Journal of Membrane Science 377 (2011) 214220

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Journal of Membrane Science

journal homepage: www.elsevier.com/locate/memsci

Study on membrane characteristics of alginatechitosan microcapsule with cell growth

Weiting Yu a , Huiyi Song a , Guoshuang Zheng a , Xiudong Liu b, , Ying Zhang a , Xiaojun Ma a,
a b

Laboratory of Biomedical Materials Engineering, Dalian Institute of Chemical Physics(DICP), Chinese Academy of Sciences(CAS), Dalian 116023, PR China College of Environment and Chemical Engineering, Dalian University, Dalian Economic Technological Development Zone, Dalian 116622, PR China

a r t i c l e

i n f o

a b s t r a c t
Alginatechitosan (AC) microcapsule with polyelectrolyte complex (PEC) membrane has been attractive in biotechnological area such as cell immobilization fermentation. The PEC membrane has been known important to control microcapsule performance, but the knowledge was mainly obtained with empty or drug-loaded microcapsules. In this paper, the emphasis was to explore the inuence of cell growth on PEC membrane characteristics, that is, to study the membrane characteristics in dynamic process. AC microcapsule entrapping yeast cells (Saccharomyces cerevisiae BY4741) was used as model, and the PEC membrane characteristics including membrane thickness, swelling behavior, permeability, and component were studied during cell culture process. It was found that both the membrane thickness and volume swelling degree of AC microcapsule increased, however, membrane permeability decreased with the growth of entrapped yeast cells. Fluorescence-labeling studies suggested the concentrated alginate layer squeezed by the proliferated cells and macromolecular metabolites layer secreted by proliferated cells contributed to above increment. The former PEC membrane, together with the new layers, constituted the apparent membrane, which increased the diffusion resistance of substances. 2011 Elsevier B.V. All rights reserved.

Article history: Received 7 March 2011 Received in revised form 17 April 2011 Accepted 26 April 2011 Available online 5 May 2011 Key words: Alginatechitosan microcapsule Membrane characteristics Microencapsulated cell culture Apparent membrane

1. Introduction Microencapsulation is a kind of membrane technology, which encloses chemicals, drugs, enzymes or cells within a semipermeable membrane to protect the bioactivity or to realize controlled release or to produce substances of value [1,2]. Among the reported microcapsule systems, alginatechitosan (AC) microcapsule has been arousing interest due to the inherent properties such as biocompatibility, nontoxicity, and biodegradability [35]. It has been widely reported in the studies on drug delivery systems [6,7], enzyme immobilization [8], cell immobilization culture [912], and cell transplantation [1316]. Alginate is an anionic linear polysaccharide containing 1,4linked d-mannuronic acid and l-guluronic acid residues [17], which can form hydrogels in the presence of some multivalent metal ions such as calcium ion. Chitosan, the only naturally cationic polysaccharide [18], can diffuse into three-dimensional (3D) alginate gel network. Under simultaneous electrostatic interaction between protonated amino groups of chitosan and carboxyl groups of algi-

Corresponding authors. Tel.: +86 411 84379139/87402448; fax: +86 411 84379096. E-mail addresses: liuxd@dicp.ac.cn (X. Liu), maxj@dicp.ac.cn (X. Ma). 0376-7388/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2011.04.053

nate [19], polyelectrolyte complex (PEC) microcapsule membrane is formed [20]. The PEC membrane has been demonstrated to play an important role in controlling the performance of AC microcapsules in biomedical applications. So far, it has been found that many parameters such as molecular weight (Mw ), chain exibility, and charge density of both polysaccharides have affected the membrane structure, mechanical strength, substance permeaselectivity. However, the above understanding mainly results from the researches based on empty [19,2125] or drugs-loaded AC microcapsules [2628], which only provide the static characteristics of membrane. When AC microcapsules are used into immobilized cell culture and fermentation, the growth and metabolism process of entrapped cells were thought to have effect on the structure and characteristics of PEC membrane, which will in return affect the cell growth and metabolism. Therefore, the PEC membrane characteristics of AC microcapsules with cell growth were concerned with the purpose of obtaining the dynamic membrane characteristics in this paper. Yeast cell (Saccharomyces cerevisiae BY4741) was used as model, calcium alginate gel (CAG) beads entrapped cells were produced rstly, followed the complex reaction with chitosan to form the PEC microcapsule membrane, and then AC microencapsulated cell culture was carried out. The PEC membrane thickness, swelling behavior, substance permeability, and membrane component were

