Abstract
Enzymatic detachment of cells might damage important features of cells
and could affect subsequent function of cells in various applications.
Therefore, non-enzymatic cell detachment using thermosensitive polymer
matrix is necessary for maintaining cell quality after harvesting. In
this study, we synthesized thermosensitive
PNIPAm-co -AAc-b -PS and PNIPAm-co -AAm-b -PS
copolymers and LCST was tuned near to body temperature. Then, polymer
solutions (5% w/v, 10% w/v, and 20% w/v) were spin coated to prepare
films for cell adhesion and thermal-induced cell detachment. The
apha-step analysis and SEM image of the films suggested that the
thickness of the films depends on the molecular weight and concentration
which ranged from 206 nm to 1330 nm for PNIPAm-co -AAc-b -PS
and 97.5 nm to 497 nm for PNIPAm-co -AAm-b -PS. The contact
angles of the films verified that the polymer surface was moderately
hydrophilic at 37°C. From cell attachment and detachment studies,
RAW264.7 cells, were convincingly proliferated on the films to a
confluent of >80 % within 48 days. However, relatively
more cells were grown on PNIPAm-co -AAm-b -PS (5%w/v) films
and thermal-induced cell detachment was more abundant in this
formulation. As a result, commercial cytodex 3 microcarrier was coated
with PNIPAm-co -AAm-b -PS (5%w/v) and interestingly
enhanced cell detachment with preserved potential of recovery was
observed at low temperature during 3D culturing.
Thus, surface modification of microcarriers with
PNIPAm-co -AAm-b -PS could be vital strategy for
non-enzymatic cell dissociation and able to achieve adequate number of
cells with maximum cell viability, and functionality for various
cell-based applications.
Keywords : surface coated microcarriers; thermosensitive
polymer; non-enzymatic cell detachment
Introduction
Maintaining adequate number of cells with preserved functionality and
potency is the major challenge of cell culturing and significantly
influence the subsequent outcomes in different medical applications such
as cell therapy, tissue engineering, and vaccine production. In this
regard, microcarrier-based cell culture has been realized as a
propitious method for achieving high cell density by providing large
surface-to-volume ratio in a suspension culture system (3D) compared to
conventional planar culture (2D) for the growth of anchorage-dependent
cells. This also advantageous to overcome several hindrances encountered
in conventional culture system, including requirement of large space and
amount of culture media, heterogeneity of pH and nutrient concentration
in the medium and inefficient exchange of gas, nutrients, and
metabolites (C. Li, Qian, Zhao, Yin, & Li, 2016; YekrangSafakar et al.,
2018).
However, attachment and detachment of cells to and from microcarriers
are basic concerns that determined the final cell productivity and
quality. In this regard, the surface and size of microcarriers play
important role for efficient cellular attachment and harvesting maximum
yield of cell recovery while maintaining cell viability, potency, and
functionality with respect to the final applications. Particularly, for
the advent of tissue engineering and cell therapy in which cells are the
final product themselves, a reliable strategy of cell harvesting should
be chosen in a way to maximize cell recovery while preserving important
cell features and functionalities(S. Derakhti, S. H. Safiabadi-Tali, G.
Amoabediny, & M. Sheikhpour, 2019; A. C. Tsai, R. Jeske, X. Chen, X.
Yuan, & Y. Li, 2020).
Although the typical enzymatic detachment method has been used for a
wide range of cells and microcarriers, the harmful nature of enzymes on
cells such as disrupting the structure of plasma membrane and a
prolonged exposure of cells to enzymes inhibits cell growth and alters
their indispensable characteristic (Tamura, Kobayashi, Yamato, & Okano,
2012). Thus, recently a non-enzymatic method such as mechanical forces,
development of degradable microcarriers, and incorporation of
thermosensitive or cleavable materials onto the surface of microcarriers
are among the alternative strategies which have been of great interest
to researchers.
Thermally induced detachment of cells from microcarriers has been
reported to better preserve cell characteristics and functionality
compared to the proteolytic enzyme induced cell detachment methods
(trypsinization). For instance, thermally induced detached hBM-MSCs and
CHO-K1 cells had comparatively lower cell death and apoptosis with
better reattachment ability and secretion of functional proteins(Tamura,
Nishi, et al., 2012; H. S. Yang, Jeon, Bhang, Lee, & Kim, 2010). As a
result, it is appeared as an attractive non-invasive method for the
detachment of cells from microcarriers. In this technique, a
thermo-responsive material is grafted on the outermost surface of
microcarrier which allows for harvesting cells with decreasing
temperature below the low critical solution temperature (LCST). So far,
various PNIPAAm-grafted microcarriers of different materials such as
dextran, polystyrene (PS), silica, alginate, and glass have been
fabricated (Hanga & Holdich, 2014; Park, Nabae, Surapati, Hayakawa, &
Kakimoto, 2013; Song et al., 2016).
