the design of polymer microcarrier surfaces for enhanced cell growth

Biomaterials 24 (2003) 4253–4264

The design of polymer microcarrier surfaces for enhanced cell growth

Dai Katoa, Masahiko Takeuchia, Toshihiko Sakuraia, Shin-ichi Furukawab,Hiroshi Mizokamib, Masayo Sakataa, Chuichi Hirayamaa, Masashi Kunitakea,*

aDepartment of Applied Chemistry and Biochemistry, Faculty of Engineering, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, JapanbThe Chemo-Sero-Therapeutic Research Institute, Kumamoto 860-8568, Japan

Received 23 February 2003; accepted 8 April 2003

Abstract

A variety of neutral and cationic polymers based on polyamino acids were prepared and investigated as microcarriers for cell

attachment and growth. Among neutral polymer particles including the alkylated poly(g-methyl l-glutamate) (PG) particles, inwhich the hydrophobicity changes as a function of the length of the alkyl groups, and hydroxy terminal PG particles, the PG particle

with the longest alkyl chain (PG-C12) demonstrated the highest cell attachment rate and highest rate of cell growth. Moreover, the

introduction of hydroxyl groups (PG-OH) led to a deterioration of cell growth. Cell growth on cationic particles having primary

amino groups was drastically dependent upon the anion exchange capacity (AEC). A higher AEC for aminated PG microcarriers

inhibited cell growth. In contrast, a higher AEC for cross-linked poly(e-lysine) (PL) microcarriers facilitated cell growth. Cell growthon cationic particles clearly showed a good correlation with the pKa;app of the microcarriers, but not with their AEC. The particles

with low and high pKa values possessed toxically acidic and basic pH microenvironments near the surface, respectively. These

microenvironments had cytotoxic effects. On the other hand, no correlation between attachment rate constants and high cell growth

was observed. The aminated particles, in which pKa were controlled at neutral pH, and PG-C12 produced obviously higher cell

growth than did a commercially available microcarrier.

r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Cell proliferation; Fibroblast; Hydrophilicity; Microsphere; Polyamino acid

1. Introduction

Many important pharmaceuticals, such as interferon[1,2] and viral vaccines [3–8], which are required forhuman and veterinary use, are currently produced byanchorage-dependent cells. In cell culture technologyinvolving in vitro cellular engineering, especially thelarge scale cultivation of anchorage-dependent mamma-lian cells, the microcarrier technique has proven to bemore effective than cell culture on flat substrates such asculture dishes [9]. In the microcarrier technique ofcultivation, cell attach and spread onto solid microbeadssuspended in growth medium, and gradually propagate.The early application of microcarriers employed micro-beads composed of N;N-diethylaminoethanol(DEAE)-derivatized Sephadex A50 by van Wezel [10].

The interactions between synthetic surfaces and cellshave been investigated, because cell-compatible materi-als are thought to be very important in many biomedicalapplications, such as the development of prostheticdevices [11–13] and tissue engineering [14–16]. In thedesign of microcarriers for cell attachment and growth,surface properties (e.g., chemical nature, charge density,roughness, wettability, rigidity and so on) [17–41] areimportant considerations. Among these properties, theexchange capacity (amino-group content) has beenconsidered a critical factor for cell attachment andgrowth. Cytodex 1 (Pharmacia), which is a commerciallyavailable microcarrier, has cross-linked dextran matricessubstituted with positively charged DEAE groups.Previous studies have indicated that the optimal chargedensity is necessary for cell attachment and growth. Forexample, Levine et al. have reported that in the vicinityof 2.0meq g�1 dry DEAE-Sephadex particles, the cellconcentration could be increased several-fold withoutthe loss of cell viability or the appearance of anytoxic effects [17]. Kiremitci et al. reported that the

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*Corresponding author. Tel.: +81-96-342-3675; fax: +81-96-342-

3679.

E-mail address: [email protected] (M. Kunitake).

