biocompatible smart microcapsules based on chitosan-poly(vinyl alcohol) copolymers for cultivation...

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DOI: 10.1002/adem.201180014
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Biocompatible Smart Microcapsules Based onChitosan-Poly(vinyl alcohol) Copolymers for Cultivation ofAnimal Cells**

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By Daria Zaytseva-Zotova, Vera Balysheva, Anna Tsoy, Maria Drozdova,
Tatiana Akopova, Leonid Vladimirov, Isabelle Chevalot, Annie Marc,Jean-Louis Goergen and Elena Markvicheva*

In this study, two novel chitosan-graft-poly(vinyl alcohol) copolymers are synthesized and used aswater-soluble at physiological conditions polycations for preparation of smart microcapsules. Themicrocapsules provide growth and proliferation of eight mammalian cell lines, including hybridomaand tumor cells, at long-term cell cultivation in vitro. The microcapsules are stable in cell culturemedium but can be dissolved by changing pH value of the medium (up to 8.0–8.2), thus making possiblea simple release of the entrapped cells. Monoclonal antibody production by encapsulated hybridomacells is demonstrated. Cultivation of tumor cells within the microcapsules allows the formation of 3Dmulticellular spheroids, which can be proposed as an in vitro model for anticancer drug screening.

Presently cell microencapsulation is widely employed in

biomedicine and biotechnology.[1] Biocompatible polymer

microcapsules for animal cell entrapment were proposed by F.

Lim and A. M. Sun in the beginning of the 1980 s.[2] To form

semipermeable polyelectrolyte membranes of the microcap-

sules, they chose natural polysaccharide alginate (polyanion)

and positively charged synthetic polypeptides (polycations),

for instance poly(L-lysine) (PLL), poly(L-arginine), poly(-

L-ornithin), etc.

[*] Prof. E. Markvicheva, D. Zaytseva-Zotova, A. TsoyM. DrozdovaPolymers for Biology Laboratory, Shemyakin & OvchinnikovInstitute of Bioorganic Chemistry of Russian Academy of Sciences,Miklukho-Maklaya str., 16/10, Moscow, 117997, (Russia)E-mail: [email protected]

Prof. V. BalyshevaAll-Russian Research Institute for Veterinary Virology andMicrobiology, Russian Academy of Agricultural Sciences,Pokrov, Vladimir region 601120, (Russia)

Dr. T. Akopova, Prof. L. VladimirovN.S. Enikolopov Institute of Synthetic Polymer Materials,Russian Academy of Sciences, Profsouznaya str., 70, Moscow117393, (Russia)

Prof. I. Chevalot, Prof. A. Marc, Prof. J.-L. GoergenLaboratoire Reactions et Genie des Procedes, UPR CNRS3349, ENSAIA-INPL, 2, Avenue de la Foret de Haye, BP 172,Vandoeuvre-les-Nancy, 54505, (France)

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It is well known today that the polyelectrolyte micro-

capsules provide a free exchange of nutrients, gases (O2 and

CO2), and a release of metabolites and cell products through

semipermeable polymer membrane of the microcapsule.[3]

Being implanted into the body, microcapsules can play a dual

role: they can work as microreactors providing release of

therapeutics (recombinant proteins and peptides) secreted by

cells through the membranes, and at the same time they can

protect the encapsulated cells against patient immune cells.[4]

[**] This work was partially supported by the bilateral Russian-French program (the joint CNRS-RFBR PICS project N.10-04-91056) and also by RFBR project N. 10-03-01022.The authors are grateful to Dr. J. McGuire (Roswell ParkCancer Institute, Buffalo, USA) for the kind gift of humanleukemia cell lines and to Dr. T. Erokhina (Shemyakin &Ovchinnikov Institute of Bioorganic Chemistry of RAS, Russia)for mouse myeloma Sp2/0 cells. The authors are also thankful toProf. E. Vodovozova (Shemyakin & Ovchinnikov Institute ofBioorganic Chemistry of RAS, Russia) and to Prof. A. Bartko-wiak (Westpomeranian University of Technology, Poland) fortheir kind support of this research.

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D. Zaytseva-Zotova et al./Biocompatible Smart Microcapsules Based on Chitosan-Poly(vinyl alcohol) . . .

