsorption amount.BSA adsorption amount was compared with those of cell culture dish and pre support3.In thisfigure,polymer particle having grafted epoxy and hydroxyl chain could adsorb BSA immediately within 20min and BSA adsorption amount attained equilibrium value.In contrast,BSA adsorption amounts of pre support3and polystyrene dish(60mm)were almost zero or little.This was because epoxy group of graft polymer chain immediately reacted with amino group of BSA,such as lysine,and BSA was covalently immobilized with polymer particle.
According to the maximum immobilization amount (4.00×10−9mol cm−2)of BSA on support3and the surface area(1.91×10−2m2(g-particle)−1)of support3,BSA immobi-lization amount was calculated as7.
64×10−7mol(g-particle)−1 and only30–35of59lysine residue of BSA are accessible for carrier binding(Van Regenmortel et al.,1988).Since maximum 2.67×10−5mol(g-particle)−1of epoxy group was consumed after BSA binding,≥97.4%of epoxy group was remaining(support3: 1.03×10−3mol(g-particle)−1of epoxy group).This estimation indicated that majority of epoxy group having an reactivity with amino group of proteins,extracellular matrix,glycoprotein,and so on,were remaining and extracellular matrix secreted from cell would bind after adsorption of BSA existing in culture media.
Time [min]
A
m
o
u
n
t
s
o
r
b
e
d
B
S
A
t
o
s
u
p
p
o
r
t
9
[
m
o
l
·
c
m
-
2
]
Fig.2.Adsorption rate of BSA against support3.Five milliliter of BSA(1.5mg ml−1) in10mM of phosphate buffer(pH7.2)and0.1g of polymer particle were mixed and incubated at4◦C.Aliquot of supernatant was collected from mixture and remaining BSA was measured.In the case of poly(styrene)dish,10ml of BSA aqueous solution was added to100mm of cell adhesive polystyrene dish and BSA concentration in aqueous solution was measured.
Next,we compare occupied area of BSA calculated from max-imum immobilization amount and outer surface area of support 3.When BSA diameter in10mM of acetate buffer was measured at7.9nm by zeta-sizer,4×10−9mol of BSA occupied area was calculated as1.18×103cm2.According to this estimation,about 1200-fold of BSA amount which can cover with plane polymer par-ticle surface was existed on the surface.This was because area of surface roughness and the pore surface inside particle was about 380-fold of that of outer surface area.Therefore,we would conclude that the introduction of graft epoxy polymer chain is effective for quickly trapping proteins including extracellular matrix secreted from cell and enhances the cell adherence.
3.3.Cultivation of various cells in the presence of polymer particle
A suspension of murine osteoblast cell line,MC3T3E1,was added to the cultivation media in the presen
ce of support3in15ml of PP tube and the mixture was incubated in CO2incubator for24h. MC3T3E1cell was adhered on support3.However,when there is no graft epoxy chain(support15,16,and17)and epoxy group was incorporated into base polymer network(support14),MC3T3E1 cell could not adhere on these supports.This result supposed that epoxy group incorporated in graft polymer chain was required for cell adherent.In the case of MS-5cell and HeLa cell,same results were obtained.Fig.3shows micrographs of various cells in the pres-ence of support3.After24h incubation in15ml of PP tube,the mixture was transferred into100mm polystyrene dish.Once cell was immobilized on the surface of polymer particle,cell grew on the surface and did not peel or break off the surface of polymer particle.The arrows in pictures show cell bridge between particles gap.Cell was individually separated by trypsin–EDTA treatment before cell culture in the presence of polymer particle.Therefore, the formation of cell bridge indicated that cell was adhered on polymer particle and grew up on its surface.Epoxy group intro-duced into graft chain can covalently react with active hydrogen in amino group of amino acid as described above and their reaction rate was quite high.Therefore,epoxy group of polymer particle may immobilize extracellular matrix.Extracellular matrix adsorbed on particle surface enhanced adherence and growth of cell.Fig.4 shows atomic force microscope of topography of support3after
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