0142-9612/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0142-9612(03)00319-3

introduction of amino groups into the microcarrierssignificantly increased the number of attached cells, butrendered the culture media toxic when used in largeamounts [20]. Hakoda and Shiragami have also reportedthere is an optimum ion exchange capacity (18meqm�2)for cell attachment and growth in petri dish culture [23].Likewise, Ishida et al. have claimed that a moderateamount of net charge on poly(a-amino acid)s wasimportant for growth of L cells on poly(a-amino acid)-coated dishes [19].Several groups have focused on the effect of hydro-

phobicity (wettability) on cell attachment and growth.Reuveny et al. have reported the importance of diaminealkyl chain length (NH2–(CH2)n–NH2; n ¼ 2; 4, 6 and 8)for cell growth of BHK cells on polyacrylamide particlesmodified with alkyldiamine [18]. Among the alkyldia-mines, butyl and hexyldiamine modified polyacrylamideparticles were reported to produce optimal cell growth.In a previous letter, we reported the cultivation of

mouse L-929 cells on aminated poly(g-methyl l-gluta-mate) (PG) and cross-linked poly(e-lysine) (PL) particlescontaining variable amounts of primary amino groups[42]. Comparisons of cell growth on aminated PGparticles and cross-linked PL particles, have revealedthat cell growth is strongly dependent on the apparentpKa (pKa;app) of the particles but not on their amino-group content. In this study, the polymer beads withpKa;app 7.4–7.6 provided the most suitable physiologicalmicroenvironment for increasing attached cell growth.Here we report in detail the cell attachment and

growth of mouse L-929 cells on various polymermicrocarriers, cultured on PG, PL and polyallyamine(PAA) particles. A variety of polymer particles wereprepared and investigated on the basis of polymer typeand the introduction of various modifiers to obtainfurther information about the most suitable surfaceenvironment on microcarriers for cell attachment andgrowth.

2. Materials and method

2.1. Materials

We used 10% (w/w) PG in ethylenedichloride,supplied by Ajinomoto Co., Inc. (Tokyo, Japan) whichhas a degree of polymerization (DP) of ca. 1000. Thenumber-average molecular weight (Mn) of the PG wasabout 1.4� 105. A 25wt% PL (DP, 25;Mn; 4.0� 10

3) inaqueous solution, produced by Streptomyces albulus,was obtained from Chisso Co., Ltd. (Tokyo, Japan). A20% (w/w) PAA (DP, 1750; Mn; 1.0� 10

5) waspurchased from Nitto Boseki Co., Ltd. (Tokyo, Japan).Chloromethyloxirane (CMO), ethylenediamine, n-pro-pylamine and n-heptylamine were purchased fromNacalai Tesque (Kyoto, Japan). Mono-ethanolamine

was purchased from Wako Pure Chemicals Ind., Ltd.(Osaka, Japan). N-laurylamine was purchased fromKishida Chemical Co., Ltd. (Osaka, Japan). All otherchemicals were of analytical reagent grade.

2.2. Preparation of the polymer microcarriers

PG particles were prepared by the suspension-evaporation method [43,44], as described previously.PG particles with a diameter of 100–300 mm were sievedout and used for further modification. Amino, alkyl andhydroxyl terminal groups were introduced into PGparticles by an ester-amido exchange reaction withethylenediamine, alkylmonoamines (n-propylamine, n-heptylamine and n-laurylamine) and ethanolamine toobtain aminated PG, alkylated PG (PG-C3, PG-C7,PG-C12) and hydroxyl terminated PG (PG-OH),respectively. PG particles (30 wet-g) and a large excessof amino compounds (150ml) in methanol (150ml) werereacted with stirring at 25�C or 60�C.Cross-linked PL [42,45] and cross-linked PAA parti-

cles were prepared by suspension copolymerization withCMO in liquid paraffin at 80�C. The cross-linked PLand PAA particles with various amino contents weresynthesized by controlling the composition ratio. Afterthe reaction, the cross-linked particles were washedextensively with n-hexane, ethanol and pure water, andthen filtered. All particles with a diameter of 100–300 mmwere sieved out and used as microcarriers.Reaction conditions for cationic and neutral micro-

carriers are summarized in Tables 1 and 2, respectively.The preparation and characterization of the PG and PLparticles have been previously reported in detail [42,46].A similar series of polyamino acid-based particles havebeen also developed for the selective adsorptionof lipopolysaccharides (LPS) from a protein solution[46–48].

2.3. Characterization of the polymer particles

Particle size and morphology were confirmed byoptical microscopy and scanning electron microscopy(SEM). The ratio of neutral PG-based particles intro-duced by control of reaction times and the ratio of cross-linked PL and PAA particles were calculated from theresults of elemental analysis. The AEC and pKa;app ofparticles with amino groups were quantified by pHtitration [47].

