Characterization and evaluation of chitosan matrix for in vitro growth of MCF-7 breast cancer cell lines
Harpreet K. Dhiman
a,
b,*, Alok R. Ray
a,
b, Amulya K. Panda
ca Centre for Biomedical Engineering, Indian Institute of Technology, New Delhi-11 0016, India
b All India Institute of Medical Sciences, New Delhi-110029, India
c National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India Received 20 July 2003; accepted 8 December 2003
Abstract
Biodegradable polymer scaffolds were prepared from chitosan with varying degree of deacetylation for in vitro culture of human breast cancer MCF-7 cell lines. These polymers were characterized in terms of functional groups by FTIR and swelling properties.
Polymers having high degree of deacetylation showed better swelling properties irrespective of the molecular weight. These polymers were biocompatible and non-toxic towards human epithelial MCF-7 cell lines. Attachment kinetics of MCF-7 cell lines on to polymer scaffold was investigated and it was observed that polymer having high degree of deacetylation favored better cell attachment. In CPIII polymer scaffold having 80% degree of deacetylation, a maximum of 1 millions cells per mg pf polymer were adsorbed within 1 h. It appears that high swelling and high degree of deacetylation of chitosan helped in better adsorption of cancer cell lines. The cellular morphology of the attached cells on chitosan matrix was similar to that observed with regular plastic culture with the difference that, cells grew as three-dimensional clumps on chitosan matrix. Polymer having high degree of deacetylation not only favored better adsorption but also showed improved cell growth kinetics. Maximum cell concentration of 6.5 x 105 cells/ml was achieved in 5 days culture on CPIII polymer scaffold. The glucose consumption and lactate production pattern of the MCF-7 cell lines on chitosan polymer matrix were similar to that observed on cell growth on tissue culture flask. These results indicate that chitosan scaffold having high degree of deacetylation can be used for three-dimensional growth of MCF-7 cancer cell lines. Such in vitro 3D culture of cancer cells can thus be used as a model for the cytotoxic evaluation of anticancer drugs.
Keywords: Cell attachment; Chitosan; Degree of deacetylation; Growth kinetics; MCF-7 cell lines
1. Introduction
Scaffolds of biodegradable materials find numerous applications in tissue repair and regeneration [1]. Besides this, DNA delivery from polymers is currently being applied to the multidisciplinary science of gene therapy and tissue engineering. Advancements in technology have made it possible to use synthetic (non-viral) delivery systems for cancer gene therapy. The tissue engineering approach to repair and regeneration is founded upon the use of polymer scaffolds, which serve to support, reinforce and in some cases organize the
regenerating tissues [2-7]. Growth of cells on polymer scaffold utilizing the principles of tissue engineering provides a viable alternative to the in vivo model for biological experimentation. In vitro grown cells in polymer scaffold in three-dimensional culture provide a better alternative model than the conventional monolayer flask culture for understanding animal cell biology. Further culture of cells in three-dimensional polymer scaffold resembles very close to the in vivo tissue situation and thus is a better model for the pharmacological evaluation of therapeutics. The scaf- fold may be required to release bioactive substances at a controlled rate or to directly influence the behavior of incorporated or ingrowing cells. Desirable aspects of scaffold chemistry may include specific interaction with, or mimicry of, extra-cellular matrix components, growth factors, or cell surface receptors [8]. Several recent studies have demonstrated greater cell attachment and
cell spreading on hydrophilic, positively charged amine- modified surfaces relatively to hydrophobic surfaces, both in the presence and absence of serum [9-11].
Whereas much attention has focused on understanding the effect of surface properties such as surface charge and energy in cell attachment, relatively little work has been done to elucidate the role of surface morphology on cell behavior [12,13,14].
A number of natural and synthetic polymers are currently being employed as tissue scaffolds [15-16].