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investigated during the process of microencapsulated cell culture. Meantime, the effect of the degree of polymerization (DP) of chitosan on the membrane characteristics was also studied with cell growth in AC microcapsules. 2. Materials and methods 2.1. Cells and materials Wild type Saccharomyces cerevisiae strain BY4741 (S. cerevisiae BY4741) was acquired from EUROSCARF (European Saccharomyces cerevisiae Archive for Functional Analysis, Frankfurt, Germany). They were cultured in yeast peptone dextrose (YPD) medium, composed of 10 g/L yeast extract, 20 g/L bacto-tryptone, and 20 g/L glucose, respectively. Chitosan samples were degraded by gamma ( ) rays from raw material (Yuhuan Ocean Biomaterials Corporation, China) by Key Laboratory of Nuclear Analysis Techniques, Chinese Academy of Sciences. The average DP of degraded chitosan samples was determined by gel permeation chromatography (GPC) [29,30], which gave the values of 125, 245, and 370, respectively (Fig. 1). The polydispersity index (PDI) values were correspondingly 1.7, 1.9 and 2.2 suggesting Mw distribution of samples was close to each other. Therefore, DP125, DP245 and DP370 were used to denote the different chitosan samples in the study. Sodium alginate was purchased from the Chemical Reagent Corp (Shanghai, China). The viscosity was over 0.02 Pa s when dissolved to form a 1.0% (w/v) aqueous solution at 20 C. Fluorochrome uoresceinamine labeled alginate was prepared according to the method developed by Strand et al. [31]. Rhodamine-B labeled chitosan was prepared according to the method developed by our laboratory. Bovine serum albumin (BSA) was purchased from SigmaAldrich Chemical Co. with Mw of 66 kDa. All other reagents were analytical grade and used as received. 2.2. Preparation and culture of AC microencapsulated yeast cells AC microencapsulated yeast cells were prepared according to the method developed in our lab [32]. Sodium alginate was dis700.00 650.00 600.00 550.00 500.00 450.00 400.00 350.00 300.00

solved in 0.9% (w/v) NaCl solution to form nal concentration of 1.5% (w/v), and was sterilized by ltration through a 0.22 m membrane lter. The sodium alginate solution was stored overnight before use to facilitate deaeration. Yeast cells S. cerevisiae BY4741, in late exponential phase, were centrifuged and suspended in sodium alginate solution. Then the suspension was extruded through a 0.50-mm needle into calcium gelling solution using electrostatic droplet generator (YD-06, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China) to form CAG beads. The CAG beads were immersed in 0.5% (w/v) chitosan solution to form alginatechitosan microcapsule membrane, followed by rinsing with 0.9% (w/v) NaCl solution. After being liquidized for 5 min using 0.055 mol/L sodium citrate and rinsed three times with 0.9% (w/v) NaCl solution, AC microencapsulated yeast cells were formed and stored for further use. 0.8 mL AC microcapsules (12 104 microcapsules) enveloping yeast cells were added in 9.2 mL YPD medium. Then AC microencapsulated cells were cultured in a shaking incubator at 28 C and 170 rpm for 24 h. All experiments regarding cells were carried out in triplicate samples. 2.3. Characterization of the morphology, membrane thickness of AC microcapsules enveloping yeast cells during cell culture process After preparation and culture, the morphology of AC microencapsulated yeast cells was observed with a Nikon Eclipse TE2000 Inverted Research Microscope (Nikon Corp., Japan). Different visual elds were randomly selected and recorded to obtain 810 microscopic images of microcapsules for each chitosan sample. The membrane thicknesses of at least forty AC microcapsules were measured from microscopic images using software ImageJ, and presented as mean standard deviation. 2.4. Characterization of cell growth in AC microcapsules during cell culture process After 0.1 mL AC microcapsules (1.52.5 103 microcapsules) were broken up using a chemical method [33], yeast cells were col-