2.4. Cell culture experiments on the polymer particles

Fibroblastic mouse L-929 cells were originally ob-tained from the Chemo-Sero-Therapeutic ResearchInstitute and maintained in 25-cm2 tissue flasks (Corn-ing, USA) at 37�C in a humidified 5% CO2 environ-mental incubator. The cell-growth medium was

ARTICLE IN PRESSD. Kato et al. / Biomaterials 24 (2003) 4253–42644254

Dulbecco’s modified Eagle’s medium (D-MEM; Gibco,USA) supplemented with 10% (v/v) fetal bovine serum(FBS, Gibco), penicillin G (5000 IUml�1) and Strepto-mycin sulfate (5000 mgml�1)(Gibco). The cells weremaintained by routine methods [5].The growth behavior of the cells on the particles was

studied in stationary culture conditions as follows [42]:2.5ml of wet particles was washed extensively usingphosphate buffer saline (PBS, Wako) without Ca2+ andMg2+, and then autoclaved at 121�C for 20min. Afterwithdrawing the supernatant, the particles were washedtwice with D-MEM and then transferred to 45� 10mm2

glass laboratory dishes. Five milliliter of D-MEM wasadded to each dish, and the particles were equilibratedwith D-MEM for 30min in a 5% CO2 environment at37�C. Cells trypsinized from tissue flasks were inocu-

lated at a concentration of 2.0� 104 cellsml�1. Allmicrocarrier cultures were maintained in a 5% CO2environment at 37�C, and cultured for up to 6 days.Cytodex 1 was used as a control material in the cell

culture experiments. It was prepared according to themanufacturer’s introductions. All glassware possiblycontacting the Cytodex 1 was siliconized with sigmacote(Sigma, USA) prior to use to prevent the microcarriersfrom adhering.The cell culture of L-929 cells onto microcarriers was

evaluated as the cell attachment rate constant at lessthan 30min, maximum cell attachment at 60min andcell growth (SS�1

0 ) 2–6 days after the inoculation. Forcell enumeration, all particles were transferred to acentrifuge tube. After the particles had settled bygravity, the supernatant was carefully removed and the

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Table 1

Preparation and characteristics of the cationic microcarriers used

Microcarrier Abbreviation Amination time (h) Molar ratio (mol%) AEC (meq/g) pKa;app

PL or PAA CMO

Aminated PGa PG-0.2 0.3 — — 0.2 7.6

PG-0.5 0.5 — — 0.5 7.7

PG-1.2 0.8 — — 1.2 7.8

PG-3.3 34 — — 3.3 8.2

Cross-linked PLb PL-1.3 — 30 70 1.3 5.0

PL-2.0 — 50 50 2.0 5.5

PL-3.7 — 70 30 3.7 6.7

PL-4.6 — 80 20 4.6 7.0

Cross-linked PAAb PAA-2.3 — 49 51 2.3 8.7

PAA-3.7 — 64 36 3.7 8.9

PAA-5.1 — 70 30 5.1 8.9

PAA-7.9 — 80 20 7.9 8.9

PAA-9.1 — 90 10 9.1 8.9

Cytodex 1 — — — 0.3 7.5

aThe reaction temperature; 60�C.bThe reaction temperature; 80�C.

Table 2

Reaction conditions, characteristics and cell growth for the neutral-based microcarriers

Microcarriers Reaction conditions C/N Introduction ratio (%) S6S�10

Time (h) Temperature (�C)

PG 5.19 0 13.5

PG-OHa 24 25 4.62 26.2 10.5

48 25 4.61 26.5 10.2

PG-C3 24 60 4.83 20.7 13.8

48 60 4.74 25.6 15.2

PG-C7 48 60 5.17 37.8 15.3

PG-C12 48 60 5.40 10.0 15.2

144 60 5.92 34.6 18.3

aOnly these particles were reacted at 25�C, because of avoiding the reaction with hydroxyl group on ethanolamine.

