On The Origin of Shear Stress Induced Myogenesis Using PMMA

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On The Origin of Shear Stress Induced Myogenesis Using PMMA Based Lab-on-Chip Sharmistha Naskar, V Kumaran, and Bikramjit Basu ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017

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ACS Biomaterials Science & Engineering

On The Origin of Shear Stress Induced Myogenesis Using PMMA Based Lab-on-Chip Sharmistha Naskar a, V. Kumaran a, b, Bikramjit Basu a, c

Author Address a

Center for Biosystems Science and Engineering, Indian Institute of Science, Bangalore- 560012, India b Department of Chemical Engineering, Indian Institute of Science, Bangalore-560012, India c Laboratory for Biomaterials, Materials Research Center, Indian Institute of Science, Bangalore560012, India E-mail: [email protected]

1 ACS Paragon Plus Environment


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Abstract One of the central themes in cell and tissue engineering applications is to develop an understanding as how biophysical cues can influence cell functionality changes. The flow induced shear stress is regarded as one such biophysical cue to influence physiological changes in shear-sensitive tissues, in vivo. The origin of such phenomenon is however poorly understood. While addressing such issue, the present work demonstrates the intriguing synergistic effect of shear stress and spatial constraints in inducing aligned growth and differentiation of myoblast cells to myotubes. In a planned set of in vitro experiments, the regulation of laminar flow regime within a narrow window was obtained in PMMA-based Lab-on-Chip (LOC) device, wherein the murine muscle cells (C2C12), chosen for its phenotypical differentiation stages, were cultured under graded shear conditions. The two factors of shear stress and spatial allowance were decoupled by another two sets of experiments. This aspect has been conclusively established using PMMA device having fixed width microchannel with varying shear and identical amount of shear with different width of channels. Based on the extensive analysis of biochemical assays (WST-1, picogreen) together with gene expression using qRT-PCR and cell morphological changes (fluorescence/confocal microscopy), extensive differentiation of the myoblasts into myotubes is found to be dependent on both shear stress and spatial allocation with a maximum at an optimal shear of ca. 16 mPa. Quantitatively, the mRNA expression of myogenic biomarkers i.e. myogenin, MyoD and neogenin exhibited 10 to 50 fold changes at ca. 16 mPa shear flow, compared to that under static condition. Also, myotube aspect ratio and myotube density are modulated with shear stress and are incommensurate with gene expression changes. The flow cytometry analysis further confirmed that the cell cycle arrest at G1/G0 phase triggers the onset of myogenesis. Taken together the present study unambiguously establishes qualitative and


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quantitative biophysical basis for the origin of myogenesis towards the critical shear stress of murine myoblasts in a microfludic device, in vitro. KEYWORDS: Microfluidics; Myotube; Myogenin; qRT-PCR

1. Introduction Microfluidic technology has paved the way towards attaining the accuracy in perfusion based culturing of cells with high efficacy than the existing conventional culturing systems, together with the capacity to simulate closely the tissue- and organ-level physiology.1 The problem of the fallacious outcome of conventional petridish culture can be now addressed with the narrow regulation of parameters by the Lab-on-Chip (LOC) application of microfluidics. Along with the reduction of spatial occupancy, this emerging technology enables experiments to run with a very small amount of biological sample and to perform the assays accurately as they essentially have negligible or nil dead volume.2 Hung et al. reported the culturing of 10×10 array of different cell types within a single chip.3 The success of these devices lies in the absence of manual errors as the functional components are serially arranged according to the design of experimental steps. The most fundamental criteria of any new technique to be acknowledged would lie in its reproducibility. While comparing LOC designed culture system to conventional cell culture system, one can realize that the latter cannot recapitulate the structure, function or physiology of living tissues.4 Also, the conventional petridish culture is incompetent to create the highly complex and dynamic three dimensional environments around the living cells, in vivo. The physiological environment in vivo is dynamic due to continuous flow of different body fluids, like blood, lymph, cerebrospinal fluid, cell interstitial fluid etc.5 Such flow exposes the cells and