The main advantages of microencapsulation as a three-

dimensional (3D) growth technique are chemically and

spatially defined 3D network of extracellular matrix compo-

nents, cell-to-cell and cell-to-matrix interactions which govern

differentiation, proliferation, and cell function in vivo. These,

in fact, are lost under the simplified two-dimensional (2D)

conditions.[5]

All the mentioned issues allow to consider microencapsu-

lation as a promising technique for a number of biomedical

applications, including diabetes treatment,[6] somatic gene

therapy,[7] tissue engineering,[8] and anticancer drug screen-

ing using microencapsulated multicellular tumor spheroids

(MTS).[9]

Cell microencapsulation was widely used to support

growth of cells, such as insulin-producing pancreatic

beta-cells (Langerhans islets),[10] parathyroid cells,[11] hepa-

tocytes,[12] stem cells,[13] and hybridoma cells.[14]

Howbeit, cell microencapsulation technique requires an

improvement of procedures, materials, and devices for

microcapsule preparation.[15] Chitosan-based polymers seem

to be a good alternative to replace commonly used PLL or

other rather expensive polypeptides, since natural poly-

saccharides (e.g., chitosans) are cheaper. Moreover, a direct

comparison showed that the toxic effect of alginate-PLL

membrane was significantly higher than those for chito-

sans.[16] However, as well known, high molecular weight

chitosans are soluble only at acidic pH value while cell

encapsulation procedure should be carried out under

physiological conditions (pH 6.8–7.2). The grafting of

chitosans with biocompatible water-soluble synthetic poly-

mers, e.g., poly(vinyl alcohol) (PVA), could be a promising

approach. Unlike chitosans, such copolymers can be soluble at

neutral pH and seem to be promising for animal cell

encapsulation.

Thus, the aim of the current research was to fabricate

biocompatible smart microcapsules based on a novel

chitosan-g-PVA copolymers and to demonstrate capability

of these microcapsules to support growth of various

mammalian cells at long-term cultivation in vitro.

Results and Discussion

Chitosan-Based Polycations

Chitosans belong to a family of natural biocompatible

cationic pH-sensitive polysaccharides produced by partial

deacetylation of chitin from reprocessing of seafood waste.

However, high molecular weight chitosans are soluble at

acidic pH values, and therefore, cannot be used for animal cell

encapsulation while oligochitosans (Mw¼ 3.0–10.0 kDa) are of

great interest, since they are soluble at physiological pH.[17]

Moreover, oligochitosans have been recently reported for

preparation of microcapsules to entrap cells.[9]

On the other hand, PVA is a biocompatible water-

soluble synthetic polymer widely employed for biomedical

applications, e.g., for controlled drug delivery and tissue

engineering.[18]

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To combine the desirable properties of chitosan and

PVA, in particular, chitosan pH-sensitivity and PVA

water solubility at physiological conditions (pH 7.2), we

synthesized PVA-g-chitosan copolymers. These copolymers

were prepared by a novel solid-state reactive blending (SSRB)

method.

The uniqueness of this method relies upon a variety of

chemical (e.g., grafting, polymer-analogous transformation,

polymerization, and polycondensation reactions) and physi-

cal transformations of solid polymers and monomers which

occurred at joint action of high pressure and shear deforma-

tion. The method is based on the reactive blending of all

compounds at plastic flow conditions by applying an external

mechanical energy to polymeric solid mixtures. Currently,

organic reactions that occur under these conditions, so called

‘‘solvent-free reactions,’’ are of great interest. These reactions

have numerous advantages over common reactions in

solution, since they are infinitely high concentration reactions.

Furthermore, in many cases they proceed faster, more

selectively, and much more efficiently compared to solution

reactions.[19] Treatment of polymers under these conditions

results in substantial changes of their structure, viz. crystal-

linity, degree of particle dispersability, reactivity of the

functional groups in heterogeneous reactions, etc.[20]

Thus, this novel technique seems to be promising to modify

infusible and poorly soluble polymers substances like

polysaccharides (e.g., chitin).[21] For example, chitosan (with

�100% yield) can be produced by alkaline solid-state

deacetylation of chitin in the extruder.[22]