D. Kato et al. / Biomaterials 24 (2003) 4253–4264 4255

microcarriers were washed twice with PBS. The particleswere again allowed to settle by gravity and the super-natant was discarded. 2ml of 0.25% trypsin in PBS wasthen added to the tube containing the microcarriers.After incubation for 5min at 37�C, the suspension wasdivided using a Pasteur pipette to detach the cells fromthe microcarriers. The detached cells were stained with0.2% trypan blue in PBS and counted using ahemocytometer. The cell-growth activity on the particleswas estimated as the ratio of total cell counts attached tothe particles after inoculation for 2–6 days (S) toinoculum (S0).The cell attachment experiment was performed using

Lab-Tek tissue culture slides, each with 8 chambers(Miles Laboratories, USA). To each chamber, 0.2ml ofautoclaved particles was added and then each chamberwas inoculated with 0.5ml of a cell suspension (5.0� 105

cellsml�1). Cell attachment was estimated by measuringthe decrease in the number of unattached cells.The concentration of lactate, a metabolic product,

was monitored in the supernatant of the culture mediumafter cell culture by enzymatical determination using the‘‘Determiner LA’’ kit (Kyowa Medex Co., Ltd., Japan).

3. Results and discussions

3.1. Preparations of polymer microcarriers

Fig. 1 outlines the schemata for the preparation of thethree types of microcarriers. The microcarriers based onpolyamino acids, PG-based and PL-based particles,

were prepared using various quantities of amino groups.The amount of amino groups on the particles wasdefined as the anion exchange (AEC), which wasdetermined by pH titration. The AECs of aminatedPG particles were adjusted by controlling the aminationconditions (reaction time and concentration of ethyle-nediamine). In the case of PL particles, AECs wereadjusted by control of the ratio of PL to CMO, whichwas used as a cross-linker. As non-polyamino acid basedparticles, cross-linked PAA particles with primaryamino groups, were also prepared using a methodsimilar to the synthetic method used for PL particles inorder to provide a means of comparison with PLparticles. All synthesized particles were confirmed to bespherical in shape (100–300 mm) by SEM. In allparticles, the surfaces were relatively smooth and therewere no large pores, in which cells could be buried,although the surface morphologies were different atsubmicron scale.As we mentioned in a previous report, the pKa;app of

microcarriers is crucial with respect to cell growth. ThepKa;app values of cationic microcarriers are stronglyinfluenced by chemical structure, density of aminogroups in the microcarriers, which corresponds to theAEC, and the hydrophobicity of the microenvironment.The preparation conditions and basic properties of thecationic microcarriers are summarized in Table 1. Fig. 2shows the relationship between the AECs and theirpKa;app range for cationic polymer particles. Themicrocarriers gave lower pKa;app values than those ofthe original polymers in solution; the pKa values forPAA and PL in solution were, 9.1 and 7.6, respectively

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Fig. 1. Reaction schemata for the preparation of modified PG, cross-linked PL and PAA particles.

D. Kato et al. / Biomaterials 24 (2003) 4253–42644256

[49,50]. In addition, the pKa1 values for ethylenediamineand n-propylamine were 7.1 and 10.6 as models ofaminated PG, respectively [51].Among the three polymer particle series, PAA

particles showed the highest pKa;app: From highest tolowest pKa values, the order of particles was PAA,aminated PG and PL particles. However, the pKa;app isshifted by the AECs and the pKa;app of all the particlesincreased with increasing AEC. In the case of aminatedPG particles, the pKa;app values are shifted from 7.4 to 8.2,when the AECs were increased from 0.2 to 3.3meqg�1.The pKa;app of PL particles increased from 5.0 to 7.5,when AECs were increased from 1.3 to 4.8meqg�1. ThepKa;app of PAA particles remained almost constant at 8.8as AECs increased from 2.3 to 9.1meqg�1.The higher pKa values associated with increased AEC

may be attributable to increased amino density, whichfacilitates ionization of amino groups by proton sharing.The pKa;app of PL is always lower than that of aminatedPG, irrespective of the AEC, because the carbonylgroups of PL have an electron-accepting effect on itsfree amino groups. In addition, the free amino groups ofPL and aminated PG are situated at the a-and g-positions in these structures, respectively.One of the advantages, that PG-based particles

proffer, is very easy modification of the particle surface.Various other chemical units including amino groupscan easily be introduced onto the surface of PG-basedparticles by an ester-amido exchange reaction [52].Therefore, hydroxyl-terminated particles and a seriesof methyl terminated PGs with different chain lengthswere also prepared and examined for cell attachmentand growth in terms of hydrophobicity [18].