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tissues to a physiologically determined shear stress, which is indispensable for normal cellular functionality. In living systems, the muscles are exposed to physiological shear which also has an effect on angiogenesis through enhancing the synthesis blood vessel sprouting factors. The physiological need behind the continuous flow of interstitial fluid (IF) is to maintain supply of the oxygen and nutrient to the tissue cells as well as removal of waste. The interstitial fluid originates by transcapillary filtration and again collected back through lymphatic system into the blood circulation.6 It flows into the interstitium of extra vascular tissues including skeletal muscles. Hence, skeletal muscles are exposed to continuous convection of IF flow like any other cell in the body. As mentioned by the authors, 10% of the IF bathes the muscles. Therefore the muscles are under constant shear stress due to the flow, even in its resting state. This has been the motivation to probe into the origin of shear stress on the cell functionality modulation, in vitro. Several researchers have reported the role of shear stress in the maintenance of physiological homeostasis by influencing the cellular behavior.7 For example, Cucullo et al. investigated the effect of shear on the endothelial lining of blood-brain-barrier, which is crucial for the normal functioning of brain.8 They reported the significant upregulation of tight junction, adherens junction proteins and genes as a result of trans-endothelial electrical resistance and high selectivity of the blood-brain-barrier permeability. Schwann cells are also reported to be shear sensitive in terms of its functionality on the substrate.9 In addition to the mechanical cues, the gradient of the soluble factors created by the flow of body fluids is one of the critical parameters determining the cellular responses, like cell differentiation, apoptotic pre-programming, proliferation etc.10 To this end, the static microscale cell culture has been successful in enabling


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novel experiments that take advantage of micro patterning technologies to control cell–cell and cell–matrix interactions.11 Such influence is reported to be dependent on cell type. Multiple studies related to mechanotransduction within muscle cells have been carried out by applying tensile strain. It has been found to be detrimental for cell survival, phenotype, metabolism or protein expression. For example, Juffer et al. have attempted to show that fluid shear is more effective than applied mechanical tensile strain in expressing growth factors in myotube differentiated C2C12 cells.12 Kook et al. have mentioned in their studies that cyclic mechanical stretching can suppress myogenesis.13 The shear stress by pulsating fluid flow (PFF; mean shear stress 0.4, 0.7 or 1.4 Pa, 1 Hz) increases the anabolic response of the C2C12 myotubes by increasing effectively the nitric oxide (NO) production along with several growth factors.12 NO elevates the activity of the satellite cells, hence results in hypertrophy of the muscle.14 In a different study, Kurth et al. also have reported that pulsatile shear exposure given at 10-3 N m-2 to 9 × 10-3 N m-2 with a pause of 10 minutes, can result in vanilloid 2 receptor mediated shear-stress responses in C2C12.15 Tourovskaia et al. have investigated the process of myogenesis in short term and long term microfluidic culture on a micropatterned surface.16 In long term culture, they had used a shear of 1.29 × 10-3 N m-2 whereas, short term culture was performed under a shear of 1.16 × 10-2 N m-2. Low shear stress was chosen by the investigators because 5 to 10 N m-2 of shear has been found to be detrimental for cell survival, phenotype, metabolism or protein expression.17-18 Anene-Nzelu et al. have mentioned about the cellular alignment in the microgrooves which has functioned as a cue for myogenesis.19 The investigators have used shear flow to enhance the alignment, henceforth increasing the myotube formation. Parallel and perpendicularly oriented microgrooves in respect to the flow where used in their study. They have used flow rate of 0.05 ml/h to show the synergistic effect of topography and



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shear in a microgroove. The cells at the periphery of the 3D cellular construct experienced shear stress ranging from 0.3 to 15 dyne cm-2, depending on their distance from the micro-piller array. In the present work, we aim to study the differentiation of muscle cells as a synergistic response to the flow induced continuous shear stress along with confinement within PMMA fabricated microfluidic devices. This contained range of different dimension of channels and a gradient of shear values. We intended to investigate the optimal combination of spatial cues with the shear stress among all the varying values of width and shear. To determine the synergistic effect of optimal width and shear from all the combination, the alignment and orientation of myoblast together with the myogenic marker expression were considered to be the parameters. It is worthwhile to mention that myogenic process produces myotubes which show syncytism and formation of nano-vesicles towards elongation of nuclei which were also additionally considered as the parameters. Our experimental framework can be rationalized by the fact that with integrating perfusion flow parameters for cells confined inside the microchannels, one can create in vivo like conditions. The novelty of our approach can be realised as the studies so far are conducted using pulsatile shear.12, 15 Also, the amount of applied shear is comparably higher with shear being applied for shorter durations (ranging in hours) in earlier studies. In the present study we have planned prolonged exposure of shear for three consecutive days on undifferentiated C2C12 cells. As the cells are subjected to continuous shear stimulation for 3 consecutive days in the present study, we have preferred to keep a low shear stress in the range of ca. 3 mPa to 42 mPa.20 The control experiments to understand the ‘shear’ effect and the ‘spatial constraint’ effect were carried out independently by growing the cells in an identical PMMA microfluidic device without shear flow and on PMMA disc in conventional petridish culture, respectively. The flow parameter within the microfluidic channels are determined using first principle calculation of