In our work, two chitosan-g-PVA copolymers were

prepared by SSRB method using a coextrusion of chitin with

PVAc in the presence of sodium hydroxide. FTIR spectro-

scopy analysis showed that deacetylation degree of both

copolymers was almost complete (up to 95–98%). It was also

revealed that the polymer-analogous transformations under

these conditions were accompanied by a formation of an

extensively grafted copolymeric system. Water-soluble at

room temperature fractions of the obtained products (� 20%

of entire systems) were characterized. Both FTIR and

elemental analyses were used to evaluate the chitosan content

in the obtained fractions. FTIR spectra of the samples after

copolymer purification and precipitation from aqueous

solutions contained characteristic absorption PVA and

chitosan bands (Fig. 1 and 2). Figure 1 shows FTIR spectra

for chitosan-g-PVA1 copolymer (curve 1) and chitosan (curve

2). As can be seen in Figure 1 copolymer spectrum contains the

band of OH stretching (3 400 cm�1) along with the presence of

CH2� bending at 850 cm�1. Figure 2 demonstrates FTIR

spectrum for chitosan-g-PVA20 sample (curve 2) compared to

a model chitosan/PVA (20:80 w/w) mixture (curve 1). The

calculations showed that the chitosan contents in the obtained

copolymers were 80 and 15 wt% for chitosan-g-PVA1 and

chitosan-g-PVA20, respectively. Glucosamine contents for

chitosan and both copolymers were also calculated from

the results of elemental analysis using C/N ratio (5.29 in

the case of initial chitosan). Elemental analysis data for

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Fig. 1. FTIR spectra of chitosan-g-PVA1 copolymer (curve 1) and chitosan (curve 2).Chitosan-g-PVA1 copolymer contains 80 wt% of chitosan.

Fig. 2. FTIR spectra of the chitosan/PVA (20:80 w/w) model mixture (curve 1) and thechitosan-g-PVA20 (15 wt% of chitosan) copolymer (curve 2).

chitosan-g-PVA1 sample are (found/calculated, %): C, 46.83/

46.69; N, 6.83/6.94; O, 39.02/39.07; H, 7.32/7.28; for

chitosan-g-PVA20 sample: C, 52.96/53.05; N, 1.40/1.32; O,

36.91/36.87; H, 8.73/8.74. Calculations from the results of

elemental analysis using C/N ratio for both pure chitosan and

chitosan-g-PVA copolymers were in a good agreement with

FTIR data. It was also revealed that the chitosan content in the

prepared copolymers was a function of PVAc/chitin molar

ratio in the initial mixtures. The increase of PVAc content in

the initial reactive mixture resulted in the decrease of chitosan

content in the obtained copolymer. Thus, in the case of

chitosan-g-PVA1 sample PVA/chitosan molar ratio was 0.92

when the initial PVAc/chitin ratio was 1.4. It was increased up

to 20.5 (in the chitosan-g-PVA20 sample) when the initial

PVAc/chitin ratio was enhanced up to 3. It could be suggested

that PVA in chitosan-g-PVA1 copolymer was statistically

distributed in the form of relatively short chains grafted onto

chitosan. This result is in a good agreement with a theory of

mechano-induced destruction of polymer chains. We suppose

that PVAc as a component with a flexible chain can act as a

surfactant, thus providing an increase in plasticity of the

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systems under coextrusion. The values of Mw of the obtained

copolymers determined by GPC method were 124 and

190 kDa for chitosan-g-PVA1 and chitosan-g-PVA20 samples,

respectively. Both obtained chitosan-g-PVA copolymers were

water-soluble at physiological conditions (pH 7.2, room

temperature).

Microcapsules

To form stable microcapsules of the desired size and

polyelectrolyte membrane thickness, alginate (polyanion)

and two chitosan-g-PVA copolymers or oligochitosan (poly-

cations) were used. The microcapsules were prepared in three

steps: (1) a dropwise addition of a NaAlg solution with

suspended cells to a CaCl2 solution to get CaAlg microbeads;

(2) an incubation of the obtained CaAlg microbeads in a

polycation solution to form a polyelectrolyte membrane on the

CaAlg microbead surface; (3) a treatment of the obtained

microbeads with a chelating agent solution (EDTA), in order

to dissolve the alginate core and to get hollow microcapsules.

The microcapsule shape, diameter, and size distribution

depended on initial characteristics of CaAlg microbeads.