3.2. Cell growth on neutral PG-based microcarriers

Many researchers have mentioned the importance ofsurface wettability in cell culture [28–41]. In order to

examine the influence of the hydrophobicity of surfaceson cell growth, three types of neutral PG-based particles(unmodified PG, alkylated PG and hydroxyl terminatedPG particles) were prepared and compared in terms ofcell attachment and growth. In order to facilitatecomparisons, the ratio of modified ester moiety intro-duced onto the particle surfaces was varied between 10–38mol% by control of the reaction conditions.The type of modifier and the ratio of neutral modifier

on the PG particles had a marked influence on cellgrowth. Logarithmic cell growth was observed on eachtype of neutral particle until 6 days after inoculation.Therefore, comparisons of neutral based-PG microcar-riers were conducted using cell growth at 6 days afterinoculation, S6S

�10 ; as shown in Table 2.

Modification with the introduction of n-alkyl terminalgroups onto PG surfaces increased cell growth. PGmicrocarriers with long alkyl chains, PG-C7 and PG-C12, revealed higher cell growth than the microcarrierswith short chains like PG-C3 and PG. In addition, thehigher introduction ratio seemed to yield greater cellgrowth. For instance, cell growth on PG-C12 particleswith an introduction ratio of 10.0% was about 17%lower than that for the particles with a 34.6%introduction ratio. The greatest cell growth wasobtained with the PG-C12 with the highest introductionratio of 34.6%, and cell growth was 1.8 times greaterthan that on unmodified PG.On the other hand, hydroxyl-terminated PG (PG-OH)

particles showed 23% lower cell growth compared withunmodified PG particles. Even for PG-OH particles, themaximum cell attachment exceeded 95% of S0 estimatedat 60min after inoculation, as was similarly observed forother alkylated PG microcarriers. Reduced cell attach-ment with the introduction of terminal hydroxyl groupshas been also reported on copolymer films consisting ofhydroxyethylmethacrylate and ethylmethacrylate(HEMA-EMA) using mouse Swiss 3T3 cells [29] andpolyethylene terephthalate (PET) films using humanumbilical vein endothelial cells [30]. This is likely due toprevention of adsorption of extracellular matrix, such asfibronectin, vitronectin, collagen and laminin on thesurface. It is well known that the polymer surfaces withterminal hydroxyl groups, such as HEMA, preventprotein adhesion. The flexibility of long alkyl chainscould have a positive influence on cell culture. Theseresults suggest that a hydrophobic surface is favorablefor growth of cultured cells on neutral polymer surfacesbut not charged surfaces.

3.3. Cell growth on cationic polymer microcarriers

Many articles have reported the effects of theintroduction of chargeable groups on cell growth [17–23]. Four types of cationic microcarriers containingamino groups, aminated PG, cross-linked PL, cross-

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Fig. 2. Relationship between AEC and apparent pKa (pKa;app) of the

cationic polymer particles.


linked PAA particle series and Cytodex 1 (Fig. 1) wereexamined by the same methods as used for neutral basedPG particles. In contrast with the neutral particles in thePG series, the introduction of amino groups ontoparticles dramatically affected cell growth.Fig. 3 shows the cell growth curves for the cationic

microcarriers. In the case of aminated PG (Fig. 3a), theparticles with the highest AEC (PG-3.3) showed no cellgrowth, whereas the particles with a relatively low AEC(PG-0.5) revealed high cell growth. Over 2–6 days ofculture on PG-0.5 particles, cell growth increased from1.10 to 19.1 SS�1

0 ; which was approximately 40% higherthan that for unmodified PG. The cell growth on PG-3.3was from 0.80 to 0.30 SS�1

0 over the culture period from2 to 6 days. In contrast, the PL particle seriesdemonstrated the opposite tendency, cell growth wasenhanced with increased AEC, as shown in Fig. 3b. PL-3.7 and PL-4.8 revealed high cell growth (2–6 days) from0.88 to 14.50 and from 1.35 to 13.3 SS�1

0 ; respectively.Moreover, the cell growth on PL-1.3, which has thesmallest AEC, was from 0.35 to 0.15 SS�1

