Studying the Influence of Electroporation on HT29 Cell Line Interaction with Fibronectin and Collagen Protein Micro-Patterned Surface

Micro-contact printing (MCP) is a scheme that allows a substrate or surface to be functionalized freely with extracellular matrix (ECM) protein such as fibronectin and collagen, in a well-defined manner. MCP can be used to regulate cell adhesion geometry on a substrate and in controlling wound healing process by facilitating directed cell migration. In this study, human colon cancer cell line, HT29 were grown on a micro-contact printed pattern of fibronectin and collagen protein with repeat gratings of 25μm, 50μm, and 100μm wide, for 48 hours. The cells alignments to the patterned substrates were then computed, where 0° means 100% alignment to the pattern. This was done with the purpose of finding those pattern that stimulated the best degree of cell alignment. Best alignment and elongation were obtained on 50μm of the two ECM proteins. The quantitative analysis of the results revealed that HT29 cells aligned most readily to the 50μm width pattern with a mean angle of alignment of 5.0° ± 1.3 and 16.1° ± 4.6, respectively, on fibronectin and collagen pattern surfaces. Contrarily, the cells aligned poorly on the 25μm width pattern of fibronectin, collagen and the control substrates with a mean angle of 33.4° ± 8.4, 36.2° ± 8.9 and 54.5° ± 6.0, respectively. Furthermore, the 50μm stamp pattern was used to investigate the influence of pulse electric field (PEF) on the HT29 alignment to the patterned substrate. The result revealed that there was significant improvement (P < 0.05) in the cell alignment between the electrically treated and the untreated cells. The alignment angles of the electrically treated cells were 4.0° ± 1.2 and 11.2° ± 3.5, respectively, on the 50μm pattern surface of fibronectin and collagen. Therefore, the result of the study revealed that micro-contact printing technique together with pulse electric field could offer a potentially fast method of controlling directed cell migration for wound healing application.


Introduction
The Extracellular matrix (ECM) protein is the main regulators of many cellular functions such as cell to cell adhesion, cell to ECM adhesion, cell communication and cell division [1]. Additionally, ECM controls cell migration and cell shape. Understanding the process that controls cell function such as proliferation, adhesion and migration is very significant for wound healing and tissue engineering application and in the development of new tissue in vivo [2,3]. Fibronectin and collagen are the major components of ECM protein. Each of this protein bind to a specific integrin molecule. The secretion of this protein in the course of development and the level of their expression control many cellular functions and properties [4].
There are different methods used for the deposition of proteins on a surface. This includes plasmainduced micropatterning, soft lithography, ultraviolet radiation micro-patterning and micro-contact printing methods [5]. However, micro-contact printing (MCP) is chosen in this research because it offers a cheap and simple surface patterning technique without denaturing the proteins as in the case of other methods. Additionally, a sub-unit of 0.1µm patterns of protein can be produced with MCP [6]. In this study, MCP was used to produce 25, 50 and 100µm micro-pattern of two different ECM proteins, namely, fibronectin and collagen. The aim of this study is to determine the stamp pattern that will induce the best degree of HT29 cell alignment on different ECM proteins. Subsequently, the best pattern on each protein will be used to examine the influence of PEF on the cell alignment.
Development in multicellular organisms in the course of wound healing process depends on the cell adhesion process which can be influenced by electrostatic charges [7,8]. Therefore, manipulation of substrate charge, for instance, by patterning the substrate or by application of external electric field to enhance cell adhesion, is a promising scheme that can control cell assembly and migration in wound healing applications [9]. It has been revealed that this surface charge can speed up or slow down cell adhesion and growth on the charged surfaces through repulsion or attraction of positive ions, especially, divalent cations in the cell culture medium [7]. These cations enable interactions between the surface and negatively charged cell membrane and play an important role in creation of focal adhesions [10]. The changes in the substrate charge may modify other properties of the surface, for example, surface chemistry, which in turn can promote cell adhesion process [11]. Thus, in this study, the influence of PEF on the alignment of HT29 cells on guidance cues was investigated which in turn could be used for facilitating cell guidance and directed cell migration in wound healing application [12,13].