Most synthetic biodegradable materials such as poly- glycolides and polylactides have high strength, while natural products like collagen are low in mechanical strength but exhibit excellent cell adhesion [1]. Since the range of potential tissue engineered systems is broad, there is a continuous ongoing search for materials which either possess particularly desirable tissue specific properties, or which may have broad applicability and can be tailored to several tissue systems. The amino polysaccharide chitosan (poly 1,4 d-glucosamine) is one such broadly applicable material. Chitosan is a partially deacetylated derivative of chitin [17,18], the primary structural polymer in arthropod exoskeletons. Chitosan is a natural cationic linear polymer that has recently emerged as an alternative non-viral gene delivery system. Chitin, a natural polymer, is the second most abundant organic resource on the earth next to cellulose [17]. Chitin is an inert polymer but deacetylation of chitin yields chitosan, which is relatively reactive and can be produced in numerous forms such as powder, paste, film, fiber, porous scaffold [8,18], etc. Both chitin and chitosan are copolymers of b; ( 1 - 4 ) linked N- acetyl-d-glucosamine and d-glucosamine units. The proportion of N-acetyl-d-glucosamine units in total number of units determines the degree of deacetylation.
The degree of deacetylation has an inverse relationship with the number of N-acetyl-d-glucosamine units, thus deacetylation of chitosan is achieved by removing N- acetyl group. In chitosan, the degree of deacetylation is more than 50%. This value also determines the solubility limit of polymer in dilute acidic acid solutions ( 2 o p H o 6 ) . The degree of deacetylation is a structural parameter, which influences physicochemical properties such as the molecular weight, the elongation at break and the tensile strength. It also influences biological properties, namely the biodegradation by lysozyme, the wound-healing properties and the osteogenesis enhance- ment.
Chitosan and some of its complexes have found numerous biomedical applications. These include wound dressing [19], drug delivery systems [20-22], bioactive coating [23] and space filling implants [24].
Advantages of chitosan as 3D scaffold for cell culture include biocompatibility, biodegradability, low immu- nogenicity, and low cost. However, little has been done to explore the use of chitosan within tissue engineering
paradigm. Since Chitosan appears to be a good candidate to act as a biodegradable material for polymer scaffold and tissue engineering, it seemed interesting to investigate the possibility of tissue culture on chitosan polymer scaffold. In this study, we considered the preparation and characterization of polymer scaffold with different degrees of deacetylation for the growth of MCF-7 breast cancer cell lines. MCF-7 breast cancer cells serve as an excellent in vitro model for studying the mechanism tumor response to endocrine therapy as well as complex relationships between binding and biological actions of the hormones [25]. The attachment and growth kinetics of MCF-7 cell lines on chitosan polymer scaffold were studied and the possibility of growing MCF-7 cell lines in three-dimensional form has been discussed.
2. Materials and methods
2.1. Preparation of chitosan
Chitin, from prawn shells, was obtained from Central Institute of Fisheries Technology, Cochin, India. Chit- osan was prepared from chitin by deacetylation process using 50% (w/w) sodium hydroxide solution at 110°C for different intervals of time (Table 1). After this treatment, flakes separated from alkali layer were extensively washed with MQ water to remove the traces of alkali. The resulting flakes were dried in a vacuum oven at 50°C for 72h. Chitosan flakes were dissolved in 1% aqueous acetic acid solution and filtered through a sintered glass filter. Chitosan was precipitated from the resulting solution with 10% aqueous sodium hydroxide solution. The precipitate was washed several times with MQ water to remove the traces of alkali. Chitosan flakes were purified using the method described by Muzzarelli et al. [10] by Soxhlet extraction with methanol, MQ water, petroleum ether and acetone in that order, each for 24 h. Finally, chitosan powder was dried in vacuum oven at 50°C for 20 h and stored in desiccator. During precipitation by NaOH, a homogenous product was obtained as supported by IR data given in Table 2.
Table 1
Physicochemical characteristics of different chitosan samples Chitosan
sample
CP-I C P - CP-III
Preparation time (h)
2 3 5
Degree of deacetylation (%) (IR method) 59.372.5 69.0 7 2.0 80.9 7 2.7
Molecular weight x 10~4
(Da)
47.6 7 0.6 41.8 7 0.8 28.5 7 0.5
Degree of swelling (Q)
0.76 7 0.027 0.85 7 0.028 0.95 7 0.026 Data represents mean7maximal errors in three experimental readings.