0 for the 2–6day culture period. PAA particles prepared with variousAECs (2.3–9.1) were examined, as shown in Table 1.However, no cell growth was observed on any of thePAA particles. The cell growth on each PAA particletype after 6 days was less than 0.2 (S6S

�10 ). Typical cell

growth behavior on PAA-2.3 is shown in Fig. 3b.It is important to emphasize that attached cell

numbers estimated at 60min. after inoculation for allparticles used reached value exceeded 92% of S0; asshown in Table 3. There was no correlation between thecell growth estimated at more than 2 days and theattached cell numbers estimated at 60min. The attachedcells reached almost 100% of S0 even on PG-3.3 or PL-2.1, which severely inhibited cell growth. These observa-tions suggest that cell attachment was completed onwhole particles and that the cell growth is inhibitedduring a propagation phase on the surfaces.The tremendous differences in cell growth with

various cationic microcarriers were also confirmed byoptical microscope observation. Fig. 4 shows phasecontrast micrographs of attached cells on typical‘‘good’’ (PG-0.5 and PL-4.8) and ‘‘poor’’ (PG-3.3 andPL-2.1) particles with respect to cell growth. The photos(A and B) obtained at 2 h after inoculation for the poormicrocarriers PG-3.3 and PL-2.1 revealed that certaincells were attached onto the surfaces. At this ‘‘cellattachment’’ stage until 2 h, there were no essentialdifferences in images between PG-0.5 and PG-3.3 andbetween PL-4.8 and PL-2.1. However, after 2 days, cellswere rarely observed on the surfaces of PG-3.3 (C) andPL-2.1 (E). After 6 days, no cells could be found on theparticles, as shown in photos (G) and (I). On the otherhand, on the particles PG-0.5 and PL-4.8, whichrevealed good performance, propagated cells coveredthe entire microcarrier surface at 6 days. These

observations indicated that the poor particles acted assterilizers rather than prohibitors of multiplication andthat initial cell attachment was not related to extinctionof cells on poor particles.Output of the metabolic product lactate in the culture

media was also measured in order to evaluate cellgrowth on microcarriers. Fig. 5 shows the changes inconcentrations of lactate produced during cell culturefor the good and poor particles based on aminated PGand cross-linked PL. The lactate concentration overtime, which directly reflected metabolism, obviously

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Fig. 3. The cell growth (SS�10 ) of mouse L-929 cells on aminated PG

(a), cross-linked PL (b), cross-linked PAA (b) and Cytodex 1 (a)

microcarriers in D-MEM supplemented with 10% FBS. S: total cell

count attached to microcarriers after inoculation for 2–6 days; S0:

inoculum (1� 105 cells/dish).


resembled the corresponding cell growth curves. Goodparticles such as PG-0.5, PL-4.8 and Cytodex 1 revealedlogarithmic curves, attributable to active metabolism. In

the culture media of the poor particles, such as PG-3.3and PL-2.0, no additional production of lactate wasobserved at all. These results clearly suggest cell

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Table 3

Typical numerical data for the cell attachment and growth of L-929 cells onto the neutral PG, aminated PG, cross-linked PL and Cytodex 1

microcarriers

Microcarrier Abbreviation Maximum cell attachment (%) Attachment rate constant (min�1) S6S�10

Neutral-based PG PG-OH 93 0.077 10.2

PG-C12 100 0.130 18.3

Aminated PG PG 100 0.096 13.5

PG-0.5 98 0.093 19.1

PG-3.3 97 0.078 0.30

Cross-linked PL PL-2.0 100 0.126 1.20

PL-4.8 95 0.116 13.3

Cytodex 1 92 0.079 16.0

The cell attachment rate constant, the maximum cell attachment and cell growth were evaluated at less than 30min, 60min and 6 days after

inoculation, respectively.

Fig. 4. Phase contrast micrographs of mouse L-929 cells on PG-3.4 (A, C and G), PG-0.5 (D and H), PL-2.1 (B, E and I) and PL-4.8 (F and J)

microcarriers. The micrographs A–B, C–F and G–J were captured at 2 h, 2 days and 6 days after inoculation, respectively. The only cells attached to

the circumference of the microcarriers could be visualized because of the relatively low transparency of the polyamino acid based particles. Original

magnifications of the images were 400� (B, E and J) and 200� (others).


extinction on the poor particles as observed in themicrograph observations.

