Cells and cell cultures
Human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex Bio Science Walkersville. These cells were cultured in Endothelial basal medium-2 (EBM-2, Lonza, Walkersville, MD, USA) supplemented with 2% fetal bovine serum (FBS, Lonza) and endothelial cell growth factors (Lonza, Hydrocortisone 0.2 ml, hFGF-B 2 ml, VEGF 0.5 ml, R3-IGF-1 0.5 ml, Ascorbic acid 0.5 ml, hEGF 0.5 ml, GA-1000 0.5 ml, Heparin 0.5 ml). Before cells were seeded, 18 mm round coverslips (Fisherbrand, Leicestershire, United Kingdom) were coated with 2% gelatin (Gelatin from porcine skin Type A, sigma). Before using the coverslip, it was dipped into 100% ethanol and flame sterilized. 500 μl of 2% gelatin solution in phosphate buffered saline (PBS) was added to 18 mm coverslips and incubated for 2 hours at 37°C. After 2 hours, 2% gelatin solution was suctioned and dried in air. For individual cell migration model (nonconfluent), 8 × 103 cells in 600 μl were plated on the coverslip at 30% confluency. The cells were plated onto gelatin-coated coverslips and were incubated for 24 hours before exposure to flow. HUVEC were studied before passage 10 in all experiments.
Parallel plate chamber system
We used the parallel plate chamber system (Figure 1) to apply shear stress to HUVEC. The parallel plate chamber system consisted of two parts, incubator system installed with the microscope to observe live cells and the flow chamber to apply shear stress to the cells. The incubator was regulated by temperature and gas composition controlling program (CCP ver. 3.8) under proper environment for cell (5% CO2, 37°C). The flow chamber was made up by main body with inlet and outlet for tubing (inner diameter, 2 mm), bottom plate and silicon gasket. Gelatin-coated coverslip seeding HUVEC was mounted on the bottom plate and put the main body and the silicon gasket (200 μm in height, 2 mm in width) together. Medium was taken out at least for 1 hour before starting experiments to prevent bubbles. The shear stress (dyne/cm2) was calculated by this equation.
Q is the volumetric flow rate (ml/s), μ is the viscosity of the medium (dynes/cm2), W is the gasket width (cm) and h is the gasket height (cm) [13]. It was known that physiological levels of venous and arterial shear stresses are 1–5 and 6–40 dynes/cm2, respectively [12]. Thus, we selected 4 and 8 dyne/cm2 in the physiological level of shear stress. It caused cell damage to apply shear stress of 10 dyne/cm2 and over to the cells.
Image acquisition
The cells were cultured in the incubator placed on the microscope stage and cell images were recorded every 5 minutes for 8 hours by the change-coupled device (CCD) camera (Electric Biomedical Co. Ltd., Osaka, Japan) attached to the inverted microscope (Olympus Optical Co. Ltd., Tokyo, Japan). Images were conveyed directly from a frame grabber to computer storage using Tomoro image capture program and memorized them as JPEG image files.
Cell tracking and evaluation of cell migration
For data analysis, captured images were imported into ImageJ (ImageJ 1.37v by W. Rasband, National Institutes of Health, Baltimore, Md). Image analysis was carried out by manual tracking and chemotaxis and migration tool plug-in (v. 1.01, distributed by ibidi GmbH, Mnchen, Germany) in ImageJ software. We obtained the datasets of XY coordinates by using manual tracking. Then, these datasets were imported into chemotaxis and migration tool plug-in. The tool computed the cell migration speed, directionality and X forward migration index (XFMI) of HUVEC and plotted cell migration pathway. The migration speed was calculated as an accumulated distance of the cell divided by time. The directionality of the cell was defined as Euclidean distance divided by accumulated distance. The Euclidean distance means the straight-line distance between the start point and the end point. The closer the directionality was to 1, the straighter the cell moved. The XFMI of the cell was defined as an XFMI divided by accumulated distance. Cells undergoing division, death, or migration outside the field of view were excluded from the analysis.
PLLA scaffold
We were provided with the scaffold from Ehwa Womans University. Poly (L-lactic acid) (PLLA) (intrinsic viscosity 0.63 dl/g, Mw = 2.5 × 105 g/mol) was provided by Purac Biochem (Gorinchem, Netherlands). Dichloromethane (MC) and acetone were purchased from Duksan Chemicals Co. (Seoul, Korea). In brief, 8% w/v PLLA solutions were prepared with the solvent mixture composed of MC and acetone (90:10 v/v). The polymer solution was poured into a 10-mL glass syringe, attached to a 25-gauge blunt end needle. A syringe pump was set at a volume flow rate of 0.1 ml/min. The distance between the needle tip and the collector was 15 cm. The electrospinning process was carried out in a sterile environment at high voltage. A voltage between 8 and 20 kV was used for all solutions. Prior to usage, the electrospun scaffolds were dried for three days under a vacuum at 70°C to remove the solvents.
The flow perfusion system
We used the peristaltic pump that produces 500 ml/hour to circulate the medium (Figure 2A). The chamber was tapered to ensure flow from the outer edges of the scaffold as well as the center to the exit port of the chamber. Screw caps were fitted with O-rings for a tight seal and prevention of leakage. The peristaltic pump pulled medium from the reservoir and provided it to the chamber including cell-seeded scaffold via 6 mm inner diameter silicone tubing. Equipment was sterilized by steam autoclave (tubing, chamber). The apparatus was assembled under sterile conditions in a laminar flow biosafety cleanbench. In order to incubate the cells in the chamber, a CO2 mini-incubator (150 × 130 × 40 mm) was designed and fabricated with a double-layered acrylic plate. The mini-incubator was connected with a CO2 gas mixing system (FC-5, Live cell instrument Inc., Seoul, Korea) and supplied 5% CO2.
Observation of cells distribution in scaffold
After 4 hours seeding the cells, the scaffolds were washed 2 times with phosphate buffered saline (PBS) and the cells were fixed with pre-cooled (−20°C) 70% ethanol for 5 minutes. Then, the cells were stained with propidium iodide (Sigma, Steinheim, Germany). The migration of cells into the scaffold was calculated by a confocal microscope (LSM 510, Carl Zeiss Micro Imaging Inc., North America), using horizontal and vertical sections through the scaffolds every 10 μm.
Immunostaining
After applying shear stress for 30 minutes and 5 hours, actin cytoskeleton was visualized by immunostaining. Each step for immunostaining was as following. Cells were fixed with 3.7% paraformaldehyde for 15 minutes at room temperature and were washed two times with PBS. Cells were permeabilized with 0.25% Triton X-100 in PBS for 5 minutes at room temperature and rinsed 3 times with PBS. Nonspecific bindings to cells were blocked with 1% bovine serum albumin (BSA) for 30 minutes at room temperature. In dark, they were treated with Alexa (488)-conjugated phalloidin (5 U/ml, Invitrogen) for actin cytoskeleton staining for 30 minutes at room temperature. The monolayers were mounted under a coverslip with aqueous mounting medium (Dako Faramounts, Dako North America Inc., CA, USA) and were observed by a fluorescence inverted microscope.
Statistical analysis
All statistical analyses were completed with SPSS Software version 12.0 (SPSS, Chicago, IL, USA). Non-normal distributions in the data were not allowed to use Analysis of variance (ANOVA) and t-tests. Therefore, comparisons between groups were carried out using the nonparametric Kruskal-Wallis test. Comparisons between subgroups used the Mann–Whitney U test with Bonferroni correction for multiple comparisons, thus yielding statistical significance if p < 0.0167. All data were presented as mean values and standard deviation (SD).