Table 2
Characteristic absorption bands as seen in FT-IR spectra of different chitosan preparations used for the culture of MCF-7 cell lines Chitosan sample Characteristic
absorption band (cm"1)
Presence of group
CP-I CP-I CP-I CP-I C P U C P - C P - C P - CP-III CP-III CP-III CP-III
3442 2923 1653 1375 3430 2922 1653 1378 3420 2921 1653 1375
-OH group -CH2 group -C Q O group
-C-O stretching of-CH2-OH -OH group
-CH2 group -C Q O group
-C-O stretching of -CH2-OH -OH group
-CH2 group -C Q O group
-C-O stretching of -CH2-OH
2.2. Preparation of porous chitosan micro-carriers For preparation of gelled chitosan micro-carriers, droplets of a 1 wt% chitosan solution of different degrees of deacetylation were collected from a 23-gauge needle in stirred 0.1 M NaOH to induce gellation. After rinsing briefly with MQ water, the gelled chitosan beads were then transferred to freezing bath. The gelled micro- carriers were cooled to —78°C, lyophilized [12]. The chitosan micro-carriers of different degrees of deacetyla- tion were sterilized by autoclaving micro-carriers in PBS for 15min at 121°C.
The size of chitosan micro-carriers for chitosan samples of different degrees of deacetylation (reported in Table 1) varied from 200-300 mm (200-230 mm for polymer sample CP-I, 240-280 mm for polymer sample CP-II, 270-310 mm for polymer sample CP-III). Gelled chitosan micro-carriers prepared with this method yield highly porous, fibrous micro-structure with effective pore diameters in 1-5 mm range [12]. The difference in micro-carrier size can be attributed to viscosity differ- ence in CP-I, CP-II and CP-III chitosan samples.
Viscosity measurements were performed to determine the molecular weight of chitosan samples. The mole- cular weight and hence viscosity of CP-I was highest (resulting in low micro-carrier size), followed by that of CP-II and CP-III (Table 1).
2.3. Characterization of chitosan 2.3.1. Infrared spectroscopy
Infrared spectra of chitosan powder were recorded on Fourier transform infrared (FTIR) spectrophotometer (Nicolet-5DX) in 4000-400 cm"1 ranges. The freeze- dried powders were dried over P2O5 in vacuum before recording spectrum .The degree of deacetylation was calculated by the base line method developed by Roberts
and Moore [26] using the relationship:
% Deacetylation = [1 - (Ai665/A3340) x (1/1-33)] x 100; where A is the logarithmic ratio of absorbance at 1665 and 3340 cm"1.
2.3.2. Determination of molecular weight
Viscosity measurements were performed to determine the molecular weight of chitosan samples using ubbel- hode viscometer (model Schott Gerate AVS-310) using 0 . 1 M acetic acid containing 0.2 M sodium chloride as solvent. Measurements were made at 25°C + 0.1 and flow times were reproducible within 0.01 s. The viscosity average molecular weight (Mv) of chitosan samples was calculated using Mark-Houwink equation:
M = KMva
where [t]] is the intrinsic viscosity of solution, 'K and V are constants and their values are at 25° C are 1.81 x l O ^ c n r ' g -1 and 0.93, respectively [27].
2.3.3. Swelling studies
The swelling of chitosan samples in phosphate buffer solution (pH 7.4, 0 . 0 5 M) was studied to determine the influence of alkaline pH on swelling behavior of the scaffold, until equilibrium was obtained. Measured amounts of chitosan scaffold were taken (Wd) and were immersed in the above buffer for 24 h. The swollen chitosan scaffold was centrifuged at 500 rpm in the presence of filter paper, which adsorbed the excess water from it. The centrifuged scaffold without the filter paper was weighed to get the swollen weight of the polymer
Ws).
The degree of swelling (Q) of these samples was calculated using the following equation:
Degree of swelling ðQ = (Ws - WdÞ=Wd;
where Ws and Wd are the weights of swollen and dry samples, respectively.
2.4. Cells
MCF-7 breast cancer cells were maintained in RPMI- 1640 supplemented with 10% FCS in T-flask in an incubator kept at 37°C and 5% CO2. Chitosan polymer scaffold was inoculated with MCF-7 cells and percent viability was calculated for 6 days to check biocompat- ibility and toxicity of the polymer preparation.