DP370 DP245 DP125

Mv

250.00 200.00 150.00 100.00 50.00 0.00 -50.00

-100.00 -150.00 -200.00 -250.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

Elution time (min)

Fig. 1. GPC chromatograms of chitosan samples (DP of chitosan is DP125, DP245, and DP370).

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lected and re-suspended. The OD600 values of cell suspension were measured at 600 nm using a UV-2550 visible spectrophotometer (Shimadzu Co., Kyoto, Japan). Then the cell density was obtained according to the established standard curve. 2.5. Characterization of the swelling behavior of AC microcapsules enveloping yeast cells during cell culture process The size and size distribution of AC microcapsules enveloping yeast cells were determined with laser diffraction particle analyzer (LS100 Q, Beckman-Coulter Corp., USA). The volume swelling degree (Sw ) was used to characterize the swelling behavior of AC microcapsules [34]. The Sw of microcapsules is dened as: Sw (%) = 100 Dt D0
3

3. Results and discussion 3.1. Characterization of the morphology, membrane thickness of AC microcapsules enveloping yeast cells during cell culture process AC microencapsulated yeast cells were made by two-stage procedure. Firstly, alginate solution containing yeast cells, with inoculum cell density of 1 107 /mL microcapsule, was dropped into calcium chloride solution to produce CAG beads. Secondly, the beads were immersed in chitosan solution (DP125, DP245 and DP370) for 20 min to form PEC microcapsule membrane. After being liquidized, AC microencapsulated yeast cells were formed. After preparation and cultured for 24 h, AC microcapsules were observed with optical microscope. Fig. 2 presented the representative microscopic images of prepared and cultured AC microcapsules. It can be seen that AC microcapsules were spherical and intact with smooth surface. The bright outer layer of microcapsules demonstrated the PEC membrane (Fig. 2, arrow indicated). AC microcapsules after preparation (Fig. 2A) showed obvious thinner PEC membrane than that of microcapsules cultured for 24 h (Fig. 2B). Moreover, at least forty microcapsules for both condition were randomly selected for membrane measurement using software ImageJ, which provided membrane thicknesses of 5.6 1.4 m (Fig. 2A) and 18.9 2.4 m (Fig. 2B), a three-fold increase after culture. Yeast cells uniformly scattered in microcapsules after preparation (Fig. 2A), while lled the inner space of microcapsules after cultured for 24 h demonstrating obvious cell growth (Fig. 2B). AC microcapsules with cells cultured at 12 and 24 h were broken up, respectively, the densities of yeast cells in microcapsules were also measured and shown in Fig. 3. In comparison with the inoculum cell density of 1 107 /mL microcapsule, yeast cells in microcapsules

