Three-dimensional gastric cancer cell culture using nanofiber scaffold for chemosensitivity test

International Journal of Biological Macromolecules 45 (2009) 65–71

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Three-dimensional gastric cancer cell culture using nanofiber scaffold for chemosensitivity test Young-Jin Kim a,∗ , Han-Ik Bae b , Oh Kyoung Kwon c , Myung-Seok Choi d a

Department of Biomedical Engineering, Catholic University of Daegu, 330 Geumnak 1-ri, Hayang-eup, Gyeongsan 712-702, South Korea School of Medicine, Kyungpook National University, Daegu 700-422, South Korea Department of Surgery, Gumi CHA General Hospital, Gumi 730-040, South Korea d Department of Materials Chemistry and Engineering, Konkuk University, Seoul 143-701, South Korea b c

a r t i c l e

i n f o

Article history: Received 17 March 2009 Accepted 8 April 2009 Available online 16 April 2009 Keywords: Chemosensitivity Electrospun nanofiber Three-dimensional culture

a b s t r a c t A three-dimensional (3D) culture of cancer cells has long been advocated as a better model of the malignant phenotype that is most closely related to tumorigenicity in vivo. To investigate the sensitivity of cancer cells to anticancer drugs, nanofiber scaffolds composed of PHBV and collagen peptide were fabricated by electrospinning. A 3D culture of cancer cells was successfully achieved by the use of nanofiber scaffolds. From the result of a chemosensitivity test, it was found that higher concentrations of anticancer drugs were required to achieve a comparable cytotoxic effect in 3D culture due to their structural architecture. These data demonstrate that the electrospun nanofiber scaffolds can provide a 3D model particularly appropriate for investigating mechanisms involved in cancer cell sensitivity to anticancer drugs. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Gastric cancer is the fourth-most common type of cancer and the second leading cause of cancer-related death in the world [1]. Surgery continues to have an essential role in the management of this disease, although the extent of surgery continues to be a subject of debate. Recently, postoperative chemoradiotherapy has become a standard of care in the treatment of localized gastric cancer [2]. Chemotherapy plays an important role in cancer therapy today. The majority of patients with cancer will require treatment with chemotherapeutic agents at some point in the course of their disease. While determining the sensitivity and resistance of an individual organism prior to treatment has been the standard for infectious diseases for years, in chemotherapy the drug regimen is still defined by tumor histology rather than a tumor’s sensitivity to a given drug. Due to individual susceptibilities, tumors of the same histology do not necessarily respond identically to the same drug regimen. A chemosensitivity test that is capable of predicting a response to a given chemotherapeutic agent could therefore help to improve the clinical outcome of cancer patients. There have been many attempts at an in vitro chemosensitivity test on the basis of a number of different technologies [3,4]. However, so far such a chemosensitivity test is not broadly accepted by oncologist because of no accurate prediction of in vivo drug response [5].

∗ Corresponding author. Tel.: +82 53 850 3443; fax: +82 53 850 3292. E-mail address: [email protected] (Y.-J. Kim). 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.04.003

Two-dimensional (2D) cell culture models of cancer cell lines are usually used to evaluate the efficacy of anticancer drugs. These standard monolayer in vitro assays determine the capacity of an anticancer regimen to inhibit cell growth or the potential to induce cancer cell apoptosis [6,7]. However, these assays can neither stratify cancer cells according to their potential for invasion nor evaluate the anticancer drug efficacy with regard to cancer cell invasion and metastasis [8]. In addition, most cancer cells are supported by an extracellular matrix (ECM) microenvironment which plays an important role in the development of anticancer drug refractoriness [9]. Thus, the development of in vitro 3D culture assays that simulate the extracellular microenvironment can provide important information about the cancer cell potential for invasion and the efficiency of anticancer drugs. Even though extensive work has been reported using 3D culture for understanding tissue architecture, very little has been published on the use of 3D culture as an in vitro model for the cytotoxic evaluation of anticancer drugs. The electrospinning technique has attracted a great deal of attention as it is an effective means to produce a nonwoven membrane of nanofibers. The interconnected porous nanofiber networks in electrospun membranes, having a very high surface-to-volume ratio, are particularly useful for biomedical applications such as scaffolds used in tissue engineering, wound dressing, drug delivery and vascular grafts [10–13]. In addition, electrospinning provides a mechanism to produce nanofibrous scaffolds from a variety of polymer materials including synthetic and natural polymers. Principally, the scaffold should be designed by mimicking the structure and biological function of native ECM proteins, which provide