In this study, poly(N-isopropylacrylamide) (PNIPAm) was copolymerized
with acrylic acid (AAc) or acrylamide (AAm) and then further polymerized
with styrene to prepare poly (NIPAm-co -AAc)-b -polystyrene
(PNIPAm-co -AAc-b -PS) and poly
(NIPAm-co -AAm)-b -polystyrene
(PNIPAm-co -AAm-b -PS), respectively. The incorporation of
hydrophilic AAc and AAm could improve the LCST behavior of PNIPAm
towards body temperature and enhanced cell adhesion by creating charged
surface as well. Polystyrene could also increase the coating efficiency
of the copolymer on commercial Cytodex 3 microcarrier through
hydrophobic interaction. Thus, the commercial Cytodex 3 microcarrier was
coated with these copolymers for efficient cell attachment and
detachment in response to temperature change during 3D culturing.
Because temperature triggered reversible hydrophobic to hydrophilic
phase transition of the thermosensitive copolymers
(PNIPAm-co -AAc-b -PS and PNIPAm-co -AAm-b -PS)
promote cell adhesion and detachment, respectively (Kumashiro, Yamato,
& Okano, 2010; N. Li et al., 2021; Sakulaue et al., 2018). The presence
of repeating hydrophilic amide and hydrophobic isopropyl groups in
PNIPAm chains allowed the copolymers to undergo reversible
conformational change with temperature alterations in aqueous
environments. When the temperature is above the LCST, the copolymer
chains form a compacted globule form which is suitable for cell
adhesion, while under the LCST, they acquire a swelled shape which
causes cell detachment from the surface (scheme 1 ) (Kim, Witt,
Oswald, & Tarantola, 2020; Nagase, Yamato, Kanazawa, & Okano, 2018;
Patel & Zhang, 2013). Moreover, PNIPAAm has been chosen due to its
quick phase transition and its LCST is near physiological temperature
(Higuchi et al., 2014). So herein, RAW264.7 cells were thoroughly grown
on polymer films as well as polymer coated cytodex 3 microcarriers at
physiological temperature and successfully dissociated with lowering the
temperature. Interestingly, temperature induced cell detachment was
relatively abundant on PNIPAm-co -AAm-b -PS (5% w/v) coated
Cytodex 3 microcarriers as well as the polymer films fabricated from
PNIPAm-co -AAm-b -PS (5% w/v) than other formulations.
Materials and Methods
Materials/Chemicals
N -Isopropylacrylamide (NIPAM), styrene, acrylic acid (AAc),
acrylamide (AAm), Cellulose triacetate (CTA),
2,2′-Azobis(2-methylpropionitrile) (AIBN), Deuterated chloroform
(CDCl3), Dimethyl sulfoxide-d6(DMSO-d6), 1,4-dioxane, N, N-dimethylformamide anhydrous
(DMF) and Cytodex® 3 microcarrier were purchased from
Sigma-Aaldrich. Dulbecco’s modified Eagle’s medium (DMEM),
3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT),
fetal bovine serum (FBS), penicillin-streptomycin, trypsin, and
phosphate-buffered saline (PBS) were obtained from GIBCO Invitrogen
Corp.
Synthesis of PNIPAm based copolymers
In order to improve the LCST of PNIPAm towards body temperature, it was
copolymerized with hydrophilic acrylic acid (AAc) or acrylamide (AAm) at
different ratios. Because, as the hydrophilic component in the copolymer
increased, the LCST value could increase.
Synthesis of poly (NIPAm-co-AAc)
Poly(N-isopropylacrylamide)-co-poly (acrylic
acid) (PNIPAm-co -AAc) was synthesized by reversible addition
fragmentation transfer (RAFT) using AIBN as initiator. Briefly,
3.36 g (29.7 mmol) NIPAm, 21.62 mg
(0.3 mmol) AAc, 83.81 mg (0.3 mmol) CTA and 9.85 mg (0.06 mmol) AIBN
were dissolved in 30 mL of 1,4-dioxane in a 50 mL round bottom flask.
After the mixture was purged with nitrogen for 30 min, it was stirred at
a temperature of 70 °C for inducing polymerization (Scheme 2a). The
reaction was carried out for 24 h and the crude product was purified by
precipitating in cold diethyl ether. The residual solvents were removed
by drying overnight under a vacuum (45 °C).
Synthesis of poly (NIPAm-co-AAm)
To synthesize poly (N-isopropylacrylamide)-co-poly
(acrylamide) (PNIPAm-co -AAm), 3.36 g (29.7 mmol) NIPAm, 205.31 mg
(1 mmol) AAm and 27.2 mg (0.1 mmol) CTA was added into 25 mL round
bottom flask and dissolved with 9.8 mL of 1,4-dioxane. After 0.2 mL of
AIBN solution in 1,4-dioxane (3.284 mg, 0.02 mmol) was added, the
mixture was purged with nitrogen in an ice-water bath for 30 min.