where D0 represents the diameter of AC microcapsules before cell culture, and Dt represents the diameter of AC microcapsules after cell culture. The larger the volume swelling degree is, the easier AC microcapsule swell. 2.6. Analysis of microcapsule membrane component with cell growth in AC microcapsules Fluorochrome uoresceinamine labeled alginate and Rhodamine-B labeled chitosan were used to prepare AC microcapsules enveloping yeast cells according to the method in Section 2.2. Then the alginate and chitosan distribution in microcapsules during cell culture was observed by confocal laser scanning microscope (CLSM) (Leica, TCS-SP2, Germany), equipped with laser sources both blue (Ar 488 nm/5 mW) and green (HeNe 543 nm/1.2 mW), and an inverted microscope (Leica, DMIRE2, Germany). 2.7. Characterization of proteins secreted by cells in AC microcapsules during cell culture process When AC microencapsulated yeast cells were cultured for 0, 12, and 24 h, respectively, the microcapsules were collected and washed by 0.9% (w/v) NaCl. Then 0.1 mL AC microcapsules were broken up. The nal volume was adjusted to 5 mL, and the broken solution was centrifuged to eliminate yeast cells. The protein concentration of supernatant was measured by the Bradford method. 2.8. Characterization of membrane permeability of AC microcapsules enveloping yeast cells during cell culture process Bovine serum albumin (BSA) was used as standard protein to characterize membrane permeability of AC microcapsule. The higher concentration of BSA in microcapsules, the higher permeability of membrane is. When AC microencapsulated yeast cells were cultured for 0, 12, and 24 h, respectively, the microcapsules were collected and washed by 0.9% (w/v) NaCl. Then 0.1 mL AC microcapsules were put into a 0.5 mL tube, and 0.1 mL physiological saline was added to re-suspend microcapsules. 0.2 mL BSA solution (1 mg/mL) was added into the tube to characterize membrane permeability. After 6 min, the residual BSA concentration in supernatant was measured by the Bradford method. The protein diffusing into AC microcapsules was calculated using the following equation: 0.2 0.3Ci Vi

C(mg/mL) =

where Ci is the protein concentration in the supernatant after diffusion, and Vi is the volume of AC microcapsules.

Fig. 2. Optical images of AC microencapsulated cells with chitosan of DP245 before culture (A), and after culture for 24 h (B), bar = 100 m.

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DP125

Cell density(10E7 per ml microcapsules)

220 200 180 160 140 120 100 80 60 40 20 0 12 *

DP245 DP370

24

Culture time (hour)

Fig. 3. Cell densities in AC microcapsules after culture for 12 and 24 h (n = 3, * represents p < 0.05).

grew remarkably with cell density over 100 107 /mL microcapsule at 12 h (an increment with two order of magnitude), and over 170 107 /mL microcapsule at 24 h (an increment of 70% compared to that at 12 h). 3.2. Characterization of the swelling behavior of AC microcapsules enveloping yeast cells during cell culture process According to the described method in Section 2.5, the swelling degrees of AC microcapsules with cells during culture process were calculated and shown in Fig. 4. The Sw values of AC microcapsules with cells displayed increment trend in 24-h culture period, which demonstrated that the volume of AC microcapsules increased obviously during cell culture process. Before cell culture, yeast cells were distributed in alginate sol transformed by the liquefaction process. When AC microencapsulated cells were incubated, yeast cells gradually grew (Fig. 3) and occupied the inner space of AC microcapsules (Fig. 2). As a result, the dissolved alginate molecules from hydrogel network were squeezed outward to microcapsules. However, only a small fraction of the dissolved alginate molecules can permeate out of the microcapsules due to the selective permeation of the membrane pores