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mechanical support and regulate cell activities. Nonwoven membrane composed of electrospun nanofibers is architecturally similar to the collagen structure of ECM, in which the collagen multifibrils of nanometer scale are composed of a 3D network structure together with proteoglycans [14]. Therefore, biomimetic matrices can be fabricated by electrospinning, which facilitate cell attachment, support cell growth and regulate cell differentiation [15,16]. In the native tissues, the structural ECM proteins (50–500 nm diameter fibers) are 1–2 orders smaller than the cell itself. This allows the cell to be in direct contact with many ECM fibers, thereby defining its 3D orientation. Electrospun nanofiber scaffolds can also provide a successful opportunity for 3D cell culture [17,18]. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a biodegradable, biocompatible and thermoplastic polyester produced by various microorganisms, which has received much attention as a suitable material for various applications in medicine due to its attractive properties [19,20]. It was reported that the porous PHBV materials were adequate for use as substrates for the cell cultures [19,21]. PHBV sustained the fibroblast cell proliferation rate similar to that observed in the collagen sponges. In addition, no acute inflammation, abscess formation or tissue necrosis was observed in tissues adjacent to the implanted materials. Collagen is a structural ECM protein occurring in most connective tissues such as skin, tendon and bone, and is a suitable compound for a variety of biomedical applications [22,23]. The present wide interest in collagen is mainly due to its merits of wealth like biological origin, nonimmunogenicity, biodegradability and biocompatibility. As a biomaterial, collagen has been predominantly used after being processed into a dry powder or slurry, a hydrogel after solution phase cross-linking, or as a porous matrix after freeze drying. However, in native connective tissues, collagen molecules exist in a 3D network structure composed of multi-fibrils. Functionally, collagen fiber networks act to resist high strain deformation and prevent premature tissue mechanical failure [24]. The 3D culture of cancer cells allows to explore many basic questions related to cancer biology, as receptors for growth factors which play an important role in tumor development are expressed in different ways in comparison to the standard 2D culture [25,26]. Recently, it was reported that the cancer cells cultured in 3D showed behaviors similar to that observed in natural endogenous matrices, which formed colonies and secondary tumor-like structures [27]. From the standpoint of exactitude of chemosensitivity test for gastric cancer cells, it may be useful to culture cancer cells in 3D. In the present study, the fabrication of nanofiber scaffolds composed of PHBV and collagen peptide by electrospinning was systematically examined. The influence of the mixing ratio of two components on the morphology of the resulting nanofibers was investigated. Furthermore, an attempt was made to develop a 3D culture system of gastric cancer cells using nanofiber scaffolds in order to exactly evaluate the efficacy of anticancer drugs.

2. Experimental 2.1. Materials Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) containing 5 wt% of hydroxyvalerate and 2,2,2-trifluoro ethanol (TFE) were purchased from Sigma–Aldrich Co. and used without further purification. Collagen peptide (CP, Mw = 5000) from calf skin was kindly supplied by Highgel Co., Ltd. RPMI-1640 medium, fetal bovine serum (FBS) and penicillin–streptomycin were obtained from Gibco BRL. Other reagents and solvents were commercially available and were used as received.