Subsequently, the solution was stirred for 24 h at a temperature of 70
°C for polymerization and then the flask was submerged into liquid
nitrogen to terminate the reaction (Scheme 2b). Finally, the crude
product was precipitated in cold diethyl ether to remove the unreacted
RAFT agent and the residual monomers. The copolymer was dried under a
vacuum oven overnight.
Synthesis of poly (NIPAm-co-AAc)-b-polystyrene
To enhance cytodex 3 microcarrier coating efficiency and cell anchoring,
Styrene was further block copolymerized with poly (NIPAm-co-AAc) or poly
(NIPAm-co-AAm) was further copolymerized with hydrophobic styrene
monomer. Therefore, poly (NIPAm-co -AAc)-b -polystyrene
(NIPAm-co -AAc-b -PS) was synthesized by reversible addition
fragmentation transfer (RAFT) reaction as follow. First,1 g (0.125 mmol)
PNIPAm-co -AAc, 130.19 mg (1.25 mmol) styrene and 4.11 mg (0.025
mmol) AIBN were dissolved with 10 mL DMF. Then, the mixture was
introduced into a 25 mL round bottom flask and purged with nitrogen for
30 min. The reaction was carried out at 70 °C with continuous stirring
for 24 h. The copolymer was purified through precipitation in cold
diethyl ether and the unreacted RAFT agent and the residual monomers
were removed along with the supernatants (Scheme 2c). Finally, the
residual solvents were removed from the precipitate polymer using vacuum
oven at a temperature of 45 °C overnight.
Synthesis of poly (NIPAm-co-AAm)-b- polystyrene
Similarly, poly (NIPAm-co-AAm)-b- polystyrene
(PNIPAM-co -AAm-b -PS) was prepared via RAFT polymerization
in the presence of AIBN initiator. In brief, 1 g (0.125 mmol)
PNIPAm-co-AAm, 130.19 mg (1.25 mmol) styrene and 4.11 mg (0.025 mmol)
AIBN were dissolved in 10 mL DMF and the mixture was poured into a 25 mL
round bottom flask. Then, the mixture was purged with nitrogen for 30
min, and then heated to 70 °C to induce polymerization (Scheme 2d).
After 24 h, the reaction was terminated by immersing the flask into cold
water bath and then the copolymer was purified by precipitating in cold
diethyl ether. Ultimately, the copolymer was dried under vacuum oven
overnight to remove the residual solvents. After all, the successful
polymerization of series of copolymers were confirmed using1HNMR, and GPC analytical techniques.
LCST turbidity measurement
The turbidity or transmittance of polymer solution was measured at 500
nm with a UV-visible spectrophotometer (JASCO-V-650). Series of PNIPAm
based copolymer solutions (0.1 wt. %) were prepared in distilled water
and the UV-vis absorbances of the polymer solutions were measured from
10 to 50 °C. The temperature was controlled by Water Thermostatted Cell
Holder with Stirrer (STR-773) and each experiment was started with an
initial stabilization at 10°C for 10 min and the heating rate was
1°C/min. The 50% of visible light transmittance curve was considered as
the LCST transition temperature.
Thin film preparation and characterization
Copolymer films were fabricated through spin coating. Initially
different concentration of polymers in ethanol (5% w/v, 10% w/v and
20% w/v) were prepared and 150 μL aliquot of an ethanolic polymer
solution was depositing onto a slowly spinning
SPL coverslip (25 mm Ø), (300 rpm)
on a Yotec SC-80R+ spin coater. The final spin speed
was 4000 rpm, for 30 s. After the polymer solutions coated on the
coverslips, all coverslips were housed in 35 mm petri dishes and drying
in a vacuum oven at 40°C overnight to ensured that the residual solvent
was eliminated (Fig.1 ).
For SEM analysis of the film thickness, the silicon wafer was used as
the substratum and it was coated with platinum before SEM imaging has
been taken. The alpha-step profilometer (Surfcorder ET3000) which uses a
diamond-made sharp probe to scan the surface of objects at a speed of
0.1 mm/s and stylus force of 5 gm was also employed to measure the
thickness of the prepared polymer films. The variation of height during
scanning was detected and recorded with a conductive sensor and used to
estimate the thickness of the film with an accuracy of 0.1 nm.
Contact angle measurement
The contact angle was determined by Drop Shape Analyzer using Sindatek
Instruments (Model 100E). First, the coverslip was placed on the stage
and the whole system was adjusted at room temperature. Then, the DI
water was kept at 37 °C, which is above the LCST of the polymers we
intended to measure.
