INTRODUCTION
In vitro culture
of cells is traditionally done on two–dimensional (2–D) surfaces of tissue
culture plastic. But in recent times, three–dimensional (3–D) scaffold systems
are mostly preferred because of their wide application in biomedical and
biological sciences. This is mainly due to the fact that 3–D culture systems
using scaffolds can influence the physical, chemical and biological environment
surrounding a cell population and their porous structures can be used to
sustain cell adhesion/ attachment and proliferation (Voytik–Harbin, 2001; Yang
et al., 2002; Zhu et al., 2002; Cui et al., 2003, Carletti et al., 2011).
Further, 3–D cell culture mimics the in vivo microenvironment and
increase cell–cell interactions compared to 2–D culture (Harrington et al.,
2013). Several types of synthetic
biodegradable polymers such as Poly–L–lactic–acid–biopolymer (PLLA), poly (lactic–co–glycolic) acid
(PLGA), Poly–caprolactone (PCL),
Polybutylene terephthalate (PBT)
etc. are used as porous scaffolding materials in different systems.
Amongst, poly–lactic acid (PLA), polyglycolide (PG), and their co–polymers are
most widely used in the fabrication of polymeric scaffolds (Mikos et al., 1994a
& b; Murphy et al., 2002; Sachlos and Czernuszka, 2003, Rasal et al., 2010)
because of their biocompatibility and biodegradability (Mikos et al., 1993;
Mooney et al., 1995; Mooney, 1999). However, the polymeric matrix composition
and structures must be considered carefully when designing a 3–D cell culture
system (Voytik–Harbin, 2001; Chung and King, 2011) so that they provide
adequate mechanical strength and hydrophilicity.
Like its application in other
fields of biological sciences, this technology also has tremendous potential in
fishery science. This is mainly due to the requirement of short term and continuous
cell cultures from economically important fish species for their application in
various in vitro studies. Further, most of the primary organ cells are
anchorage dependent and require specific environments that very often include
the presence of a supporting material to act as a template for cell growth
(Yang et al., 2002). But primary culture is mostly done in 2–D environments in
which variable culture conditions and choice of methods, such as procedure for
enzymatic digestion and coating of culture vessels, often affect the quality of
the primary culture (Garrick, 2000; Mothersill et al., 1995) resulting in low
proliferation and growth of cells in fish model. Thus, frequent preparation of
cell cultures is necessary, if primary cells are routinely used for
experimental purposes. As a result of the inadequacy of 2–D cell culture, there
is a growing interest in culturing adherent eukaryotic cells in 3–D culture.
3–D cultures within a polymeric matrix (also termed scaffold) are proposed as a
better model to study complex biological processes than cells grown monolayer
(Abbott, 2003), as they mimic properly the in vivo tissue state.
As scaffolds provide the
structural support for cell attachment and subsequent proliferation beyond any
doubt and have certainly huge scope in fish cell culture practices, the
hypothesis of our current experiment is to use polymeric scaffolds for
evaluating their efficacy in terms of adhesion, growth and proliferation of
fish cell. In this regard, we have formulated and screened PLA scaffold blended
with four different natural polymers such as alginate (PLA–A), dextran (PLA–D),
gelatin (PLA–G) and pectin (PLA–P) to find out a suitable polymeric scaffold by
evaluating the adhesion, attachment, growth and proliferation of anchorage dependent fibroblast–like cells
from heart of Indian Major Carp, Cirrhinus mrigala.
MATERIALS AND METHODS
Materials
Used
PLA with intrinsic viscosity 0.17
dL/g was purchased from Birmingham Polymers, Inc. (Birmingham, AL). Poly vinyl
alcohol (PVA) (average MW 30,000–70,000 Da), bovine serum albumin (BSA,
Fraction V), sucrose, chitosan from crab shells (85% deacetylated), alginate, dextran,
gelatin and pectin were purchased from Sigma Aldrich (St. Louis, MO, USA).