2.2. Electrospinning The electrospinning setup utilized in this study consisted of a syringe and needle (ID = 0.41 mm), a ground electrode, and a high voltage supply (Chungpa EMT Co., Korea). The needle was connected to the high voltage supply, which could generate positive DC voltages up to 40 kV. For the electrospinning of PHBV or PHBV/CP nanofibers, PHBV or a mixture of PHBV and CP was first dissolved in TFE at a concentration of 8% (w/v). The weight ratio of PHBV and CP in the mixed solutions was either 7:3 or 5:5. PHBV or PHBV/CP solution held in a 10-ml syringe was delivered into a needle spinneret by a syringe pump (KDS 100, KD Scientific Inc., USA) with a mass flow rate of 1.0 ml/h. The steel needle was connected to an electrode of a high voltage supply and a grounded stainless steel plate was placed at 12 cm distance from the needle tip to collect the nanofibers. The positive voltage applied to the polymer solutions was 13 kV. All experiments were carried out at room temperature and below 60% RH. 2.3. Characterization of nanofibers The morphology of electrospun PHBV and PHBV/CP nanofibers was observed by a field emission-scanning electronic microscope (FE-SEM, S-4800, Hitachi). Prior to SEM observation, all of the samples were coated with osmium. The average diameter of nanofibers was determined by analyzing the SEM images with an image analyzing software (Image-Pro Plus, Media Cybernetics Inc.). ATR–FTIR spectra of the samples were obtained with a Nicolet 5700 spectrophotometer (Thermo Electron Co.). X-ray diffraction (XRD) measurements were carried out to characterize the crystalline phase of PHBV and PHBV/CP nanofibers with a Rigaku X-ray diffractometer D/MAX 2500 with Cu K␣ radiation at 40 kV/30 mA. The diffractograms were scanned in a 2 range of 10–50◦ at a rate of 2◦ /min. 2.4. Degradation of nanofibers The PHBV and PHBV/CP nanofiber scaffolds were cut into a size 3 cm × 3 cm and immersed into warm phosphate-buffered saline (PBS, pH 7.4, 37 ◦ C) for different periods of time (1, 4, and 7 days) to test their degradation behavior. After each degradation period, the samples were washed and subsequently dried in a vacuum oven at room temperature for 24 h. An SEM observation of the scaffolds was performed to understand the change in nanofiber’s morphology during this period. This experimental condition was selected in order to simulate a real situation of PHBV and PHBV/CP nanofibers in physiological applications such as scaffolds for tissue engineering. 2.5. Gastric cancer cell culture To study the cell proliferation on different nanofiber scaffolds, the cell viability was evaluated based on a gastric cancer cell line, MKN28. All cultures were made in RPMI-1640 medium with 10% fetal bovine serum (FBS) and antibiotics in a humidified atmosphere under 5% CO2 at 37 ◦ C. Prior to cell seeding, the samples were sterilized under UV for 3 h, followed by rinsing with PBS and a culture medium 5 times, respectively. Then, cancer cells (5 × 104 cells/well) were seeded on the nanofiber scaffolds in a 24well microplate. The cell proliferation on different substrates was monitored for 1, 3, 5 and 7 days by an MTT assay with microplate reader (OPSYS-MR, Dynex Technology Inc.). The mechanism of this assay is that metabolically active cells react with tetrazolium salt in the MTT reagent to produce water-insoluble formazan dye that can be observed at 570 nm.

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Fig. 1. SEM micrographs of electrospun (a) PHBV, (b) PHBV/CP73 and (c) PHBV/CP55 nanofibers.

2.6. Measurement of the IC50 concentration of anticancer drugs in 2D culture system In order to compare between 2D and 3D culture systems, the IC50 values, which means the concentration of drugs producing a 50% inhibition of cell proliferation as compared to untreated cell control, was first determined in a 2D culture system. In this study, 5 types of usual anticancer drugs such as 5-fluorouracil (5-FU), oxaliplatin, paclitaxel, cisplatin and irinotecan were used, which were dissolved in DMSO at 100 mM as stock solutions and diluted to proper concentrations with culture medium. For the measurement of IC50 , MKN28 cells were seeded in a 24-well microplate without using the nanofiber scaffolds at a cell density of 5 × 104 cells/well and allowed to grow for 24 h in a culture medium before treatment. Then, the culture medium was replaced with a fresh medium containing various concentrations of anticancer drugs ranged from 1 to 1000 ␮g/ml. After treatment, cells were incubated for another 24 h in a humidified atmosphere under 5% CO2 at 37 ◦ C and viability was determined by MTT assay. 2.7. Chemosensitivity test in 3D culture system using nanofiber scaffolds MKN28 cells were seeded on the nanofiber scaffolds in a 24well microplate at a cell density of 5 × 104 cells/well and allowed to grow for 24 h in a culture medium before treatment. Then, the culture medium was replaced with fresh medium containing the IC50 concentration of anticancer drugs determined in the 2D culture system. Chemosensitivity was evaluated by the examination of cell viability with MTT assay after following the incubation of cells for 24, 48 and 72 h. 3. Results and discussion