Cell viability test (MTT Assay)
The biocompatibility of the copolymers was evaluated by assessing
RAW264.7 cell viability which was determined using MTT cell
proliferation assay following the protocols of the previous studies
(Birhan et al., 2020; Fentahun Darge et al., 2021). Briefly, RAW264.7
macrophages were seeded on 96-well plates at a density of
1x104 cells/well in complete DMEM medium supplemented
with 10% FBS, and 1% streptomycin and maintained in a humidified
incubator at 37 °C under 5% CO2. After 24 h, the medium
was replenished with fresh medium containing copolymers at different
concentration (ranged from 0.0005 to 5 mg/mL) and incubated at 37 °C for
24 h. Then, the cells were washed with PBS (pH 7.4) and added MTT
reagent to each well at a concentration of 1 mg/mL of medium. Following
4 h incubation, the MTT containing medium was replaced with 150 µL DMSO
to each well and the plates were incubated at 37°C for 25 min until all
the formazan crystals were dissolved. Finally, the absorbance of reduced
formazan in each well (triplicate samples) was measured with microplate
reader at 570 nm and the cell viability was estimated using the
following equation (equation 1).
\(\ Cell\ viability\ (\%)=\ \frac{\text{Absorbance\ of\ Test}}{\text{Absorbance\ of\ control}}x100\)(1)
Cell attachment and thermally induced detachment from polymer
films and polymer coated cytodex 3 microcarriers
The cellular attachment on the surface of thermosensitive copolymer
films coated on coverslips or cytodex 3 microcarriers and their
subsequent thermally induced detachment was examined using RAW 264.7
cells. First the coverslips were coated with copolymer solution and then
RAW 264.7 cells were seeded (4x104cells/cm2) on the copolymer films and bare coverslips
as a control group followed by incubation for 48 h at a humidified
atmosphere (95% air and 5% CO2) at 37 °C. Since the
LCST of the copolymers is above 37 °C, keeping the cells at this
temperature allowed to be attached on it. Similarly, for suspension cell
cultures (3D cell culture) on Cytodex 3 microcarriers or copolymer film
coated Cytodex 3 microcarriers, RAW 264.7 cells were used. First the
Cytodex 3 microcarriers were hydrated and coated with copolymer solution
as follow: Cytodex 3 was hydrated in PBS (1g/100 mL) at room temperature
and 2 to 3 drops of Tween80 was added. After 3 h, the supernatant of the
Cytodex 3 was decanted and washed by fresh PBS (50 mL/g of Cytodex 3)
for 3 min. Then, the Cytodex 3 was sterilized in an autoclave at 115°C,
15 psi (pounds per square inch) for 30 min. Following the removal of the
supernatant, the cytodex 3 was briefly rinsed with warm culture medium
(50 mL/g of Cytodex 3) and then mixed with the polymer solution (10 %
w/v medium) for 24h. Finally, it was transferred into the culture medium
(3 mg/mL medium). Prior to use, the supernatant was discarded and the
microcarriers were transferred to the new culture medium (33 mL) at 37°C
in the spinner flask. Then, the cells were transferred into the spinner
flask (4×105 cells/mL) and spinner flask was gently
agitated at 25 rpm for 2-3 days. At every 2 days, the agitation was
temporarily stopped for 5 min to allow the microcarriers to be
precipitated and about 66% of the culture supernatant was replenished
with fresh medium.
To perform thermally induced cellular detachment from the polymer film
coated coverslips or microcarriers, the cells were put at a low
temperature (20 °C) for different time intervals (0, 10, and 20 min) and
compared the effective detachment of cells.
The cells were also re-seeded
after harvesting to assess the functionality of cells after thermally
induced detachment. Films were sterilized under UV light for 12 h prior
to cell culture experiments.
Results and Discussion
Synthesis of PNIPAm-AAc and PNIPAm-AAc copolymers
Poly (NIPAm) is a thermosensitive
polymer which exhibits LCST behavior in response to temperature change.
However, its LCST value is lower than body temperature which ranges from
⁓30 to 32 °C, irrespective of polymer concentration. In order to raise
the LCST of the polymer, frequently hydrophilic polymers are
incorporated in the copolymer or increase the proportion of hydrophilic
to hydrophobic components in the copolymer and have been shown to
elevate the LCST of PNIPAm (Jain, Vedarajan, Watanabe, Ishikiriyama, &
Matsumi, 2015; Koc & Alveroglu, 2016; Osváth & Iván, 2017). Therefore,
in this study, NIPAm was copolymerized with hydrophilic acrylic acid
(AAc) or acrylamide (AAm) at different ratios to prepare a
thermo-responsive PNIPAm-co -AAc with optimized LCST value near to
body temperature. It was synthesized by reversible addition
fragmentation chain transfer polymerization (RAFT) which is important
for getting the desired polymers owing to a higher control on the final
molecular characteristics, like polydispersity and molecular weight
(scheme 2a ) (García-Peñas et al., 2019).