as demonstrated with alginatepoly-lysine microcapsule [3537]. The outward movement of alginate molecules and prevention by membrane resulted in the concentrated alginate molecules at the inner layers of PEC membrane, together with high concentration of sodium ions, which caused the difference of osmotic pressure at both sides of microcapsule membrane. Therefore, the inuent of water for balancing the osmotic pressure inside and outside the membrane contributed to the swelling behavior of microcapsule. It was also noticed that Sw values showed positively dependent on the DP of chitosan samples. In general, chitosan with low DP means short molecular chain and low steric hindrance, many chitosan molecules can easily diffuse into deep area of Caalginate hydrogel network to form thick PEC membrane with small inside space [30]. The thick PEC membrane has strong anti-swelling ability [34], and the small inside space means the dissolved alginate molecules squeezed by cells were relatively lower. Therefore, both reasons contributed to the lowest Sw values for chitosan sample with lowest DP. On the contrary, it was difcult for chitosan with high DP to diffuse into hydrogel network, the complex reaction occurred mainly at CAG bead surface to form thin membrane but leave big inside space. The weak anti-swelling ability of thin PEC membrane and more alginate molecules squeezed by cells make it easy for microcapsule to swell during cell culture process. 3.3. Analysis of microcapsule membrane component with cell growth in AC microcapsules Both membrane thickness and swelling results demonstrated that the growth of entrapped cells clearly have effect on the PEC membrane characteristics of AC microcapsules. To uncover the reasons beyond these results and phenomena, the uoresceinamine-labeled alginate (green) and rhodamine-labeled chitosan (red) were used to display the membrane and to investigated the changes of membrane during cell culture process. Fig. 5 showed CLSM images of uorescence labeled AC microcapsules with cells before culture (A), after culture for 24 h (B), and uorescence intensity after culture (C). Both Fig. 5A and B contained two images providing the information from transmitted light channel (left), and the overlay of transmitted and uorescence light channels (right). It can be seen that the membrane of AC microcapsules in optical image was thinner before culture (Fig. 5A, left) than that after culture (Fig. 5B, left). While the membrane thickness in merged optical and uorescence images was almost same (right of Fig. 5A and B, yellow uorescence layer represent the real PEC membrane formed by green uorescence labeled alginate and red uorescence labeled chitosan). By drawing a straight line through the observed section of AC microcapsule with cells (Fig. 5B), the change of uorescence intensity along the straight line (Fig. 5C) could be plotted via software. The peak width representing the membrane thickness in transmitted light image (last curve of Fig. 5C) was wider than that representing uorescence labeled membrane (rst and second curves of Fig. 5C). Considering the cell growth, it was deduced that the accumulation of macromolecular metabolites such as proteins secreted by entrapped cells contributed to the difference of membrane thickness between optical and uorescence images. To validate above deduction, AC microcapsules with yeast cells were broken after culture for 12 and 24 h, respectively. The suspension was centrifuged, and the supernatant was analyzed by the Bradford method for protein quantication. Fig. 6 showed that protein concentration increased with the culture process. For example, protein concentration after culture 24 h was over 10 mg/mL. It proved that cells secreted plenty of proteins during culture process, but most of these macromolecular metabolites accumulated in microcapsules due to the retention effect of PEC membrane. According to the fact that cells grew and almost occupied the

20 18 16 DP125 DP245 DP370

Swelling degree (%)

14 12 10 8 6 4 2 0 12 24

Culture time (hour)

Fig. 4. Swelling behavior of AC microcapsules with cells after culture for 12 and 24 h.

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Fig. 5. CLSM images of uorescence labeled AC microcapsules with cells before culture (A), after culture for 24 h (B), and the corresponding uorescence intensity curves (C).

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Protein of cell secreting in microcapsule(mg/ml)

14 12 10 8 6 4 2 0 12

DP125 DP245 DP370

24

Culture time (hour)

Fig. 6. Concentration of proteins secreted by cells and accumulated in AC microcapsules after culture for 12 and 24 h (n = 3, * represents p < 0.05).

inner space of microcapsules (Fig. 2), the ever existed inner solution was squeezed out of microcapsules. The metabolized proteins diffused toward the membrane with solution. As a result, these proteins with higher Mw could not permeate outward and gradually accumulate at the inner surface of membrane to form new layer, which displayed the similar optical refraction property with PEC membrane under optical microscope. Therefore, it was concluded that the component of cultured microcapsule membrane was composed of the former alginate/chitosan membrane, concentrated alginate layer squeezed by cells, and macromolecular metabolites layer. In fact, it is no longer a strictly dened microcapsule membrane but an apparent membrane or microcapsule wall. Furthermore, the substance permeability of the apparent membrane was also investigated during cell culture process. Because the culture medium is too complicated to analysis, bovine serum albumin (BSA) was used as model protein to evaluate the inward substance diffusion to the apparent membrane. At culture time of 0, 12 and 24 h, AC microcapsules were collected, rinsed and immersed into BSA solution. The inward diffusion of BSA was calculated by analyzing supernatant. It was shown in Fig. 7 that BSA permeability at 12 h was somewhat same as that at 0 h, but decreased almost
0.35