Formulation
of Scaffold
Natural polymer blended PLA
scaffolds (PLA–A, PLA–D, PLA–G and PLA–P) were prepared by the multiple solvent
evaporation method with slight modifications (Sahoo et al., 2005). In brief, 800 ml
of aqueous phase (W1) was emulsified into the polymer solution (O)
(200 mg of PLA polymer in 4 ml of dichloromethane) using a homogenizer
(Biospacte Product Inc, Bartlesville, OK) at 121 × g to form a primary (W1/
O) emulsion. The internal aqueous phase (W1) contained 2.5 % w/v
BSA, sucrose (10 % w/v), chitosan solution (1.25 % in 0.01 % v/v lactic acid), PVA (5 % w/v) and any one of the
natural polymer from alginate, dextran, gelatin and pectin (5 % w/v). BSA was
added into the internal aqueous phase to enhance stability of the primary
emulsion. The primary emulsion was added drop wise into a 1 % w/v aqueous
solution of PVA containing 10 % w/v sucrose (W2) under constant
magnetic stirring on a stir plate to form a multiple emulsion (W1/O/W2).
The emulsion was stirred overnight on a magnetic stir plate to evaporate the
organic solvent. Scaffolds were recovered by centrifugation at 27,377 × g,
washed three times with distilled water, and then lyophilized for 48 h
(LABCONCO Corporation, USA) to get the lyophilized powder for further study.
Characterization of Scaffold
The shape and surface morphology
of scaffolds were characterized by scanning electron microscopy (SEM). The
powdered scaffolds were stuck on to a brass stub through double adhesive tape
and gold–coated using a sputter gold coater at 20 KV (Hitachi, E–1010, Ion
Sputter). The stub was fixed into the sample holder and placed in vacuum
chamber of SEM (Hitachi S–3400N, USA) and observed under low vacuum. The
average particle diameter was determined from the SEM pictures of different
formulations of scaffold. To calculate the mean particle diameter, 50 particles
were taken for measurement.
Swelling Property
The swelling properties of the
different formulated scaffolds were determined by incubating them in phosphate
buffered saline (PBS, pH 7.2) at room temperature. A known weight (Wo) of the
individual scaffold formulation (in triplicate) was placed in 5 ml PBS for 5 h.
The wet weight (Wt) of the scaffolds was determined immediately after the
surface–adsorbed water was removed by filter papers.
The PBS uptake ratio of the
individual scaffold formulations (Pa) was calculated as per the following
formula–
Pa= (Wt–Wo) / Wo
Primary
Culture of Heart Explants of Cirrhinus mrigala
Primary culture was obtained from
explants of the heart tissue of C. mrigala fingerlings following the
method described by Nanda et al. (2009). Briefly, the heart tissue of C.
mrigala fingerlings (45 ±
5.0 gm) was excised aseptically and
immersed immediately in Dulbecco's Modified Eagle's Medium (DMEM).
Prior to cultivation, the tissues were minced to a size of approximately 1–2 mm3
and repeatedly washed with DMEM. Explants of heart tissues were
distributed over 25 mm2 flasks pre–coated with 0.5 ml gelatin (2 %
w/v) and kept for 3 h under semi–dried condition. After incubation, the flasks
were fed with 5 ml of DMEM growth medium supplemented with glutamine (0.3 %
w/v), non–essential amino acids solution (100 X), 10% fetal bovine serum (FBS)
and antibiotic–antimycotic solution (Sigma–Aldrich, St. Louis, MO, USA) and
incubated at 26 ± 1ºC. The
flasks were routinely observed for growth, proliferation and formation of
monolayer under phase–contrast microscope (Olympus, Japan). The
fibroblast–like cells from the explants started proliferating within 3–4 d and
reached confluency (80–90 %) after 12–14 d. Sub–culture was done as per the method described by Wolf and Quimby (1976) using
non–enzymatic cell dissociation solution (Sigma, USA) and the total viable
cells were counted by trypan blue dye exclusion method.
Effect
of Various PLA Scaffold Formulations on Growth of Fibroblast–Like Cells
The following protocol was used to
determine the suitability of various scaffold formulations on adhesion and
growth of fibroblast–like cells from heart tissue of C. mrigala.
Individual scaffold formulation was separately soaked in 70% alcohol (2 mg ml–1)
for overnight at 4 0C and centrifuged at 12,167 × g for 10 min at 4 0C.
Supernatant from each sample was discarded and different scaffold formulations,
each in triplicate, were aseptically transferred to six well plates using 1 ml
of DMEM. Plates without having any scaffold formulations were taken as control
(2–D) culture. Fibroblast–like cells obtained from
sub–culture of heart tissue was
added in each well at a seeding density of 2.0 × 105
cells ml–1 media supplemented
with FBS and antibiotic–antimycotic solution as described previously. Plates
were incubated at 26 ± 10C replacing medium on every alternate day.