The
successful polymerization of the copolymer was confirmed using1H NMR spectroscopy. As shown in Fig.2a ,
the
characteristic signals at 𝛿 1.21(A) and 3.95(B) ppm are attributed to
the methyl and methine protons of NIPAm, respectively. Additionally, the
signals at 𝛿 2.07 (C) and 1.63 (D) ppm which came from the methine and
methylene protons of the main polymer chain, respectively, verified the
successful preparation of the
copolymer.
Similarly, NIPAm was copolymerized with acrylamide (AAm) to prepare
PNIPAm-co -AAm and optimized the LCST of PNIPAm near to body
temperature for intended applications in cell attachment/detachment. The
characteristic peaks of 1H NMR spectrum inFig.2b below confirmed the successful preparation of
PNIPAm-co -AAm. The signals at 𝛿 3.82(a) and 1.04(b) ppm
were attributed to the methine
protons and methyl protons of NIPAm, respectively. In addition to the
characteristic peaks of methine and methylene protons of the main chain
of the copolymer at 𝛿 1.93 (c) and 1.41 (d), respectively, the signal at
𝛿 7.29(e) ppm which was attributed to the amide protons of acrylamide
further proved the effective copolymerization of PNIPAm-co -AAm.
LCST measurement of the copolymers
The LCST of poly (NIPAm-co -AAc) and poly (NIPAm-co -AAm)
were assessed with turbidimetric analytic method. The turbidity or
transmittance of the copolymer solution was measured at 500 nm with a
UV-vis spectrophotometer (JASCO-V-650). Polymer solutions (0.1 wt. %)
were prepared in distilled water and the UV absorbances were measured
from 10 to 50 °C. The solutions were kept in refrigerator at 4°C
overnight before measurement. The LCST transition temperature of the
copolymer solutions were explained by determining the cloud point at
50% of transmittance of incident visible light which is the most direct
way to observe LCST behavior. When the temperature of the solution
raised above the transition temperature, the copolymer aggregated and
exhibit a cloudy appearance or phase separation of the solution. On the
other hand, when the solution temperature reduced below LCST,
water-polymer interaction increased by hydrogen bonding and form
transparent solution, whereas at a temperature of above LCST, the
polymer-polymer hydrophobic interactions increased and the hydrogen
bonds were broken, subsequently polymer globules were formed and the
solution became cloudy (Fig. 3 ) (Sun et al., 2019). Thus, the
turbidity was measured from lower to higher temperature and 50% of
transmittance was used for deciding LCST of the copolymer solutions.
As shown in Fig . 4 a & b , when the ratio of acrylic
acid or acrylamide that copolymerized with NIPAm were increased, the
LCST temperature of the copolymer was shifted to the higher values. This
is due to the hydrophilic nature of acrylic acid and acrylamide and thus
the copolymer solution requires higher temperature for phase
transmission and enabled the copolymer to be aggregated (Katsumoto,
Tanaka, Sato, & Ozaki, 2002). The cloud point of
PNIPAm-co -AAc1,
PNIPAm-co -AAc3, and
PNIPAm-co -AAc5 was shifted to 29.5°C, 30°C and
32°C, respectively, indicating that increasing the ratio of the acrylic
acid increases the LCST of the copolymer solution. The suffix,1, 3 and 5
in the copolymer denoted to refer the ratio of AAc to NIPAm (1:1, 3:1
and 5:1, respectively). Similarly, the cloud point of
PNIPAm-co -AAm1,
PNIPAm-co -AAm3 and
PNIPAm-co -AAm5 was shifted to 28°C, 30°C and
32°C, where the LCST of the copolymer solution increased with increase
the ratio of the acrylamide in the copolymer.
Furthermore, we compared the intensity of 1H NMR
signals of the copolymer solutions at 25°C (standard) and 37°C in
D2O to verify the LCST behavior of the copolymer. As
depicted in Fig. 4c , 1H NMR signal intensity
was relatively lower at 37°C than the 25°C 1H NMR
signals. Since the polymer was aggregated at 37°C, its1H NMR signal intensity was reduced as compared with
25°C 1H NMR signals and this phenomenon proofed the
LCST property of the copolymer.