2.5. Cell attachment kinetics on chitosan polymer scaffolds
Sterilized chitosan micro-carriers (CP-I, CP-II and CP-III) were incubated in RPMI-1640 supplemented with 50% FCS for 3h. After incubation, the micro- carriers were centrifuged at 800 rpm for 10min and the
supernatant was discarded after centrifugation. The pellet of micro-carriers was inoculated with MCF-7 breast cancer cells. Typically 1 mg of polymer and 1 ml of medium were taken in 15 ml sterile tubes for all micro-carrier samples. 2 x l 05c e l l s , 3 x 105 and 4 x l 05c e l l s were added to CP-I, CP-II and CP-III micro-carriers, respectively. The cells were allowed to attach in static condition. Cells were kept at 37°C in CO2 incubator for attachment. At different time intervals the solution was centrifuged and cell concen- tration in polymer was counted. MCF-7 cell attachment kinetics was estimated by measuring the unattached cells concentrations as a function of time after inoculation.
Haemocytometer was used for counting unattached cells.
2.6. Capacity determination and inoculum
standardization of CP-III chitosan polymer scaffold Capacity of polymer is defined as number of cells attached per mg of polymer. CP-III chitosan micro- carriers were sterilized and incubated in RPMI-1640 supplemented with 50% FCS for 3h. After incubation, the micro-carriers were centrifuged at 800 rpm for 10min and the supernatant was discarded after cen- trifugation. Different concentrations of cells were inoculated per milligram chitosan micro-carrier per ml media in 15 ml sterile tubes (Table 3). The cells were allowed to attach in static condition. Cells were kept at 37°C in CO2 incubator for attachment for 1 h.
Unattached cells were counted using trypan blue dye after 1 h on haemocytometer.
For inoculum standardization, different cell concen- trations of 0.5 x 105, 1.0 x 105 and 2.0 x 105 cells per milligram chitosan micro-carrier per ml media were taken in 15 ml sterile tubes (Table 3). A total of 2mg of polymer and 2 ml of medium were taken in sterile tubes.
The cells were allowed to attach in static condition. Cells were kept at 37°C in CO2 incubator for attachment for 1 h. After cell attachment, the micro-carriers were transferred to a six well plate and kept at 37°C in a humidified 5% CO2 environmental incubator. Cell enumeration was done every day for 6 days. For cell
Table 3
Evaluation of cell attachment capacity of MCF-7 cells on CP-III chitosan scaffold
Cell/mg of polymer per ml of medium
5.0 x 106
2.5 x 106
1.0 x 106
0.5 x 106
0.25 x 106
Unattached cells (1 h incubation)
3.2 >
1.6 >
0.2 >
0.1 >
0.06 >
< 106
< 106
< 106
< 106
< 106
Attached cells (1 h incubation)
1.8 x 106
0.9 x 106
0.8 x 106
0.4 x 106
0.19 x 106 Data represents mean values of three experimental readings.
enumeration, 1 ml of sample was taken out in a centrifuge tube, and centrifuged at 800 rpm for 15min.
The micro-carrier pellet was suspended in 1 ml of 0.1 % (w/v) crystal violet in PBS containing 0 . 1 M citric acid.
The sample was incubated at 37°C for 60min. The suspension was then sheared with a Pasteur pipette to detach the nuclei from the micro-carriers. The nuclei were then counted on haemocytometer.
2.7. Growth kinetics of MCF 7 cells on different chitosan scaffolds
Chitosan micro-carriers (CP-I, CP-II and CP-III) of all polymer samples were sterilized and incubated in RPMI-1640 supplemented with 50% FCS for 3 h. Then, micro-carriers were centrifuged at 800rpm for 10min and the supernatant was discarded after centrifugation.
The pellet of micro-carriers was inoculated with MCF-7 breast cancer cells. Typically 2mg of polymer and 2ml of medium were taken in 15 ml sterile tubes for all micro-carrier samples of different degrees of deacetyla- tion. Each sample was inoculated with 2 x 105 cells. Cell concentration was around 1 x 105 cell/ml of the culture medium. The cells were allowed to attach in static condition. Cells were kept at 37°C in CO2 incubator for attachment for 1 h. After cell attachment, the micro- carriers were transferred to a six well plate and kept at 37°C in a humidified 5% CO2 environmental incubator.
Cell enumeration was done every day for 6 days by the method described earlier. 1 ml of sample was taken out in a centrifuge tube, and centrifuged at 800 rpm for 15min. The supernatant media was kept for glucose and lactate estimation. Glucose and lactate concentrations in the culture supernatants were determined by means of enzymatic assays, in which the changes in optical density were measured and compared with those of standard solutions using a spectrophotometer.