ular weight CP. As shown in Fig. 2, the solution viscosity was decreased with increasing the content of CP, which ranged from 960 to 200 cP. It was reported that the solution properties such as solution viscosity, conductivity and surface tension are the main factors influencing the transformation of polymer solution into electrospun nanofibers [28]. Among them, the solution viscosity is the most important parameter in electrospinning, which can affect the morphology of nanofibers, with the result that higher viscosity gives rise to the increase of fiber diameter. 3.2. Properties of nanofibers ATR-FTIR analysis was carried out for surface characterization of PHBV and PHBV/CP nanofibers in the range of 400–4000 cm−1 . As shown in Fig. 3, two peaks centered at 3436 cm−1 and 1724 cm−1 were observed for the PHBV nanofiber. The former one, due to the hydroxyl stretching vibration, was relatively weak and negligible, and the latter one was due to the C O stretch of the ester group present in the molecular chain of highly ordered crystalline structure [29]. In the case of the PHBV/CP nanofibers, the characteristic absorption band was observed at 3294 cm−1 associated with N H stretching vibration in CP. According to the previous report, a free N H stretching vibration occurs in the range from 3400 to 3440 cm−1 , and when the N H group of peptides is involved in hydrogen bonding, the position is shifted to lower frequencies, usually around 3300 cm−1 . From the result, it indicated that many of the N H groups of CP were involved in hydrogen bonding [30]. In addition, common bands of protein appeared at approximately 1644 cm−1 (amide I) and 1538 cm−1 (amide II), corresponding to the stretching vibration of C O bond, and coupling of bending of N H bond and stretching of C N bond, respectively. The amide I band at 1644 cm−1 was attributable to both a random coil and ␣-

3.1. Electrospinning of PHBV and PHBV/CP nanofibers The use of scaffolding materials for tissue engineering is an attempt to mimic the extracellular matrix (ECM) in connective tissues. Electrospun nanofiber scaffolds could be an ideal structural matrix that resembles the fibrous structure of collagen bundles in the ECM. Furthermore, ultrafine nanofiber structures are thought to enhance cell adhesion, migration and proliferation [15,16]. In this study, nanofibers were prepared by electrospinning from pure poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and PHBV/collagen peptide (CP) blend solutions with weight ratios of PHBV to CP of 7:3 (PHBV/CP73) and 5:5 (PHBV/CP55), respectively, at a concentration of 8% (w/v). Fig. 1 shows SEM micrographs of electrospun PHBV and PHBV/CP nanofibers. With increasing the concentration of CP in the solution, the average diameter of nanofibers was reduced from 520 ± 58 nm for PHBV nanofiber to 270 ± 63 nm for PHBV/CP55 nanofiber (Fig. 2). This result may be related to the change of solution viscosity by the use of low molec-

Fig. 2. Effect of the CP content on the average fiber diameter of nanofibers and viscosity of the electrospinning solutions.

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Fig. 3. ATR-FTIR spectra of (a) PHBV, (b) PHBV/CP73 and (c) PHBV/CP55 nanofibers.

helix conformation of CP [31]. The intensity of these characteristic absorption bands was increased by increasing the content of CP in the PHBV/CP nanofibers. The interactions between PHBV and CP may occur by hydrogen bonding. The O H groups and N H groups of CP are capable of forming hydrogen bonding with C O groups of PHBV. However, it was hard to find out the formation of hydrogen bonding between PHBV and CP by ATR–FTIR analysis. CP powder exhibited almost the same characteristic absorption bands as compared with PHBV/CP nanofibers, which were observed at 3295 cm−1 (N H stretching vibration), 1644 cm−1 (amide I) and 1538 cm−1 (amide II) (data not shown). Crystallographic analysis was performed using X-ray diffraction (XRD) for elucidating the change of crystal structure between PHBV and PHBV/CP nanofibers. It has been reported that PHBV crystallizes in ether poly(3-hydroxybutyrate) (PHB) or poly(3-hydroxyvalerate) (PHV) crystalline lattice for HV content lower or higher than

Fig. 4. X-ray diffraction patterns of (a) PHBV, (b) PHBV/CP73 and (c) PHBV/CP55 nanofibers.