Synthesis of PNIPAm-co-AAc-b-PS and
PNIPAm-co-AAm-b-PS
Once we optimized the LCST of NIPAm-co -AAc or
NIPAm-co -AAcm copolymers near to body temperature via
copolymerizing NIPAm with AAc or AAm, it was further copolymerized with
styrene by RAFT and obtained poly
(NIPAm-co -AAc)-b -polystyrene (PNIPAm
-co -AAc-b -PS) or poly
(NIPAm-co -AAm)-b -polystyrene
(PNIPAM-co -AAm-b -PS), respectively (Scheme 2c
&d ). The presence of polystyrene enhanced the coating efficiency of
the copolymers on the cytodex 3 microcarriers through hydrophobic
interaction. Incorporation of polystyrene in either PNIPAm-co -AAc
or PNIPAm-co -AAm also improved cellular attachment where serum
proteins from culture medium could be adsorbed on the surface of
polystyrene and further used for anchoring cells on it (Akiko Yamamoto,
2000; Clauder et al., 2020; John G. Steele, 1993; Zeiger, Hinton, & Van
Vliet, 2013). Hence, we prepared the copolymers as per the protocols and
the products were confirmed by 1HNMR spectroscopy. As
shown on Fig.5a, the 1H NMR signals at 𝛿 1.04
(A) and 3.84 (B) ppm were attributed to the methyl and methine protons
of NIPAm, respectively. The signals at 𝛿 1.96 (C) and 1.46 (D) ppm are
attributed to the methine and methylene protons from the main chain of
PNIPAm, respectively, proved the successful polymerization. On top of
these, the presence of a signal at 7.19 (E) ppm represented the aromatic
protons of polystyrene which further verified the polymerization of
PNIPAm -co -AAc-b -PS copolymer.
Likewise, Fig. 5b below illustrated the 1H
NMR spectrum of PNIPAm-co -AAm-b -PS where the signals at 𝛿
3.82(a) and 1.03(b) ppm indicated the methine and methyl protons of
NIPAm, respectively. The characteristic peak at 7.32(e, f) ppm was also
due to the amide protons and aromatic protons of acrylamide (AAm) and
polystyrene (PS), respectively, further proved the formation of
PNIPAm-co -AAm-b -PS copolymer.
The molecular weight obtained from GPC (Table 1 ) also used to
determine the successful preparation of the above copolymers. From the
result, we could observe that the molecular weights of
PNIPAm-co -AAc-b -PS and PNIPAm-co -AAm-b -PS
were significantly higher than the PNIPAm-co -AAc and
PNIPAm-co -AAm, respectively, suggested an effective
polymerization with styrene.
Preparation of polymer films and measurement of the thickness
The thickness of the films prepared from spin-coated
PNIPAm-co -AAc-b -PS and PNIPAm-co -AAm-b -PS at
different concentration (5% w/v, 10% w/v and 20% w/v) on coverslips
were measured using alpha-step profilometer and summarized inTable 2 . The surface was crosscut by needle and measured the
broken side of the surface to detrend the thickness of the film. SEM
imaging was also used to analyze the thickness of the copolymer films.
The solution of the copolymers at different concentration was
spin-coated on silicon wafer. In the meanwhile, copolymer coated silicon
wafer was broken and placed vertically on the carrier to measure the
thickness of the film with SEM imaging. The value of cross-sectional
thickness of the films obtained from SEM measurement are also shown inTable 2 and the corresponding SEM images are illustrated inFig. 6 . As we realized from the results, in all cases the film
thickness was directly proportional with polymer concentration which was
almost doubling as we increased the concentration of copolymer solutions
from 5% to 10% and 20%. Moreover, the thickness of
PNIPAm-co -AAm-b -PS films were much thinner than
PNIPAm-co -AAc-b -PS films in each polymer concentrations.
This might be attributed to their different molecular weights that
PNIPAm-co-AAc-b-PS had higher molecular weight (Mn=9064 g/mol) than
PNIPAm-co -AAc-b -PS (Mn=7260). As the molecular weight
increases, the polymer solution becomes more viscous and hinders the
fluid to flow during spin couating which in turn significantly affect
the final thickness and morphology of the polymer films (Na, Kang, &
Park, 2019; Reid et al., 2018).
Water contact angle
The value of contact angle of the
coated films is an indicator of the hydrophilicity of the polymer
surface. The polymer with water contact angle of less than 90° is
considered as hydrophilic while above 90° is hydrophobic (Vazirinasab,
Jafari, & Momen, 2018). The contact angle for spin-coated
PNIPAm-co -AAc-b -PS and PNIPAm-co -AAm-b -PS
copolymer films with different concentration are summarized inTable 3 . Generally, cells can attach and grow on hydrophobic
substrates, while they are resistant to attaching on very hydrophilic
substrates. Because the water associating with the hydrophilic
substrates could prohibit protein adsorption and prevent subsequent cell
attachment on the surface (Elbert & Hubbell, 1996). Although, cells are
able to grow on hydrophobic substrates, several studies realized that
neither extremes are conducive for cell attachment and growth. In other
words, super-hydrophilic surface (contact angle < 5°) and
super-hydrophobic surface (contact angle >150°) are not
suitable for cell attachment and growth. This is due to the fact that
hydrophilicity of the substrate surface affects the binding strength,
conformation and type of proteins adsorbed from the culture medium,
which in turn determine cell attachment and growth. Only on moderately
hydrophilic substrates with a water contact angle of approximately ⁓55°
achieved optimal cell attachment and growth (Cai et al., 2020; Lee,
Khang, Lee, & Lee, 1998). In this regard, the contact angle of PNIPAm
based copolymer films were relatively ideal for cell attachment (Lee et
al., 1998; Sumner et al., 2004). Particularly, the contact angle of
PNIPAm-co -AAc (5%), PNIPAm-co -AAc (10%) and
PNIPAm-co -AAm-b -PS (20%) exhibit a water contact angle of
53.0° , 53.8° and 46.6° , respectively, which
were closer to the ideal contact angle for cell attachment and growth
compared with other formulations (Table 3 & Fig.7 ).