3. Results and discussion
3.1. Physicochemical characterization of chitosan The physicochemical characteristics of different Chit- osan samples are reported in Table 1. The degree of deacetylation affects brittleness of the polymer, more the degree of deacetylation, lesser the brittleness of the polymer. This is an important factor for cell culture studies as it influences cell adhesion. The cell adhesion increases with increase in degree of deacetylation as reported in the cell attachment kinetics. The degrees of deacetylation of different chitosan samples were calcu- lated from FT-IR spectra of chitosan samples and are presented in Table 1. Functional groups present in different chitosan formulations are present in Table 2.
The presence of significant peaks at 1653 cm l in all samples (Table 2) due to CH3-C Q O group denotes the presence of acetyl group. Peaks at 3442, 3430, 3420 cm"1 (Table 2) in CP-I, CP-II and CP-III samples represent the presence of -OH group. These peaks confirm that chitosan is a partially deacetylated product. The proper- ties like solubility, viscosity, etc. of polymer is largely dependent on the molecular weight. The molecular weight of chitosan influences the properties like solubi- lity and viscosity and thus plays a key role as carrier for cell culture. The molecular weight falls as the degree of deacetylation increases.
Besides, the ability of scaffold to swell plays an important during the in vitro culture studies. This is because when the micro-carriers have swelling capabil- ity, they allow their pore size to increase in diameter in order to swell. This facilitates the cells not only to just attach but also allows the cells to penetrate inside the micro-carriers to grow in a three-dimensional fashion, during in vitro culture studies. Increase in pore size also allows cells to avail the maximum internal surface area of the micro-carriers. Phosphate buffer of pH 7.4 was selected as the swelling agent because the medium used for in vitro culture studies is of slightly alkaline pH. The sample with the highest degree of swelling will have the highest surface area/volume ratio thus allowing that sample to have maximum cell growth in a three- dimensional fashion. The increase in degree of swelling also allows the samples to avail nutrients of the culture media more effectively, during in vitro culture studies. It was observed that chitosan having high degree of deacetylation has high swelling properties. CPIII chit- osan preparation having 80% degree of deacetylation showed 90% swelling in comparison to 70% swelling shown by CPI polymer (Fig. 1). As degree of deacetyla- tion and swelling properties influences the cell attach- ment on to the polymer surface, it controls the overall growth on polymer matrix. Thus, chitosan having different degree of deacetylation were used for cell culture.
3.2. Biocompatibility
Cell attachment to the substratum is almost always mediated by extra-cellular matrix (ECM) proteins adsorbed on the culture surface. ECM proteins are present in the serum, which are used in most cell culture applications. Surface properties can affect cell attach- ment by influencing the ability of the substratum to adsorb protein and/or by altering the conformation of the adsorbed protein. Some of these proteins which are present in serum, and the extra-cellular matrix, such as fibronectin, vitronectin, collagen, and laminin, contain specific amino acid sequences that bind to cell-surface integrin receptors and influence cell behavior and gene expression. Numerous studies indicate that surface wettability and charge influence the type, quantity, and conformation of the adsorbed proteins on material surfaces [28].
All three chitosan preparations showed excellent biocompatibility as MCF-7 cells could grow in presence of serum. The morphology of cells grown on chitosan scaffold were similar to that observed for cell growth of tissue culture flasks (Fig. 2). Prolonged exposure of cells to chitosan scaffold did not result in cell death or change in morphological nature. This is in accordance with the excellent biocompatibility of chitosan on different cell lines such as hepatocytes [29], chondrocytes [30] and keratinocytes [31]. These results also confirmed that degree of deacetylation has very little effect on biocompatibility of chitosan. Cells were healthy and showed similar metabolic activity in terms of glucose uptake and lactate secretion (Table 4).
3.3. Cell attachment kinetics
Proliferation of mammalian cells takes place in three stages, first the cells attach to a suitable matrix, then they spread and finally they divide in presence of
100 90 80 - 70 -
f 60-
% 50-
# 40 30 - 20 - 10
0
tut f
I II
o
CP-I Chitosan Microcarrier - CP-II Chitosan Microcarrier
CP-III Chitosan Microcarrier
3 4 5 Time in Hours
Fig. 1. Comparative graph showing the influence of phosphate buffer (pH 7.4) on the equilibrium swelling characteristics of various chitsoan micro-carriers (CP-I, CP-II and CP-III). Each point represents the mean of the results from three experimental readings. The error bars
denote7 SD (standard deviation for n = 3). Fig. 2. Photograph of MCF-7 cell growth on chitosan scaffold.