50 mol% [32]. PHBV used in this study has an 5 wt% HV content, therefore, it crystallizes in the PHB crystalline lattice and has the same crystal structure as that of PHB showing the orthorhombic unit cell. As shown in Fig. 4, PHBV nanofiber exhibited two strong diffraction peaks at around 13.3◦ and 16.8◦ corresponding to (0 2 0) and (1 1 0) planes, respectively. In addition, two other weak diffraction peaks were observed at 25.4◦ and 26.9◦ ascribed to (1 2 1) and (0 4 0) planes. For the PHBV/CP nanofibers, the intensity of the diffraction peaks arising from PHBV increased slightly by blending PHBV with CP in the ratio of 7:3 (PHBV/CP73). On the other hand, by increasing CP content to 50 wt% in the blend (PHBV/CP55), the intensity of the diffraction peaks obviously decreased, and consequently two weak diffraction peaks observed at 25.4◦ and 26.9◦ disappeared. These results suggest that there exists an intermolecular interaction between PHBV and CP in nanofibers.

Fig. 5. Morphology changes of (a) PHBV, (b) PHBV/CP73 and (c) PHBV/CP55 nanofibers before and after a biodegradability test.

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3.3. Degradation of nanofibers The rate and mode of degradation of the polymers influence their service life, mechanical properties and the response of the biological system towards them. Cancer cells should be cultured for at least 3 days, 1 day for preculture and then 2 days for drug treatment, to examine the sensitivity of anticancer drugs. Therefore, scaffolds used in the chemosensitivity test are required to be slowly or not degrading during cell culture time. The mechanism of the biodegradation of heterochain polymers such as poly(l-lactide) and PHBV is hydrolysis (biotic or abiotic) followed by bioassimilation [33]. Fig. 5 illustrates the morphological changes in electrospun PHBV and PHBV/CP nanofibers during in vitro degradation after 1, 4 and 7 days in phosphate buffered saline (PBS, pH 7.4) at 37 ◦ C. As expected, morphological changes in PHBV nanofiber were not observed during 7 days. PHBV is hardly degraded in an aqueous condition of neutral pH in the absence of enzyme (depolymerase) [34]. In addition, as can be seen from Fig. 5(b), PHBV/CP73 nanofiber was slightly degraded after 1 day, though further degradation and significant morphological changes were not observed during 7 days. However, significant morphological changes were observed for electrospun PHBV/CP55 nanofiber. CP can be easily dissolved in water and hence by increasing CP content the degradability of PHBV/CP nanofibers increased in PBS. 3.4. Viability of gastric cancer cells An MTT assay was carried out to evaluate the cell proliferation of MKN28 cells on PHBV and PHBV/CP nanofiber scaffolds. The proliferation of cells on PHBV/CP nanofibers is higher than PHBV nanofiber (Fig. 6), indicating that the CP-containing nanofibers have accelerated the proliferation and differentiation of MKN28 cells. Among the PHBV/CP nanofibers, cell proliferation was significantly higher on PHBV/CP73 nanofiber compared with PHBV/CP55 nanofiber. The degradation rate of PHBV/CP55 nanofiber was very high in an aqueous medium as shown in Fig. 5, resulting that MKN28 cells were probably detached from the surface of nanofiber scaffolds. Thus, we selected PHBV/CP73 nanofiber for the chemosensitivity test due to its slow degradation rate and better cell proliferation. 3.5. 3D Culture of gastric cancer cells It is unanimously recognized that the tissue microenvironment is of crucial importance to metastatic tumor progression. In the natural 3D environment, the malignant cells interact not only with the stromal cells or cross-linked proteins but also are exposed to the action of several autocrine and paracrine factors such as cytokines or proteolytic enzymes [35]. In this way, the fate of cancer evolution is conditioned by complex interactions that are difficult to repro-

Fig. 6. Cell viability of MKN28 cells grown onto (a) PHBV, (b) PHBV/CP73 and (c) PHBV/CP55 nanofibers as a function of culture time.

duce in ex vivo experiments. Recent progress in tissue engineering has revealed the suitability of a polymeric 3D scaffold for ex vivo cell growth [17,18,36]. Moreover, the electrospun nanofiber scaffolds potentially provide a 3D structure for cell attachment, growth and migration. Therefore, cell morphology and the interaction between cells and nanofibers were studied for 14 days. SEM micrographs showed that MKN28 cells adhered and spread on the surface of the nanofiber scaffold and had already started to migrate through the pores and to grow under layers of nanofibers on day 14 (Fig. 7). It was previously reported that the electrospun collagen nanofibers were densely populated with smooth muscle cells within 7 days and smooth muscle cells were observed deep within the matrix and fully enmeshed within the electrospun collagen nanofibers [22]. These data strongly suggest that MKN28 cells interact and integrate with the surrounding nanofibers and grow in the direction of fiber orientation, forming a 3D structure. 3.6. Chemosensitivity test of gastric cancer cells Chemosensitivity assays work by directly analyzing effects of therapies on living cancer cells to find out potential anticancer drugs with respect to each individual type of tumors. The 2D model of in vitro growth of cancer cells has frequently been employed to determine the cytostatic effects of different drugs. In these types of culture, malignant cells are only able to proliferate and to increase their number. In contrast, the cancer cells in 3D cultures adopt a behavior similar to what is observed in a natural endogenous matrix [27]. For this reason, 2D and 3D culture systems were compared in order to select optimal in vitro conditions to better evaluate the efficacy of anticancer drugs.