The effect of various scaffold formulations on cell growth kinetics was
observed over one week.
Based on the preliminary screening
of different scaffold formulations, the best formulation (PLA–A) was processed
further to evaluate the effect of different seeding density on growth and
proliferation of fibroblast–like cells from heart of C. mrigala.
Effect
of PLA–A on the Growth and Proliferation
of Fibroblast–Like Cells at Varying Seeding Density
Table
1: SEM Size of Scaffold (µm) and
different PBS uptake ability of different formulations of PLA scaffolds
(Data as mean ± SEM, n=3)
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The effect of PLA–A on the growth
and proliferation of fibroblast–like cells from heart of C. mrigala at
varying seeding density (0.2 × 10 5 to 1.6 × 105 cells ml–1
media) was studied. Cells were seeded on to six well plates and added with
constant amount of PLA–A (2 mg ml–1 media) and the growth kinetics
was studied up to 9th day post seeding. Similarly, normal 2–D culture study was also
conducted by seeding fibroblast–like
cells from heart of C. mrigala @
2.0 × 105 cells ml–1
media in six well plates without addition of scaffolds to compare the growth. Scaffolds were
aspirated from the culture plates at different time points (3rd, 5th,
7th and 9th day) to determine the cell growth kinetics of
proliferating cells. Photomicrographs
of selected scaffold adhering cells were taken with an inverted microscope
(Olympus, Japan). The total number of cells in all the scaffold clumps was
determined following the method
described by Dhiman et al. (2004). Briefly,
the cells from individual scaffold clump was first detached by treating to cell
adhering scaffolds with 1 ml of 0.1 M citrate buffer
RESULTS
Formulation and Characterization of PLA Scaffold
In this study, four different
natural polymer blended PLA scaffolds such as PLA–A, PLA–D, PLA–G and PLA–P
were prepared by using multiple emulsion solvent evaporation method. In all the formulations of scaffolds, PVA was
used in the external aqueous phase (W2) as an emulsion stabilizing
agent and also, it was used in the internal aqueous phase (W1) of
the primary emulsion. The overall objective was to formulate scaffold with PVA
incorporated in the internal matrix structure of the scaffold as PVA in the
internal matrix has a better wetting property than scaffold without PVA (Sahoo
et al., 2005). The SEM pictures (Figure 1a–d) have confirmed the four
formulations of PLA scaffold showing a spherical shape with rough surface and
covered with a thin film of polymer. The average diameter of different
formulations of scaffold prepared in this study ranged from 145 μm
to 162 μm (Table 1).
DISCUSSION
A paradigm shift is taking place
to culture animal cells in 3–D within a polymeric matrix replacing the 2–D cell
culture systems. Out of several polymers that are being used, PLA is the most commonly used because of its
biocompatibility as well as high mechanical strength that maintains the matrix
porous structure for cell migration and growth (Stock and Meyer, 2001, Cheng et al., 2009; Madhavan et
al., 2010). Further, PLA
polymers are used due to their relatively slower degradation rates as compared
to other synthetic polymers (Wang et al., 2005). As PLA polymer is hydrophobic, coating or blending these polymeric
scaffolds with hydrophilic polymers promotes cell adhesion (Lavik et al., 2002).
Further, till date no information
is available on the use of scaffolds for evaluating their suitability in terms
of adhesion, attachment, growth and proliferation of anchorage dependent
fibroblast-like cells in fish cell culture. Keeping all these in mind, we
fabricated porous PLA scaffolds blended with hydrophilic polymers of natural
origin such as alginate (PLA-A), gelatin (PLA-G), pectin (PLA-P) and dextran
(PLA-D) for this study. Further, PVA and chitosan were included in internal
phase to promote adhesion. As PVA is amphiphilic in nature, the anchored PVA
could have provided functional OH groups that have been reported as being
helpful in cell attachment (Yang et al., 2002). In this study, chitosan was
incorporated in the internal matrix structure for better cell growth of
fibroblast–like heart cells of C. mrigala. Earlier, Chuang et al. (1999) have demonstrated
that PVA membranes modified by blending with chitosan promote fibroblast
adhesion.