Table 4
Yield coefficient of glucose consumed to lactate produced during the growth of MCF-7 cells on chitosan scaffolds Days Lactate production (mg/ml) Glucose consumption (mg/ml) 1 Lactate=Glucose
CP-I C P - CP-III Control CP-I C P - CP-III Control CP-I CP-II CP-III Control
1.57 1.34 2.26 3.90 3.57
0.96 1.94 2.10 2.58 3.68 2.49
1.27 2.00 2.50 3.96 4.03 3.27
13.20 2.06 2.58 2.90 3.53 3.51
0.28 0.74 1.42 1.69 1.98 1.99
0.30 0.61 1.28 1.6 1.96 1.97
0.28 0.60 2.00 1.691 1.96 2.00
0.28 0.67 1.29 1.77 2.00 2.00
5.61 1.81 1.59 1.75 1.97 1.81
3.20 3.18 1.64 1.55 1.88 1.26
4.54 3.33 2.50
1.64
4.29 3.07 2.00 1.36 1.77 1.76 Data represents mean values of three experimental readings.
extra-cellular matrix and nutrients. It is thus essential to analyze the cell attachment kinetics with the chitosan scaffold to evaluate it suitability for cell culture. Among the three different types of chitosan matrix, CPIII polymer showed maximum cell adhesion in 1-h time despite having high initial inoculum of 0.4 x 106 cells/ml as compared to CP-I and CP-II (Fig. 3). Chitosan scaffold having low degree of deacetylation show lower adhesion properties in comparison to the polymer scaffold having high degree of deacetylation. The process of cell adhesion in matrix consists of cell attachment, filopodial growth, cytoplasm webbing, flattening of cell mass followed by ruffling of peripheral cytoplasm. CPIII polymer probably promoted all these features and thus resulted in higher adhesion of the MCF-7 cells. High degree of deacetylation and high swelling properties of chitosan have been reported to promote excellent cell growth of many cell lines [31].
High degree of deacetylation probably provides more cationic sides on the scaffold, which helps in stronger electrostatic interaction with the negatively charged surface of MCF-7 cell lines. This is in accordance with the published information indicating the suitability of high degree of deacetylation and high swelling proper- ties of CPIII polymer for better cell attachment. The highly deacetylated polymer CPIII was used for capacity determination and it was observed that approximately 1 x 106 cells per mg of polymer could be adsorbed (Table 3). Higher initial cell concentration helped in high attachment rate to the CP III polymer but we concluded that the approximate maximum adsorption capacity was around 1 x 106cells/mg of polymer. Rapid adsorption and non-toxicity of the cells on chitosan scaffold makes it more suitable for the growth of animal cells to a high concentration.
3.4. Growth kinetics of MCF-7 cell lines on chitosan scaffold
3.4.1. Inoculum standardization
Initial inoculum concentration plays an important role in growth of animal cells in vivo. This is because a minimum number of cells are always required to
0.45 0.4 0.35
CP-I Chitosan Matrix CP-II Chitosan Matrix CP-III Chitosan Matrix
30 45 Time (Minutes)
Fig. 3. Attachment kinetics of MCF-7 cell line on different Chitosan matrices (CP-I, CP-II, CP-III). Each point represents the mean of the results from three experimental readings. The error bars denote7 SD (standard deviation for n = 3).
promote cell to cell contact for initiation of cell proliferation. MCF-7 cells were grown on chitosan scaffold with varying initial cell concentrations and the growth kinetics was monitored (Fig. 4). Use of low initial cell concentration resulted in longer lag phase of growth. Low inoculum concentration (0.5 x 105 cells/ml) also resulted in cellular stress during the growth of cells on chitosan scaffold; hence, the extent of cell increase was 2-3 fold only. It was observed that high initial cell concentration of 2 x l O5 cell/ml resulted in about 4- 4.5 fold growth (increase in cell number) in 5 days.
Initial cell concentration of 1 x 105 cell/ml resulted in highest cell growth (6-7 fold increase in cell number) in 5 days. Thus, for all subsequent experiments on MCF-7 cell growth, initial cell concentration of 1 x 105 cell/ml were used which not only promoted good growth but was also found to be most suitable to do kinetics and anticancer drug effect studies.