Usually, various dispersion techniques for fabrication of

microbeads with the diameters ranged from 150 to 900 mm are

employed. In this study a special device with a coaxial air flow

was used. It allowed to break the NaAlg solution flow thus

decreasing the NaAlg drop size just on a needle tip before the

drops fell into the CaCl2 solution. Optimal conditions for the

preparation of CaAlg microbeads by this technique were

described earlier.[9] Here, the mean diameter of the obtained

microbeads was 750� 50 mm.

On the next step, microcapsules were formed by incubation

of the CaAlg microbeads in the polycation solution. The

thickness of the microcapsule membrane depended upon the

polycation type, concentration of the polycation solution, and

the incubation time (Table 1). If the incubation time was not

long enough for membrane formation, the microcapsules were

completely dissolved at the EDTA solution treatment. On the

other hand, when the incubation time was too long, the CaAlg

microbeads were fully cross-linked by the polycation, and the

microcapsule membrane was not observed under a light

microscope. As a result of optimization, mechanically stable

microcapsules with an optimal membrane thickness were

obtained after the CaAlg beads incubation in oligochitosan,

chitosan-g-PVA1, and chitosan-g-PVA20 solutions for 5, 5, and

10 min, respectively.

The obtained microcapsules had a larger diameter

than the appropriate CaAlg microbeads, and their swelling

behavior in the physiological solution depended on the

type of the polycation we used (Table 1). This can be explained

by the difference in Mw and the polycation type. More was the

content of hydrophilic PVA in the chitosan-g-PVA copolymer,

higher swelling of the microcapsules was observed.

Nevertheless, all the microcapsules withstand centrifuga-

tion (9 000� g) for 10 min. The obtained microcapsules had a

nice spherical shape and were stable in both physiological

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Table 1. Membrane thickness of microcapsules in function of polycation type and incubation time.

Polycation sample Microcapsule meandiameter [mm]

Polycation solutionconcentration [%]

Membrane thickness as functionof incubation time [mm]

5 [min] 7 [min] 10 [min]

Oligochitosan 900� 75 0.10 78� 5 112� 6 n.d.[a]

0.20 89� 5 135� 6 n.d.

Chitosan-g-PVA1[b] 780� 50 0.20 27� 5 – 54� 5

0.25 51� 5 76� 5 n.d.

0.50 81� 5 – n.d.

Chitosan-g-PVA20[c] 1150� 80 0.75 – 37� 5 66� 5

1.15 – 42� 5 87� 5

[a] Membrane was not detected;

[b] Chitosan/PVA ratio was 80:20w/w,Mw¼ 124 kDa;

[c] Chitosan/PVA ratio was 15:85 w/w,Mw¼ 190 kDa.

Table 2. The list of cell lines grown in alginate-polycation microcapsules.

Cell line Oligochitosan Chitosan-g-PVA1 Chitosan-g-PVA20 Cell line characteristics:origin/growth properties

Time needed to fillmicrocapsule volume [days]

CCRF-CEM n.d.[a] þ þ Human/suspension 18

CEM/C1 n.d. þ þ Human/suspension 22

1D2 þ n.d. n.d. Mouse/suspension 11

Sp2/0 (Ag14) þ[b] þ n.d. Mouse/suspension 14

P388D1 þ n.d. þ Mouse/suspension 28

MCF-7 þ n.d. n.d. Human/adherent 28

M-3 �[c] – – Mouse/adherent –

BHK-21 þ n.d. n.d. Hamster/adherent 11

PSGK-60 þ n.d. n.d. Siberian mountain goat/adherent 14

[a] n.d., not determined;

[b] ‘‘þ ’’ Cell aggregates formed within the microcapsules;

[c] ‘‘� ’’ Cell aggregates did not form within microcapsules.

solution and in the cultivation medium for 4 weeks. Therefore,

we suggested that the obtained microcapsules with the

optimal membrane thickness of 80–100 mm could be used for

long-term cell cultivation.

Since the chitosan-g-PVA copolymers were pH-sensitive

due to chitosan, the microcapsules were shown to be easily

dissolved by slightly increasing pH value of the incubation

medium. For instance, the replacement of the physiological

solution for a culture medium with increased pH (8.0–8.2)

resulted in membrane dissolving.

Thus, the use of novel chitosan-g-PVA polycations makes

possible simple cell release from the microcapsules after

long-term cell cultivation by slight increasing pH of cultiva-

tion medium up to 8.0–8.2, if further manipulations with the

obtained multicellular spheroids are needed.