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fluid dynamics. After the microfluidic culture, extensive fluorescence and confocal microscopy analysis were conducted to quantitatively and qualitatively analyse the cell morphological changes under graded shear flow conditions. The gene expression changes using PCR finally confirms the evidence towards myogenesis.

2. Materials and Methods 2.1. Device fabrication The designing of a microfluidic culture system involves some critical processes, including the microfluidic layout, selection of materials, fabrication process and sterilization technique along with the non-negotiable requirements like maintenance of sterility and cytocompatibility of the device material. In the present work, microfluidic channels are designed using software CorelDRAW X6. The prototype of the channeled space was drawn using SOLIDWORKS version 24, which was later imported into the COMSOL 4.3 workspace for solving the flow problem to get the velocity profile. The microfluidic device has been fabricated using the polymethyl methacrylate (PMMA) sheets procured from Plexiglas®, USA (figure 1a). The PMMA substrates were chosen for microfluidic devices in the present case. Comparing widely used polydimethylsiloxane (PDMS) with PMMA, the former is more hydrophobic than the latter.21 Using PMMA sheets as the material for microfluidic device is preferable as the same can be dismantled apart during imaging and analysis. It is important to mention that permanent bonding of PDMS to PDMS or to any hard surface creates problem, when it is necessary to have the open channels for proper imaging and analysis. Also, PDMS substrates become opaque due to absorption of water molecules. In particular, small hydrophobic molecules are absorbed through PDMS, but it is less absorbed in case of PMMA. Comparing polystyrene (PS) with



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PMMA, PS is slightly hydrophobic than PMMA with lower hydrophobic recovery after UV/plasma treatment.22 In the present case, four layers of the PMMA sheets were involved in the device design. The unpatterned bottom most PMMA sheet with smooth surface acts as the cell growth substrate. The second sheet of PMMA substrate from bottom was cut-through using laser cutter machine by creating parallel channels of graded width starting from 2 mm to 100 μm by reducing 100 μm each time. The length of the channels was kept same as 35 mm and height as 1 mm. The third layer of the PMMA sheet from bottom contains the common bay to ensure uninterrupted fluid flow at entry and exit of the microchannel. The fourth sheet only contains inlet and outlet (both of 1mm diameter) and confines the channel in form of closed chamber, except the option for inlet and outlet for the fluid flow. To fix the sheets one upon another, 3M™ pressure sensitive adhesive (PSA) films of 100 μm were used with exactly same super-imposable patterns with the respective PMMA sheets. All the layers of PMMA and PSA sheets were stacked using the alignment screw in order to avoid mispositioning of the patterns. The laser parameters were standardized by trial and error to obtained optimized power at 80% with the pulse of 300 for the PMMA sheets and 23% with 750 pulses for the PSA sheets. The inner surfaces of the microchannels were coated with collagenous protein. Type-I collagen is the fibrillar member of the collagen protein family and is available abundantly in the tendons, muscles, bones and extracellular matrix (ECM) of various tissues. The epimysium of the skeletal muscle contains type-I collagen that facilitates cell attachment, growth, differentiation, migration, and tissue morphogenesis.23 For the present study, Type-I collagen (GIBCO® Collagen type-I Rat Tail) was procured from Thermo Fischer Scientific. The protein was solubilized in 20 mM acetic acid solutions to achieve a working concentration of 5 µg/ml. With


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slow pumping of the collagen solution, the channels were filled and kept undisturbed inside the incubator at 37ºC for 4 h. Afterwards, the channels were washed thoroughly with phosphate buffer saline (1x PBS; pH 7.2 to 7.4) and were kept in a sealed petridish for culture experiments.