3.4.2. Growth kinetics of MCF-7 cell lines
Growth kinetics of MCF-7 breast cancer cell lines was monitored using chitosan scaffolds having different degrees of deacetylation (Fig. 5). It was observed that in concomitant with the swelling properties and cell attachment studies, CPIII polymer promoted better cell
E 5
<j)
75 4 O
3
+ 0.5x100000 cells/ml 8 1 . 0 x 1 0 0 0 0 0 cells/ml + 2.0x100000 cells/ml
2.-.—
1 i
0 1 2 3 4 5 6
Time (Days)
Fig. 4. Inoculum standardization of the growth of MCF-7 cells on CP- III chitosan scaffold. Each point represents the mean of the results from three experimental readings. The error bars denote7 SD (standard deviation for n = 3).
"b 5 E 4
i
CP- I Chitosan Matrix CP-II Chitosan Matrix + CP-III Chitosan Matrix
Control (2-D Plate)
jr
y\—-^
/ / //
/ \ ///
///
//
\ \?^
\ \
\
Time (Days)
Fig. 5. Growth kinetics of MCF-7 cells on chitosan scaffolds (three- dimensional culture) and comparison to that of plastic culture plate (two-dimensional culture) i.e. control. Each point represents the mean of the results from three experimental readings. The error bars denote7 SD (standard deviation for n = 3).
growth amongst the other preparation. Maximum cell concentration of 6.8 x 105 cells/ml was achieved using CPIII polymer. Cells grew more slowly in comparison to the plastic culture even though the absolute cell concentration was higher. Longer lag phase was also observed during growth of MCF-7 cells on chitosan matrix. High growth of cells per unit volume of culture fluid indicates the suitability of scaffold-based culture for evaluation of pharmacological agents using MCF cell lines. Metabolic activities of MCF-7 cell lines in terms of glucose consumption and lactate production were monitored during the cell growth and are presented in Table 4. Cancer cell growth is always associated with high lactate production in growth medium and it was observed that irrespective of different types of chitosan scaffold, the lactate yield (Ylactate=glucose) was similar in all cases. Comparable growth kinetics and metabolic activities of MCF-7 cell lines in chitosan scaffold indicated that such matrix could be used efficiently for
three-dimensional growth of cancer cells. Growth on polymer scaffold of different dimension will help in initiating suspension culture of anchorage dependent cell lines such as MCF-7. Such matrix-type growth of cancer cell lines can be used as a better in vitro model for the screening and evaluation of anticancer drugs.
4. Conclusion
In this study, chitosan scaffolds of varying degree of deacetylation were prepared for three-dimensional growth of MCF-7 breast cancer cell lines. The objective was to develop and standardize polymeric scaffold of chitosan, so that these can be used for three-dimensional growth of cancer cell lines. With cells growing in the near tissue like morphology and structure on polymer scaffold, the chitosan scaffold can be used for cytotoxic evaluation of anticancer drugs. It was expected that cytotoxicity of the anticancer drugs would be very similar to that observed in vivo tissue condition due to three-dimensional nature of cell growth in chitosan scaffold. It was observed that chitosan having high degree of deacetylation promoted better cell growth.
MCF-7 cell growth was comparable to that observed with tissue culture flask with the added advantages of having three-dimensional growth. Cell growth in poly- mer matrix could also be helpful in its growth as suspension culture so that better control of environ- mental and culture parameters can be evaluated for understanding growth and metabolism. Metabolic activities of the MCF-7 cell lines in terms of glucose uptake and lactate production was comparable to that observed in cell culture with standard tissue culture flask. Growth of anchorage-dependent cell lines such as MCF-7 on chitosan matrix provides a better alternative to static culture experiments for the valuation of anticancer drug response. It is expected that by using cell culture models on polymer scaffold, the anticancer activities in terms of dose and action will be better understood than of two-dimensional culture in tissue culture flask. Such models can also be used to screen the activities of new drugs for anticancer effect more effectively. Easy availability, cheap source, simple preparation method and excellent biocompatibility of chitosan thus make it more attractive substratum for animal cell culture in vitro.
Acknowledgements
Authors are thankful to Dr. S.K. Basu, Director, National Institute of Immunology, New Delhi, India for providing cell culture facility to test the chitosan scaffold.
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