Growth of Cells in Microcapsules

In order to test three types of microcapsules for their

capability to support cell growth, various cells differed in their

origin (human, mouse, etc.), and growth properties (suspen-

sion culture or monolayer) were chosen for microencapsula-

tion. The optimal initial cell concentration used for micro-


encapsulation was found to be (1.0� 0.2)� 106 cells �mL�1of

the NaAlg solution. This concentration allowed to form

microcapsules with rather homogeneous cell distribution

inside them (70–150 cells per microcapsule).

Table 2 demonstrates the ability of various cell lines to grow

within the microcapsules. This ability depended upon cell

line properties but did not depend strongly on the polycation

type we used. Thus, we got multicellular spheroids within the

microcapsules for almost all selected cell lines, except M3

mouse melanoma cells. Melanoma cells grew on the inner

microcapsule membrane but did not fill the microcapsule

volume, since their growth most probably was suppressed,

although there was a lot of space inside the microcapsule.

Thus, eight different cell lines were successfully cultivated

within the microcapsules for 2–4 weeks. Typical growth

dynamics for encapsulated cells is presented in Figure 3.

Initially myeloma Sp2/0 cells distributed evenly as single cells

within the microcapsules. After being cultured for 5 d, the

cells formed small aggregates. These aggregates increased in

their sizes and filled the whole microcapsule volume in 14 d

after microencapsulation. As can be seen, the cell growth

did not depend on polycation type chosen for membrane

formation.


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Fig. 3. Myeloma Sp2/0 cell growth in alginate/oligochitosan (A–D) and alginate/chitosan-g-PVA1 (E–H) microcapsules at day 1 (A and E), day 5 (B and F), day 10 (C and G), andday 14 (D and H). The microcapsule membrane is indicated with arrows. Scale bar is 300 mm.

Fig. 4. Growth kinetics of 1D2 hybridoma cells and BHK-21 cells in the microcapsules.

Fig. 5. Titers of MAbs produced by encapsulated 1D2 hybridoma cells in the cultivationmedium. Arrows indicate the time when the culture medium was completely replaced.

Figure 4 shows the growth kinetics of microencapsulated

BHK-21 cells and 1D2 hybridoma cells for 21 d of cultivation.

Cell concentration increased 10-fold within first 5 d,

and it reached the values of 7� 106 cells �mL�1 and

4.5� 106 cells �mL�1 for BHK-21 and 1D2 cells, respectively,

by day 11th. On day 11 alive cell concentration decreased while

dead cell concentration raised. This could be explained by high

cell density within the microcapsules resulting in the decrease

of nutrients diffusion and limitation of oxygen supply.

Thus, the developed microcapsules were capable to

provide growth of eight cell lines at long-term cultivation

up to 1 month.

Production of Monoclonal Antibodies (MAbs) by EncapsulatedCells

As well known, cell microencapsulation provides a number

of technical advantages for a large-scale MAbs production as

well as it allows a sustained and controlled delivery of the de

novo produced therapeutic product. Moreover, cell entrap-

ment in microcapsules with semipermeable membranes could


be used to reduce or in some cases even to avoid chronic

administration of immunosuppressive drugs.[14]

In the current study 1D2 hybridoma cells producing MAbs

against IgG heavy chain were chosen as a model cell line. The

samples of the culture medium were collected daily and

concentration of MAbs released from microcapsules was

determined by enzyme-linked immunosorbent assay (ELISA;

Fig. 5). Suspension cell culture was used as a control. Cells

entrapped in the microcapsules presented rather higher MAbs

production after first 9 d. The total volume of the medium

containing MAbs was 800 mL within 21 d. These results

confirmed that the developed microcapsules could be used for

MAbs production by entrapped cells.

Generation of Multicellular Tumor Spheroids in Microcapsules

Cell microencapsulation is a promising approach to

develop a novel in vitro 3D model, which could rather well

mimic small size solid tumors. R. M. Sutherland was the first

to propose MTS as a more adequate model of small solid


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Fig. 6. MCF-7 cell viability in encapsulated MTS and in a monolayer culture as afunction of MTX concentration.

tumors compared to 2D suspension or monolayer cultures.[23]

MTS have been demonstrated to represent quite properly

the 3D growth and organization of solid tumors, and

consequently to better mimic cell–cell interactions and

microenvironment in a tumor tissue.[24] Due to these

similarities to a tumor xenograft, MTS have become a

valuable tool in tumor biology. Nowadays, MTS are widely

used as an in vitro model for various biomedical studies

related to cancer therapy, e.g., radiotherapy or photodynamic

therapy.[25] A classical technique for MTS formation

includes cultivation of anchorage-dependent cells at stirring

which prevents cell attachment to a surface and results in

cell growing as aggregates finally forming multicellular

spheroids.