Cytotoxicity test (MTT assay)
Biocompatibility of the copolymer is the most important feature to be
used as a cell attachment substrate. Thus, the potential toxicity of
PNIPAm-co -AAc-b -PS and PNIPAm-co -AAc-b -PS
copolymers have been evaluated on RAW264.7 cell lines using in
vitro MTT dye reduction assay (Fentahun Darge et al., 2021; Sakulaue et
al., 2018). The toxicity of the copolymers was performed by quantifying
the viability of cells treated with copolymers
(PNIPAm-co -AAc-b -PS and PNIPAm-co -AAc-b -PS)
for 24 h at a concentration range of 0.0005 to 5 mg/mL. As shown inFig. 8 , The in vitro cytotoxicity test revealed that the
copolymer exhibited negligible cytotoxicity towards RAW264.7 cells up to
a concentration of 5 mg ml/L, suggested that the material is
biocompatible for the application of cell attachment matrix.
Cell attachment and thermal-induced detachment
After RAW 264.7 cells were cultured on bare coverslips and copolymer
coated coverslips (4x104 cells/cm2)
for 48 h, cell confluent was monitored under microscope. According to
microscopic observation (Figure 9 a, b, & c ), after
48 h, RAW 264.7 cells were already grown well on the surface of both
bare coverslips and copolymer coated coverslips and the cell confluent
was almost >80% which revealed that the copolymers are
non-toxic to be used as a substrate for cell proliferation and
thermo-induced cell detachment. Moreover, from microscopic examination,
we realized that moderately dense cells were observed in
PNIPAm-co -AAm-b -PS films which may be due to the presence
of surface positive charges derived from amide groups enhanced cell
adhesion (Guo et al., 2016; Hoshiba, Yoshikawa, & Sakakibara, 2018). So
as to achieve thermal-induced cell detachment from the control group
(bare coverslips) or polymer film coated coverslips, the medium of cells
incubated for 48 h was replenished with cold PBS (20°C) and kept at
20°C. Then the cell detachment was observed at 0 min (immediately after
cold PBS was added), 10 min and 20 min and cell images have been taken
for each corresponding times.
Immediately after the addition of cold PBS, there was no cell
detachments observed in either the control group or polymer coated
coverslips. However, after 10 min in refrigerator (20°C), mass of
aggregated cells was floating in polymer coated coverslips, while very
few single cells were detached in the control group. Therefore, it was
kept in 20°C for additional 10 min and we have observed a well dispersed
detached cells in polymer coated coverslips but not in the control
group. Predominantly, PNIPAm-co -AAm-b -PS (5% w/v) showed
relatively substantial number of cell detachment than other
formulations. This might be due to relatively more hydrophilic nature of
PNIPAm-co -AAm-b -PS (5% w/v) which exhibit smaller contact
angle (40.1°) and favored more cell detachments than other formulations
with higher contact angles (Fig. 9b ). Comparatively more
hydrophilic polymers that contain polar functional groups provide
surface hydration through strong hydrogen bonding with water molecules
in aqueous solution. Therefore, a water hydration barrier formed between
the polymer and hydrogen molecules could determine the conformation and
type of essential proteins adsorbed on the polymer surface and
subsequently affect proper cell adhesion or the firmness of cell
attachment (Chen, Yan, & Zheng, 2018; de los Santos Pereira et al.,
2016; Leng et al., 2015; Wei, Zhang, Li, Zhang, & Bi, 2015). As a
result, PNIPAm-co -AAm-b -PS at a concentration of 5% w/v
provided maximum cell detachment and was chosen to coat cytodex 3
microcarriers for further 3D cell culturing and thermal-induced cell
detachment from the microcarriers.