3.4. Influence of amino moiety on cationic microcarriers

for cell growth

Many researchers have argued that adequate aminogroup content is necessary for culture microcarriers [17–23]. In this section, we will discuss the chemicalsignificance of amino group content on surfaces for cellculture. Fig. 6a shows the effects of the AEC of thevarious particles on cell growth 6 days after inoculation.Both the PG and PL microcarrier series, showedappropriate AECs for maximum cell growth. Amongthe PG series, PG-0.5 showed the highest cell growth(S6S

�10 ¼ 19:1) and had a relatively small amount of

amino groups (0.5meq g�1). However, in the case of thePL particles used in this study, high cell growth wasobserved on the particles with a large amount of aminogroups (over 3.7meq g�1). This difference in the optimalAEC for cell growth between the microcarrier seriessuggests that there are important factors other thanAEC. In a previous letter report, we argued that thepKa;app on the surface of microcarriers would be thedominant influence on cell growth rather than chemicalstructure and amino group content.Good correlation between cell growth and the pKa;app

of the various particles was confirmed for both the PGand PL series as shown in Fig. 6b. The PL-4.6 (pKa;app7.0) showed high cell growth but the PL-1.3 (pKa;app 5.0)showed extremely low growth. The optimum pKa;app forcell growth in both particle series was near neutral pH.This finding was supported by observations of cellgrowth on other particles. A commercially availableCytodex 1 with a DEAE moiety (pKa;app 7.5), showedrelatively high cell growth. On the other hand, the PAAseries with pKa;app ranging from 8.7 to 8.9, yielded no

cell growth. These observations prove that cell growth isstrongly dependent on the pKa;app but not necessarily onamino group content or the amino concentration on themicrocarrier surface, which are in rough agreement withthe AEC.Fig. 6b clearly demonstrates that cell growth is

facilitated when the pKa;app value of the particlesapproaches neutral. The surface of particles with aneutral pKa was the most suitable environment for cellculture. On the other hand, the particles with high orlow pKa values, evidenced cytotoxic properties. Theparticles with low and high pKa values would possesstoxically acidic or basic microenvironments, respec-tively. The particle surfaces with a neutral pH micro-environment did not prevent cell growth and subsequentthe introduction of amino groups facilitated cell growth.The pH microenvironment of surfaces is predominantlyregulated by pKa;app due to buffer effects as shown in

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Fig. 5. Changes in the concentration of the metabolic product lactate

in the culture medium with PG-0.5 (a), PG-3.4 (b), PL-2.0 (c), PL-4.8

(d) and Cytodex 1 (e) during culture of mouse L-929 cells.

Fig. 6. Correlations between cell growth (S6S�10 ) and the AEC (a) and

pKa;app (b) on various microcarriers (PG, PL, PAA and Cytodex1). S6:

total cell count attached to microcarriers after inoculation for 6 days;

S0: inoculum.


Fig. 7 [53]. It would be worth to note that PL is wellknown to be an antibacterial material. It is interesting tonote that the particles based on an antibacterial materiallike PL, can still yield good cell growth by adequatecontrol of pKa:

3.5. Initial cell attachment rate to the polymer

microcarriers

Overall, cultivation of anchorage-dependent cells isbroadly dependent on two factors, cell attachment andcell growth on the surface. Many researchers haveexamined the effects of surface modifications for cellattachment [18–41]. In general, cell growth is closelyrelated to cell attachment. That is, fibroblasts growthundergoes higher on substrates, which are more favor-able for cell attachment [32]. Among all of the particleswe used, the maximum cell attachment estimated at60min after inoculation was over 92% of S0; suggestingthat cell attachment was almost complete, as shown inTable 3. It is noteworthy that even the lethal particlessuch as PG-3.4 (pKa;app ¼ 8:3; S6S

�10 ¼ 0:3) and PL-2.0

(pKa;app ¼ 5:5; S6S�10 ¼ 1:2), on which cells were almost

all extinct after 6 days, performed as well for cellattachment as the particles which facilitated high cellgrowth. These results suggest that the microcarriermaterial affected cell growth but did not affect theprocess of cell attachment. However, it is still worth-while to consider the cell attachment rate constant inevaluation of the performance of various polymerparticles in cell cultures [54–56]. Fig. 8 shows the time

dependence of cell attachment within 60min afterinoculation on various microcarriers. A semilogarithmicplot of unattached cell concentration with respect totime yielded a straight line, indicating first-orderattachment kinetics. The attachment rate constants arelisted in Table 3.The order of the higher attachment rate constants was