Although many techniques are presently available to get

3D tissue models in vitro,[26] all of them have inherent

limitations. Thus, commonly used in vitro classical methods,

such as liquid-overlay, spinner flask and gyratory rotation

systems are time consuming and cannot provide production

of MTS with narrow spheroid size distribution within a

desired range. These disadvantages can be easily overcome

using microencapsulation for MTS generation. The approach

based on tumor cell encapsulation as a novel technique to

quickly and easily prepare a large number of microencapsu-

lated MTS with narrow size distribution within a desired

diameter has been reported by us quite recently.[9]

In the present study microencapsulated MTS were

obtained for four various tumor cell lines (e.g., human cell

lines CCRF-CEM, CEM/C1, MCF-7, and mouse Sp2/0 cells).

As can be seen in Figure 3, Sp2/0 cells formed a compact

multicellular clusters within the microcapsules at long-term

cultivation already on day 14.

Adenocarcinoma MCF-7 cells was chosen as a model cell

line to demonstrate that the encapsulated MTS could be

considered as a more rapid and valid in vitro model compared

to a monolayer culture for evaluation of anticancer drugs.

The MTX cytotoxicity was estimated as an inhibition rate

in cell viability. To evaluate non-specific sorption of MTX by

the microcapsules, cell viability in the monolayer culture

sample containing empty microcapsules was tested. It was

found that non-specific MTX sorption by the microcapsules

was insignificant (3–5%). The cell viability decreased with

increasing drug concentration in both encapsulated MTS

and the monolayer culture (Fig. 6). However, the inhibition

rate in encapsulated MTS was much lower than that for the

monolayer culture. Thus, the IC50 (a half maximal inhibitory

concentration) was 55 and 2.7� 10�9M for MTS and

monolayer, respectively. These results demonstrated that

microencapsulated MTS were more resistant to anticancer

drug treatment than the monolayer culture.

Conclusions

Chitosan-g-PVA copolymers were obtained by a novel

SSRB method. Biocompatible smart microcapsules based on

the polyelectrolyte complex between alginate and chitosan-


based polycations (oligochitosan or chitosan-g-PVA copoly-

mers) were elaborated. The microcapsules fabricated from

alginate and chitosan-g-PVA copolymers were stable during

long-term cell cultivation. Multicellular spheroids generated

within these microcapsules could be easily released by slightly

rising pH value of the culture medium up to pH 8.0–8.2, since

the microcapsules were pH-sensitive. The novel polyelec-

trolyte microcapsules were demonstrated to support growth

and proliferation of eight animal cell lines differed in their

growth properties and origin. Our results also indicated that

the microcapsules could be successfully used for production

of MAbs by 1D2 hybridoma cells at their long-term cultivation

for 21 d. Moreover, encapsulated tumor cells were able to

generate multicellular spheroids, which could be used as an in

vitro model to test chemotherapeutic drugs, namely MTX.

Thus, the developed biocompatible smart microcapsules

embedded with different animal cells could be proposed

for several biomedical applications.

Experimental

Chemicals

Alginic acid sodium salt (NaAlg; medium viscosity, approx.

3 500 cps at 25 8C), EDTA sodium salt, bovine serum albumin (BSA),

mouse IgG antibodies, horseradish peroxidase-linked goat anti-rat

IgG conjugate, and methotrexate (MTX) were from Sigma–Aldrich

(Germany). Calcium chloride (CaCl2� 2H2O) was purchased from

Panreac (Spain). PBS (pH 7.2), Tween-20, sodium chloride (NaCl),

and Trypan Blue were from PanEco (Russia), Serva (Germany),

REACHEM (Russia), and FlowLaboratories (Germany), respec-

tively.