The SEM studies have confirmed the
four formulations of PLA scaffold showing a spherical shape with rough surface
and covered with a thin film of polymer (figure 1). This rough surface could be
due to adsorb property of chitosan onto the matrix interface because of its
ionic interaction, as the scaffold polymer interface is anionic and chitosan is
cationic (Sahoo et al., 2005). Moreover, the ability of chitosan to support
cell attachment and survival has been attributed to its chemical structure,
which resembles glycosaminoglycans, an extracellular matrix component (Mei et
al., 2005). Further, sucrose, which is
known to increase the surface tension of water (Lee and Timasheff, 1981), was
used in the internal phase of the primary emulsion. This must have contributed
in formation of larger water droplets and formed large void structures inside
its matrix upon leaching out from scaffold materials. The porous matrix
structure could have provided a greater surface area to promote cell adhesion,
proliferation and growth as found in our study. Similarly, Particle size
is an important parameter that could affect the degradation of the polymer
matrix (Dunne et al., 2000). Therefore, to achieve scaffolds of desired
diameter, the condition for emulsification and formulation composition was
optimized.
The PBS uptake ability of
polymeric scaffold mainly depends on the three–dimensional structure and
hydrophilic nature of scaffold. It is very crucial to notice that the PBS (in
replace of media) should adsorb properly into the scaffold matrix for influencing
the biological activity of scaffold (Tan et al., 2007). Swelling of scaffold in
culture condition increases the pore diameter, thus facilitating the cell not
only to adhere but also to migrate in to the scaffolds; thus providing the
scaffold the maximum internal surface area (Zhang and Zhang, 2001). The PBS
uptake studies show that all the formulations exhibited good swelling capacity
and retained more PBS than their body weight. However, PLA–A scaffold
formulation showed more PBS retention as compared to other formulations. This
variation in the extent of swelling and water retention ability could be due to
the difference in hydrophilicity of the polymer chains and the cross–linking
density (Xu and Burg, 2007).
PLA blended natural polymeric
scaffolds were evaluated to find their suitability for in vitro growth
of fibroblast–like cells. Fibroblast–like cells were chosen because of their
high in vitro replication capacity and therefore, can be considered as
an effective tool for preliminary studies of cell adhesion and growth onto a
scaffold (Lazzeri et al., 2007).
In our study, we observed that the cells seeded on PLA–A scaffold demonstrated
better cell growth after 7th day post seeding as compared to other
formulations as well as control. This could be due to the structural topography
(many hydrophilic groups) and good medium retention ability of alginate.
Because of this, the 3–D network of PLA–A could have retained large volume of
nutrient medium and supported good swelling structure as observed by Peppas and
Sahlin (1996) thus facilitating better proliferation of fibroblast–like cells.
Cell proliferation may differ
depending on seeding density and scaffold type used. To ascertain this, PLA–A
scaffold was taken to evaluate the effect of different seeding density on
growth and proliferation of fibroblast–like cells since maximum proliferation
was found in the above formulation. At lower initial cell seeding density (0.4
to 0 .6 × 10 5 cells ml–1 media), a lag phase in cell
growth was recorded. This could be due to slow growth of cell for less
cell–cell contact (Sahoo et al., 2005). Similarly, at high seeding density (1.6
×10 5 cells ml–1 media), slow growth pattern was
observed. This is in agreement with van Gaalen et al (2010) who also recorded
that seeding more cells may not necessarily result in more proliferation. As
with high seeding density, the scaffold surface might have been covered to its
saturation capacity, and hence further cell growth may have been limited. The growth kinetics studies demonstrated that
a seeding density of 0.8 to 1.6 × 10 5 cells ml–1 media
showed good trend of proliferation compared to others. Hence, a substantial
influence of the seeding density on growth and proliferation of fibroblast–like
cell was observed on our formulated PLA–A scaffold as compared to 2–D culture
(control).
This
study revealed that among the four scaffold formulations, PLA–A could be
potentially used for in vitro culture in achieving better growth and
proliferation of anchorage dependent fibroblast–
like cells from heart of Indian Major Carp,
C. mrigala compared to 2–D cell culture. This opens up new possibilities
for studying other complex biological processes in fish using 3–D culture.
ACKNOWLEDGEMENTS
The authors are
thankful to the Director, Central Institute of Freshwater Aquaculture (ICAR),
Kausalyaganga, Odisha, India and the Director, Institute of Life Sciences (DBT)
for providing necessary facilities to carry out the present investigation.
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Research
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