Characterization of PNIPAm-co-AAm-b-PS coated
microcarriers
Attachment and detachment of cells to and from microcarriers are basic
concerns that determined the final cell productivity and quality. The
surface of microcarrier is the most important feature that determined
cell attachment and stimuli responsive detachment of cells from the
microcarriers. Therefore, surface modification or coating of
microcarriers with smart polymers helps for cell anchoring and
non-enzymatic (stimuli responsive) detachment (Sorour Derakhti, Seyed
Hamid Safiabadi-Tali, Ghassem Amoabediny, & Mojgan Sheikhpour, 2019;
A.-C. Tsai, R. Jeske, X. Chen, X. Yuan, & Y. Li, 2020). Thus, a
thermosensitive and biocompatible PNIPAm-co -AAm-b -PS
(5%w/v) copolymer was used to coat cytodex 3 microcarrier for enhancing
cell anchoring and thermal-induced cell detachment. The copolymer coated
microcarrier (cytodex 3) was confirmed by FT-IR spectra (Figure
10c ). Both the sharp signal of N-H stretching (3300
cm-1) in pNIPAm-co -AAm-b -PS and the
broad signal of O-H stretching (3400 cm-1) in cytodex3
were changed in the coated cytodex3, indicating the successful coating
of the microcarrier with pNIPAm-co -AAm-b -PS. Moreover, the
occurrence of very sharp signal at 1640 cm-1 in coated
cytodex3 was due to the specific signal of amide bending (1530
cm-1) from pNIPAm-co -AAm-b -PS further
confirmed the coating of microcarrier.
The morphology of cytodex 3 and polymer coated cytodex 3 were also
examined with SEM imaging. As shown in Figure 10 a & b , the
surface of uncoated cytodex 3 is very smooth while the surface
morphology of the coated microcarriers were rough and some wrinkles were
observed. The reason of the wrinkles is probably caused by the process
of sample preparation, which should be dehydrate before SEM measurement.
The polymer shrieked and resulted wrinkled surface of on the coated
Cytodex 3.
Cell attachment and
detachment from thermosensitive polymer coated cytodex 3 microcarrier
RAW 264.7 cells were cultured on bare cytodex 3 and poly
(NIPAm-co -AAm)-b -PS (5% w/v) coated cytodex 3 in spinner
flask. The microscopic image of cells shown in Figure 11 had no
difference in cell adhesion and morphology between commercially
available bare cytodex 3 and polymer coated cytodex 3 microcarrier
during 48 h of incubation period. This suggested that the
NIPAm-co -AAm-b -PS
(5% w/v) coated on the cytodex 3 microcarrier surface did not affect
cell attachment or viability of cells. However, interestingly a
reduction of the temperature from 37°C to 20°C induced a significant
cell detachment from coated cytodex 3 microcarriers within 20 min
(Figure 12). The thermo-responsive behavior of
PNIPAm-co -AAm-b -PS which could have hydrophobic to
hydrophilic phase transition as temperature reduced below its LCST
enhanced cellular detachment (Nguyen et al., 2019; L. Yang, Fan, Zhang,
& Ju, 2020). Therefore, unlike the bare cytodex 3, thermosensitive
coated cytodex 3 microcarriers showed spontaneous cell detachments with
reducing temperature lower than LCST. This temperature induced cell
detachment strategy might be essential to harvest adequate viable cells
with preserved cell features and functionality that are mostly lost
during enzymatic cell dissociation (Kim et al., 2020; Sakulaue et al.,
2018).
Conclusion
PNIPAm based thermo-responsive block copolymers were successfully
synthesized and the LCST of PNIPAm was optimized near to body
temperature by copolymerizing with hydrophilic AAc and AAm to be used
for cell attachment and thermal-induced cell detachment. The UV-Vis
spectroscopy measurement of turbidity with controlling the temperature
confirmed that increasing the ratio of hydrophilic AAc or AAm
copolymerized with PNIPAm, raise the LCST value (~34
°C). PNIPAm-co -AAc and PNIPAm-co -AAm were further
copolymerized with styrene for cell anchoring and enhancing coating on
cytodex 3 microcarrier. The in vitro cytotoxicity study against
RAW264.7 cells revealed that PNIPAm based copolymers were biocompatible
up to a concentration of 5 gm/ml and can be dissolved in ethanol for
spin-coating on coverslip to prepare thin films. The thickness of the
copolymers film and water contact angle were measured and have
moderately hydrophilic nature which was suitable for cell culturing.
Therefore, the RAW264.7 cells were successfully attached and
proliferated on the polymer films. Among different formulations, the
PNIPAm-co -AAm-b -PS (5% w/v) copolymer films showed
relatively better thermal-induced cell detachment and thus chosen to
coat the microcarrier (Cytodex 3) for further 3D cell culturing. Hence,
following coating of cytodex 3 microcarriers with
PNIPAm-co -AAm-b -PS (5% w/v), RAW264.7 cells were properly
attached and grown on it and subsequently abundant cells were detached
within 20 min in response to reduction of temperature (20 °C). After
detachments, the cells were reseeded and abled to attach on the surface
again without noticeable difference compared with the control group.
Apparently, PNIPAm-co -AAc-b -PS and
PNIPAm-co -AAm-b -PS copolymers were perfectly prepared and
was verified as a suitable substrate for cell culturing and can be used
for thermo-responsive cell dissociations to alleviate enzyme associated
cell damage during cell harvesting.