2.2. Computational analysis of fluid flow in PMMA microfluidic device The flow condition in the device was simulated using finite element-based software COMSOL Multiphysics v5 (COMSOL Inc, Burlington, MA) and from such analysis, the average shear stress applied on the bottom surface of the channels was determined. The algorithm, which was used to develop the solution of the fluid flow is Eulerian, where the mesh is fixed and the material particles change their position with respect to the fixed mesh. The flow regime was selected as laminar flow. The inlet flow velocity were kept same and set in accordance with the experimental volumetric flow rate of 100 µl/h, and a constant atmospheric pressure condition was used at the outlet. The perfusion medium considered as an incompressible, homogeneous and Newtonian fluid. Meshing of the fluid domain of the device had been performed using finer mesh using 75000 mesh points (figure 1d, 1c).

The assumptions

implemented for solving the Navier–Stokes equation was taken to be no slip conditions along the solid walls with constant atmospheric pressure at the outlet boundary.

2.3. Wettability The wettability of PMMA sheets was tested using contact angle goniometer, procured from Dataphysics, Germany. Sessile drop method was chosen to measure the hydrophobicity using distilled water as a probe liquid. Minimum of six independent measurements were taken and the



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values were presented as mean ± standard deviation. The water adhesion tension (T) on PMMA surface was calculated by, T = | γ cosθ |

(1)

where, θ is the measured water contact angle and surface tension of water (γ) is taken as 72.8 dyne/cm for water.24

2.4. Mouse myoblast cell (C2C12) culture C2C12 is a myoblast cell line derived from Mus musculus (mouse), which remains in undifferentiated condition unless they are cultured in differentiation initiating starvation medium.25 The rationale behind choosing C2C12 cell line includes (a) they are the unipotent cells (though BMP-1 can drive them towards osteogenic lineage26) with the ability to undergo differentiation, when suitable cues are provided and (b) they have good adherence on surfaces without compromising the reproducibility of the experiments.27 The C2C12 cell line can differentiate, forming contractile multinucleated myotubes and producing characteristic musclespecific proteins. The cells were obtained from NCBS, Bangalore. All the cell culture experiments were conducted with cells at passage of 2 to 7. Cells were revived from cryopreserved stock using complete culture media, which contained DMEM (Dulbecco Minimum Essential Medium; Invitrogen), supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% antibiotic antimycotic solution (Sigma Aldrich). The cells were maintained in CO2 incubator (Sanyo, MCO-18AC, USA) at constant 37°C temperature and 95% humidification with 5% CO2. During the microfluidic culture, the cells were not allowed to be confluent more than 70% to 80% to avoid morphological changes and depletion in number. Trypsin-EDTA (Invitrogen, 2 ml of 0.05% strength) was used to lift the cells enzymatically and re-suspended in


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known amount of media to achieve a cell concentration of ca. 104 cells per ml of media. The single cell suspension thus prepared had been used for seeding into the microfluidic LOC devices with channel inner walls coated with collagen type-I. It is worth mentioning that before the coating procedures, the PMMA substrates were rinsed with absolute ethanol (Merck, Germany) and exposed to UV for sterilization.

Two set of control experiments were carried out to

understand the ‘shear’ effect and the ‘spatial constraint’ effect. Those experiments respectively involve growing the cells in an identical PMMA microfluidic device without shear flow and on PMMA disc in conventional petridish culture.

2.4.1. Cell seeding and perfusion setup for maintenance of aseptic condition Some of the problems of using a microfluidic cell culture are air bubble inclusion, leaking as well as contamination. A syringe pump (New Era syringe Pump Systems, Inc, NY, USA; model no. NE-1002X) was used to load the cell suspension at a flow rate of 0.5 ml/h in order to avoid trapping of air bubbles as well as uneven flow pattern in the channels, especially at the constriction and sharp edge corners. The devices were kept undisturbed at an optimal cell growing environment as mentioned previously for another 24 h after loading. This allows them to get attached and to grow sufficiently on the respective surfaces. Once the cells are in their proliferating phase, the flow of the serum starved media (DMEM with 1% FBS) was started at volumetric flow rate of ca. 100 ml/h, using the sophisticated scientific syringe pump. The medium used was supplemented with 1% of FBS as it is unavoidable for differentiation. The media flowing out from the device was collected in sealed bottle with only two openings, one for collecting the spent media and the other for adjusting the air pressure as the collection bottle was getting filled with the fluid in the course of the experiment. The connection between the device



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and both the source syringe and collection bottle were done using 1mm diameter silicone tubing (figure 1b, 1c). Culture medium with 10 % FBS was also used as flow environment in a separate experiment to ensure that the device had no adverse effect on the cell growth.