Oligochitosan (Mw¼ 3.5 kDa, deacetylation degree 89%) was

kindly provided by Prof. A. Bartkowiak (Poland). For synthesis of

chitosan-g-PVA copolymers, a-chitin (Mw¼ 650 kDa) isolated from

crab shells was purchased from COMBIO (Russia), and poly(vinyl

acetate) (PVAc), Mw¼ 350 kDa, was from PLASTPOLYMER (Russia).

Sodium hydroxide (98.5%, microprills with typical grain size

distribution of 0.5–1.2 mm> 93%) was from Merck (Germany). PVA

(Mowiol 66–100; Mw¼ 100 kDa; vinyl acetate units content 1 wt%) was

from Sigma–Aldrich (Germany).


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Cell Cultures

A number of mammalian cell lines were used in this study, namely

Siberian mountain goat kidney cell line (PSGK-60); baby hamster

kidney cells (BHK-21); rat-mouse hybridoma cells (1D2) producing

MAbs against IgG heavy chain; a mouse macrophage-like cell line

(P388D1); mouse melanoma cells (M3); human breast adenocarcinoma

cell line (MCF-7). We also used myeloma cells (Sp2/0), and two

human T-lymphoblastic leukemia cell lines (CCRF-CEM and

camptothecin resistant CEM/C1 subline).

PSGK-60 cells were cultured in MEM (Sigma–Aldrich, Germany)

while other cell lines were cultivated in DMEM (Sigma–Aldrich,

Germany) in CO2- incubator (HERAEUS B5060 EK/CO2) at 37 8C.

All culture media were supplemented with 1% gentamicin from

Akrihin (Russia) and 10% fetal bovine serum (FBS) from HyClone

(USA).

Synthesis and Characterization of Chitosan-g-PVA Copolymers

Two graft copolymers of chitosan and PVA, i.e., chitosan-g-PVA1

and chitosan-g-PVA20, having PVA/chitosan molar ratio 0.92 and

20.5, respectively, were obtained by solid-state synthesis as described

earlier [27]. Briefly, PVAc and chitin solid mixture (PVAc/chitin molar

ratio of 1.4 or 3) was processed with a semiindustrial corotating

twin-screw extruder (ZE 40A� 40D UTS, Berstorff, Germany) in the

presence of sodium hydroxide at 60 8C. The extruder has a variable set

of processing elements providing a high shear strain and a powerful

dispersive action. To remove low molecular weight compounds, the

prepared blends were exposed to a water/organic mixture (ethanol/

ethyl acetate/water) for extraction for 48 h followed by electrodialysis

in an aqueous solution for 1–2 d. After filtration, the sample solutions

were frozen and lyophilized at�10 toþ30 8C up to moisture content of

2–3%. Formation of chitosan-g-PVA copolymers was confirmed by

Fourier transform infrared spectroscopy (FTIR), gel penetration

chromatography (GPC), and elemental analysis.

To analyze the samples by FTIR, thin films (5–10 mm thickness)

were prepared by casting 2 wt% copolymer aqueous solutions onto

glass substrates. The cast solutions were dried at 50 8C for 2 h. To

prepare model blend films, PVA aqueous solution (10 wt%) and

previously obtained chitosan (Mw¼ 60 kDa; N-acetylated units

content 8 wt%) solution in 0.33 M acetic acid solution (10%) were

mixed. The formed films containing chitosan acetate were incubated

in a 1 M sodium hydroxide solution for 1 h, then carefully washed with

deionized water, and dried. To estimate the chitosan content in the

obtained products, a model blend film containing 20 wt% of chitosan

was used. Glucosamine content was calculated from an intensity ratio

of the bands assigned to chitosan �NH2 bending at 1 597 cm�1 and to

PVA�CH2� bending at 850 cm�1. The IR-spectra were recorded in an

absorbance mode at a resolution of 4 cm�1 and processed with Win-IR

software v. 4 (Bio-Rad Digilab Division). All the spectra were

normalized using the composite C�O band stretching vibrations of

the pyranose ring at 1 075 cm�1 as an internal standard.

The average Mw of the samples was determined by GPC on

Agilent 1200 (PLaquel – OH 40 column, pre-calibrated with

dextran standards, a flow rate of 1 mL �min�1, 25 8C). A solution

sample (0.2 wt%) in the eluent (H2Oþ 0.02% NaN3) was used. Relative

copolymer amounts were determined by means of an online refractive

index detector.