PG-C12, PL-2.0, PL-4.8bPG, PG-0.5>Cytodex1, PG-3.3, PG-OH. The most hydrophobic particle with thelongest alkyl chain, PG-C12, demonstrated the highestattachment rate constant (0.13min�1) exceeding that ofother neutral particles, such as PG and PG-OH.However, hydrophilic particles with cationic charge,such as PL-2.0 and PL-4.8, also gave similarly highattachment rate constants. Among the aminated parti-cles, the PL series showed higher attachment rateconstants than the aminated PG series. These resultssuggest that a hydrophobic surface is advantageous interms of cell attachment, on neutral polymer surfacesbut not on charged surfaces. Several groups havereported the advantages of hydrophilic surfaces for cellattachment; these studies have focused on cell growth oncharged surfaces. Regarding cell attachment, adhesionof the extracellular matrix would be one crucialparameter, as mentioned before. Both surface chargeand wettability, reflecting a balance between hydro-phobicity and hydrophilicity, would affect adhesion ofthe extracellular matrix [28–30,32,33,37,41].Interestingly, no correlation between the initial

attachment rate constants and high cell growth wasobserved. The particles that facilitated cell growth, PL-

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Fig. 7. Schematic illustration of the surface microenvironments on the aminated microcarriers for cell attachment and growth in terms of pKa;app:


4.8 and PG-0.5, gave high and low attachment rateconstants, as did the lethal particles, PG-3.3 and PL-2.0.

3.6. Adhesion of microcarriers onto glassware

An interesting characteristic of the polymer micro-carriers developed in this work, was weaker adhesion of

the particles onto glassware. Fig. 9 shows a comparisonof the adhesion behaviors of microcarriers onto glass-ware. After the dish was filled with the microcarrierssolution, the dish was tilted to visualize adhesion ofmicrocarriers onto the bottom of glass surfaces.Aminated PG and cross-linked PL did not adhere (Figs.9a and b). However, the images of the exposed upperhalf of the dishes showed that Cytodex 1 stronglyadhered to the bare glassware (Fig. 9c). Therefore, theglassware surface must be treated by siliconization in thecase of Cytodex 1, as mentioned in the supplier’scatalogue. This would be disadvantageous for suspen-sion culture, such as cell culture in a spinner flask. Onthe other hand, most of the microcarriers we preparedshowed no adhesion to glassware, as shown in Figs. 9aand b. It is a great advantage that they can be usedwithout siliconization. The adhesion of microcarriersonto glassware would be predominantly due to electro-static interactions. Certainly, only PAA-9.1 with thehighest amino group content, showed weak adhesiononto glassware.

4. Conclusions

In this study, a variety of neutral polymer particlesand cationic polymer particles having primary aminogroups were prepared and investigated to determine thebasic principles of polymer microcarrier surface designin terms of cell attachment and growth. Neutral particlesand cationic particles revealed obviously different effectson cell growth. The hydrophobicity or flexibility of sidechains seemed to be advantageous for cell culture, onneutral surfaces. Cell growth on cationic particleshaving primary amino groups was drastically dependentupon the AEC. The surface of particles with a neutralpKa;app was the most suitable environment for cellgrowth, because of the neutral pH microenvironment. Inother words, the particles with low and high pKa valuespossessed acidic and basic pH surface microenviron-ments, which had cytotoxic effects, respectively. On theother hand, no correlation between attachment rateconstants and high cell growth was observed. All of

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Fig. 8. Initial attachment kinetics of L-929 cells onto the neutral PG-

based microcarriers (a) and cationic PG or PL based microcarriers (b).

Fig. 9. Typical photographs of glass dishes containing polymer microcarriers PG (a), PL (b) and Cytodex 1 (c) to demonstrate the adhesion of

microcarriers onto glassware.


these results are significant in furthering the under-standing and novel design of polymer microcarriers forthe culture of anchorage-dependent cells.

Acknowledgements

We are grateful to Chisso Co., Ltd. for the supply ofthe PL. We are also grateful to Ajinomoto Co., Ltd. forproviding the PG. This work was supported in part bythe CREST-JST, Japan.

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