Fig. 7. SEM micrographs of (a) surface and (b) cross-section of MKN28 cells grown on 3D nanofiber scaffold for 14 days.

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Table 1 IC50 concentration of various anticancer drugs determined in the 2D culture system. Anticancer drug

Tested concentration (␮g/ml)a

IC50 (␮g/ml)

5-FU Oxaliplatin Paclitaxel Cisplatin Irrinotecan

1–500 1–500 1–500 1–200 1–1000

100 200 100 25 500

a The concentration of anticancer drugs used in the measurement of the IC50 values.

Chemosensitivity was usually expressed as the IC50 values. Therefore, the IC50 concentration for anticancer drugs in 2D culture system was first determined. For this measurement, MKN28 cells were cultured in a 24-well microplate without using nanofiber scaffolds during 24 h for preculture and following 24 h for the treatment of anticancer drugs. The IC50 concentration of anticancer drugs was in the range from 25 to 500 ␮g/ml (Table 1). Among them, cisplatin showed the greatest inhibition activity against the growth of gastric cancer cells. In the case of a chemosensitivity test in the 3D culture system using nanofiber scaffolds, a higher concentration of anticancer drugs was needed to arrest the growth rate to a similar extent as compared to that observed in the 2D culture system. Even though the IC50 concentration of drugs determined in 2D culture was used, the inhibition activity scarcely reached 50% after 24 h of drug addition except for paclitaxel and irrinotecan, which was ranged from 7 to 35% (Fig. 8). Moreover, cisplatin exhibited the lowest inhibition activity in 3D culture system. However, the inhibition activity increased with time in all cases. These data clearly show the increased resistance of cancer cells to anticancer drugs in the 3D culture system when compared to the 2D culture system. Structural architecture in a 3D tissue model regulates differentiated cell functions through changes in cell shape, as well as increased cell–cell and cell–matrix interactions. These intrinsic changes in cell function profoundly affect the response of a tissue model on external drugs [37]. Therefore, cell growth on 3D nanofiber scaffolds exhibits a higher drug resistance as a multicellular tumor. This resistance was not a result of mass transport effects but was an intrinsic property of the cells themselves. In 3D cultures, transport limitations generate an in vivo-like response to external drugs by restricting accessibility and including cellular heterogeneity.

Fig. 8. Chemosensitivity of various anticancer drugs for MKN28 cells cultured on nanofiber scaffolds.

4. Conclusions 2D culture systems allow the study of cell biology with regards to cell differentiation, growth and function, however, they can not provide evidence for the cell–cell and cell–extracellular matrix (ECM) interactions that modulate key processes implicated in the architecture of normal and cancer tissues. One in vitro approach to the analysis of the cell interactions in a system that retains the architecture of tissue is the organ culture. However, long-term organ culture is compromised by gas and nutrient exchanges to maintain the cell viability throughout the tissue mass. A second in vitro approach is to grow disaggregated cells to high density within a 3D reconstituted ECM that closely simulates the geometric microenvironment of tissues in the body. In the present study, PHBV and PHBV/CP nanofiber scaffolds produced by the electrospinning process were introduced for the chemosensitivity test of anticancer drugs. The properties of nanofiber scaffolds were strongly influenced by the blending ratio between PHBV and CP. It was found that PHBV/CP73 nanofiber enhanced the cancer cell proliferation compared to other nanofiber scaffolds, which allowed the 3D culture of gastric cancer cells. Interestingly, cancer cells showed the increased resistance to anticancer drugs in 3D culture when compared to 2D culture, which may be due to their 3D features. It is concluded that the 3D culture system offers a new approach for in vitro screening of anticancer drugs.

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