Glucosamine contents for chitosan and the both copolymers

were also calculated from the results of elemental analysis using C/N

ratio.


Entrapment of Cells in Microcapsules and Cell Cultivation

Cell encapsulation was performed as described earlier [9], with a

minor modification. Cell precipitate (1–6� 106 cells) was mixed with a

sterilized NaAlg solution (2 wt%, 2 mL), and the mixture was added

through the needle (0.4 mm in diameter) into a CaCl2 solution

(0.5 wt%) using a special device with a coaxial air flow (2� 105 Pa) [28].

The obtained calcium alginate (CaAlg) hydrogel microbeads were

incubated with chitosan-g-PVA copolymer or oligochitosan solution

(25 mL, 0.1–1.2 wt%, 5–10 min), in order to form a polyelectrolyte

membrane on the microbead surface. Then the microbeads were

washed three times with a physiological solution (0.9 wt% NaCl).

To get hollow microcapsules, the microbeads were incubated in an

EDTA solution (50� 10�3M, 10 min), then washed again with the

physiological solution, and finally transferred to the culture medium.

The encapsulated cells were cultivated for 3–4 weeks to generate cell

aggregates (multicellular spheroids) within the microcapsules.

To measure the concentration of microencapsulated cells, micro-

capsule aliquots (0.1 mL slurry) were mechanically destroyed by

pipetting up and down rapidly. By using pipette it was possible to

destroy not only microcapsule membrane but also all cell aggregates

formed inside microcapsules and to get single cells. Then the released

single cells were stained with Trypan Blue and calculated in a

Fuchs–Rosenthal hemocytometer. All the experiments were repeated

three times, and the data were expressed as means� standard

deviations.

All the solutions for cell encapsulation were prepared in a

physiological solution.

Empty microcapsules (without cells) were prepared in the same

way as described above. The microcapsule size distribution and the

membrane thickness were calculated using optical microscopy

(Reichert Microstar 1820E, Germany).

Determination of Monoclonal Antibodies

Concentration of MAbs produced by encapsulated 1D2 hybridoma

cells in culture medium was examined by ELISA. The mouse IgG

antibody were added in 96-well plates (NUNC, Denmark) (2 mg in

100 mL �PBS �well�1) and incubated overnight at 4 8C. Then non-

specific binding sites were blocked with 200 mL of a buffer solution

(1 wt% BSA in 0.2 M PBS containing 0.05 wt% Tween-20), pH 7.2, at

37 8C for 1 h. Then the wells were washed with PBS four times. Then

aliquots (100 mL) of culture medium samples were added and

incubated at 37 8C for 1 h. After washing with PBS, aliquots

(100 mL) of a horseradish peroxidase-linked goat anti-rat IgG

conjugate (diluted to 1:10 in PBS containing 0.05 wt% Tween-20)

were added. Finally, the plates were incubated at 37 8C for 45 min.

Then 0.4 mg �mL�1 2,20-azino-bis(3-ethylbenzthiazoline-6-sulphonic

acid) was added to each well for detection. After 15 min the reaction

was stopped with 2 M H2SO4 solution, and the absorbance was

measured using microplate reader (Multiscan MCC/340) at 450 nm.

Evaluation of Methotrexate Cytotoxicity

After long-term cultivation (28 d) of MCF-7 cells in microcapsules

the obtained microcapsulated MTS were transferred into 96-well

plates (Nunc, Denmark) and incubated with a set of MTX solutions to

get final MTX concentrations of 1, 2, 10, 50, and 100� 10�9M. The

microcapsule aliquot (100 mL slurry) was mixed with the cultivation

medium (150 mL) supplemented with 10% FBS and MTX of

the appropriate concentration, and then incubated in a 5% CO2

atmosphere at 37 8C for 48 h. The monolayer cell culture


RESEARCH

ARTIC

LE


(5� 105 cells �mL�1) was used as control. The cell viability was

calculated after Trypan Blue dye staining using the Fuchs–Rosenthal

hemocytometer. The cytotoxicity was expressed as cell viability using

the following formula: Cell viability (%)¼ [viable cells concentration

in the experiment/viable cells concentration in the control)]� 100.

Received: March 1, 2011

Final Version: April 15, 2011

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biocompatible smart microcapsules based on chitosan-poly(vinyl alcohol) copolymers for cultivation...

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