2.5. Estimation of cell proliferation using WST-1 Assay Water-soluble tetrazolium salts (WST-1, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]1,3-benzene-disulfonate,Roche)28 assay was preferred over MTT (3(4,5-dimethylthiazol-2-yl)2,5- diphenyltetrazolium bromide) assay to determine the cell proliferation as WST-1 is known to have least or no interaction with the biomaterial substrate. Two separate setups with proliferation media (10% FBS supplemented) and differentiation media (1% FBS supplemented) were used during the flow conditions. The WST-1 assay was conducted on the cell samples after the prescribed culture durations of 1 day, 3 days and 5 days. It was necessary to perform the 1st day WST-1 assay to check the proliferation of the cells prior to the flow from day 2. 10% v/v solution of WST-1 in complete culture medium was used, and samples were incubated for 4 h at 37°C in 5% of CO2 atmosphere along with saturated humidity. The pooling of samples (maintained under identical conditions) was performed as the fluid volume from single channel was less for spectroscopic detection. We collected 200 μl of the reacted fluid and the optical density (OD) of the developed color was measured by a microplate reader (iMark, Bio-rad laboratories) at a wavelength of 450 nm. The absorbance value provides a direct correlation to the number of viable cells in each well. The measurements were obtained by averaging three independent data and the assay was repeated thrice. Cell proliferation was calculated by using the following equation where control is taken as the cells grown on collagen coated PMMA discs:


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% Cell proliferation =

× 100

(2)

2.6. Pico green assay for DNA content estimation The total content of the DNA of the channelized cells was estimated using Quanti-iT Picogreen dsDNA assay kit (Invitrogen) following manufacture’s protocol. After prescribed duration of culture (1, 3 and 5 days) , the samples were washed in 1x PBS and lysed with of 0.1% Triton-X for 20 min. Pooling of identical samples was done in order to get an adequate amount of cell lysate. The picogreen working solution was prepared by mixing of 1x TE buffer stock reagent at 1:300 dilution. 50 µl of lysate was then added to the prepared working reagent and after 5 minutes of incubation, the intensity was measured using multimode plate reader (Eppendorf AF2200) at excitation and emission wavelengths of 485 nm and 535 nm, respectively.29 Quantification of dsDNA was calculated according to a standard curve of dsDNA (ng/ml) plotting concentration against intensity.

2.7. Flow cytometry analysis of cell cycle using propidium iodide (PI) PI binds to double stranded DNA by intercalating between the base pairs. It has low fluorescence emission in unbound condition, but has increased fluorescent activity by several fold in when bound to DNA. The analysis of cell cycle of the myoblasts by flow cytometry was performed using hypotonic PI solution with added trisodium citrate dihydride and was run on a BD FACS Canto II flow cytometer. The pooling of the samples was carried out to obtain 104 cell/ml. The readings were obtained with flow cytometry runs for 1000 events with low speed flow. The recorded data were analyzed using BD FACSDiva version 6.1.1, with model-3 fit.



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2.8. Fluorescent imaging Fluorescent staining was performed for four different proteins along with DAPI. Two of the selected marker protein for myotube differentiation, i.e MyoD and Myogenin (MyoG) were targeted for immunostaining after the cells were cultured, maintaining the protocol where collagen coated PMMA discs were used as control. C2C12 cells were seeded approximately at a density of 2 × 103 cells/ well. The cells were cultured for 1 day, 3 days and 5 days, while on 2nd to 5th day, they were subjected to the fluid flow condition. The samples fixed using 4% paraformaldehyde (PFA; SD Fine-Chem Lit) solution Samples were permeabilized by 0.1% of triton X. Blocking with 1% Bovine Serum Albumin (BSA) for 1 h was done to prevent nonspecific binding of the dye. Primary antibodies of myogenin (MA5-11486 Invitrogen) and MyoD (M-318: sc-760, Santacruz Biotech) were added at 1:10 dilution for 1 h. Goat secondary antibody tagged with fluorophore as AF488 (A 11029) was added at 1:20 dilution and kept for 1 hr. Hoechst stain 33342 DAPI (Invitrogen) was added and kept for 15 min to visualize the active nuclei. The cells were observed under confocal microscope (Leica TCS SP5 confocal microscope) and fluorescence microscope (Nikon LV 100D, Japan).