half at 24 h. Based on above results and discussion, the decline of substance permeability could be ascribed to the increase of apparent membrane thickness during cell culture, which consequently increased the diffusion resistance of protein. Generally, AC microcapsules with low chitosan DP form thick and loose membrane, but thin and dense membrane with high chitosan DP. Although both the thickness and pore size of membrane are the barriers for substance diffusion, the latter is usually dominant factor as shown in Fig. 7 (0 h). With the time course of culture, yeast cells in AC microcapsules with chitosan DP 125 and DP 245 in rst 12 h could get much nutrients for growth, but the high diffusion resistance by membrane with chitosan DP 370 due to fast block of pores (Fig. 7, 12 h) gave signicant low cell growth (Fig. 3, 12 h). Meantime, AC microencapsulated cells secreted metabolites such as proteins, and many proteins were retained in microcapsules with chitosan DP 370 for high diffusion resistance (Fig. 6, 12 h). When AC microencapsulated cells were cultured for 24 h, the substance permeability of AC microcapsules with chitosan DP 125 and DP 245 remarkably decreased (Fig. 7). In consideration of protein analysis in Fig. 6, it was thought that the secreted proteins gradually blocked the loose membrane pores of microcapsules with low chitosan DP, and then accumulated to form new layer as part of apparent membrane. As a result, the resistance for both BSA and secreted proteins diffusion also increased correspondingly due to extension of diffusion distance and increase of compactness of apparent membrane. Furthermore, the nutrients supply for cell growth correspondingly decreased, which resulted in the slow increase of cell densities at 24 h (Fig. 3). Cell increment in AC microcapsules with chitosan DP 125 showed signicant lower than that in microcapsules with chitosan DP 245 and DP 370 (Fig. 3). The smallest inner space of microcapsule with chitosan DP 125 had been occupied by cells, which limited the further cell growth. Therefore, the cell increment was slowest in the second 12 h. However, microcapsules with chitosan DP 370 had the largest inner space, which was benecial for further cell growth though the nutrients supply was slow. Therefore, the growth space limitation became the dominant factor for cell growth in the second 12 h. As a whole, chitosan DP, as a parameter, was no longer an only factor on membrane characteristics during cell culture, and could not show denite effect tendency with cell growth. 4. Conclusion AC microcapsule entrapping yeast cells (S. cerevisiae BY4741) was used as model, and the PEC membrane characteristics including membrane thickness, swelling behavior, permeability, and component were studied with the emphasis on cell culture process. The membrane thickness and the volume swelling degrees of AC microcapsules showed obvious increment during cell culture process, however, membrane permeability decreased with the growth of entrapped yeast cells, which suggested the growth of entrapped yeast cells clearly affected the PEC membrane characteristics. The reasons on the increment of membrane thickness and swelling degree were further analyzed based on uorescence labeled technique and substance diffusion studies. The formerly alginatechitosan PEC membrane, concentrated alginate layer squeezed by cells, and macromolecular metabolites layer secreted by cells constituted the apparent membrane during culture process. Acknowledgements The authors thank the National Natural Science Foundation of China (No: 20806080, 20876018, 20736006) and the National Basic

*
BSA diffusion into microcapsule(mg/ml)
0.30

* *

DP125 DP245 DP370

0.25

0.20

0.15

0.10

0.05

0.00

12

24

Culture time (hour)

Fig. 7. The inward BSA diffusion to apparent membrane before and after culture for 12 and 24 h (n = 3, * represents p < 0.05).

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Research Program of China (No: 2007CB714305) for the nancial support. And we also thank Prof. Guozhong Wu at Key Laboratory of Nuclear Analysis Techniques, Chinese Academy of Sciences for the degradation of chitosan samples by gamma ray. References
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