2.9. Quantitative-Reverse-Transcription PCR (qRT-PCR) PCR was performed for detecting the mRNA level of three chosen myotube proteins - myogenin, MyoD and neogenin. Quantative-Reverse-transcription PCR was done using a one-step cell-toCT qRT-PCR kit procured from Invitrogen (A24571, Ambion). This specific PCR kit was chosen considering the challenge of extracting mRNA from very less number of cells even after sample pooling.

PCR protocol was set according to the manufacturer’s instruction with slight

modification to minimize the procedural loss of mRNA. As the channels could accommodate


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low number of cells, the cell lysis solution was added directly to the adhered cells instead of enzymatic lifting. Primer solution at a strength of 200nM was added to 20 μl of reaction volume, which consists of 10 μl of the qRT-PCR mix, 0.16 μl of RT-PCR mix. The volume was made upto the above mentioned desired amount by adding nuclease-free water. It is worth mentioning that for each gene, three sets were prepared to validate the outcome. β-actin was considered as the housekeeping gene against which the expression of gene is being considered to be up regulated or down regulated. Quantification of gene expression was calculated in the form of 2-

CT

(CT,

cycle threshold) values. ROX was the inbuilt reference dye to rule out the plate to plate variation of fluorescence. PCR primers were designed using NCBI based primer designing tools. The parameters like GC content, length, exclusion of complementary sequence were strictly maintained while designing the primers to get an optimum output (Table 1).

2.10. Statistical analysis All the results were expressed as mean ± standard error (SE). Student’s t-test and one-way ANOVA with post-hoc Tukey’s test were performed to reveal the statistical differences regarding the results of the cell response at different shear stress. Parametric correlation studies were performed by calculating Pearson’s r values. Non-parametric correlation statistics were performed using ordinate data by deducing the Spearman’s rho. All the statistical analysis had been performed using SPSS-16.0 software (SPSS Inc.@2010). The cut-off p-values were set at 0.05 and 0.01, lower than which were considered to be too low to accept the null hypothesis.



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3. Results This section describes the nature of fluid flow in the PMMA microfluidic devices. This will be followed by the qualitative and quantitiatve analysis of cell proliferation and functionality using a host of biochemical assay including qRT-PCR and morphological changes using fluorescence/confocal microscopy, when myoblasts are grown under differential shear flow conditions in serum-rich or serum-starved culture media. It is worthwhile to reiterate here that two sets of control experiments were independently performed to decouple the ‘shear’ effect and the ‘spatial constraint’ effect, with one by growing the cells in an identical PMMA microfluidic device without shear flow and another on PMMA disc in conventional petridish culture, respectively. Apart from establishing the statistical significance of the cell biology results, the parametric and non-parametric correlation analysis of the gene expression and the combinatorial parameters will be discussed.

3.1. Fluid flow and shear stress within microfluidic device To start with, this subsection analyses the flow characteristics, that cells would be exposed within PMMA microfluidic devices under the selected culture conditions. The cell culture medium is considered as an incompressible Newtonian fluid, whose flow inside the channels was simulated using finite element software (FES; COMSOL Multiphysics version 4.3). It is crucial to determine the flow properties of the cell culture medium as the altered behavior of the cell growth was largely claimed to be dependent on the shear stress exerted. Reynold’s number (Re) was calculated by the following equation to determine the flow regime, that myoblast experience inside the channels. Re =

´× ×

(3)

µ


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´=

(4)

×

where, ´ is the flow velocity in the channel, W is width of the channel, H is the height, ρ is the density of the cell media which was taken as 997 kg/m3,30 µ is the dynamic viscosity of the cell media of value of ca. 0.001 Pa.s.31 The Reynolds numbers were

On The Origin of Shear Stress Induced Myogenesis Using PMMA

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