Cell Culture
The human colon cell line HT29 was used in this study. The HT29 cells were grown in a 25cm 2 culture flask as a monolayer in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (antibiotic). The cells were incubated at a temperature of 37 o C in a humidified atmosphere with 5% CO 2 [14], [15], [25]. Cells were harvested and passaged with a tryple express solution (TES) whenever they reached 80-90% confluency. Spent medium was changed every 2 to 3 days [14] if cells were not confluence. For subculture, the old medium was discarded by aspiration. Thereafter, 2ml of phosphate buffer saline (PBS without calcium and magnesium chloride) was added for washing the cells. The added PBS was aspirated and discarded. Next, 2ml of TES was added for cells detachment. The cells plus the TES were incubated for 5-10 minutes at 5% CO 2 at 37 o C until all cell become rounded and fully detached from the substrate. When the cells were fully detached, an equal volume of complete growth medium was added to stop the effect of the detachment enzyme used (TES). Next, the cells were re-suspended to form a uniform suspension which is then utilized for experimentation in subsequent sections or seeded in new flasks for further propagation.

MCP Technique
The PDMS stamps and glass coverslips were washed with 70% ethanol and left to dry totally in a biosafety cabinet before commencing the MCP process. Glass coverslips were then micro-contact printed with two ECM proteins (fibronectin and collagen IV). The dilution used for fibronectin and collagen were 100 and 50µg/ml, respectively [16]. The stamping method used was as follows: a stamp was inked by dipping into different ECM protein solutions for 60 seconds duration [17]. The stamp was then removed from the protein solution and allowed to dry in air for 90 seconds. The dried stamp was then placed in contact with the glass coverslip or substrate and pressed lightly using another glass coverslip for 60 seconds. The process was repeated for each stamp width (25, 50 and 100µm) on different proteins (fibronectin and collagen). Thus, this allowed the glass coverslips to be patterned with 25, 50 and 100µm width of the fibronectin and collagen coated tracks separated by 25, 50 and 100µm width of uncoated tracks, respectively. Table 1 summarizes the micro-contact printing process. A protein free coverslip was used as a control.

Protocol For Cell Electroporation
In this study, the commercial electroporator ECM830 made by BTX Harvard apparatus was used for electroporating the HT29 cells in suspension before seeding onto the substrates. The low voltage (LV) mode of the BTX ECM 830 electroporator at a voltage of 240V with a 4mm electrode gap was used to achieve a 600V/cm electric field strength which was optimized earlier for HT29 cell line [18]. First of all, cells are detached using the subculture procedures. After neutralizing the effect of the detaching enzyme, 800µl of cells suspension at a concentration of 1×10 5 cells/ml was poured into a 4mm cuvette and then placed in BTX ECM 830 electroporator chamber. Electroporation was executed with an electric field of 600V/cm intensity for 500µs duration. Immediately after electroporation, the cuvette was moved into the biosafety hood.

Protocol of Plating Cells on Glass Coverslips
The glass coverslip was micro-contact printed with three different grating of the two types of ECM proteins. Next, the glass coverslip was placed, one in each well of a 6-well plate. As control, a protein free glass coverslip was also placed in another well. Next, 2ml of growth media was added to each of the wells. Next, 0.1ml of HT29 cells at concentrations of 100,000 cells/ml was seeded in each well. The six well plate was then incubated at 37 o C and 5% CO 2 for 48 hours. After 48 hours, cells were then imaged with standard phase contrast microscope. With the aid of DinoCapture2.0 software, cell alignment to the patterns were computed by measuring the angle between the cell long axis in relation to the stamp pattern, such that an angle of 0º represents 100% alignment to the pattern. Patterns that give the best cell alignment for each protein were also selected and seeded with electroporated HT29 cells of the same concentration as the untreated cells (0.1ml of a 100,000 cells/ml). The cells were also incubated under the same condition.

Alignment Angle and Cell Length Measurement
The DinoCapture2.0 Software was also used for measuring the alignment angle and cell length. The software is designed by the AnMo Electronic Corporation to work only with their product like the Dino camera. It is a free software program that offers users with quite a number of tools for analysis of both live video images and acquired pictures. It also has numerous features that provide users better skills and experience with a digital microscope.
Line measurement tool: This tool enables the user to measure the distance between two points. In order to measure distance or length with DinoCapture2.0 software, the followings steps were performed: 1. Acquire the images using microscope via Dino camera.
2. Open the image in DinoCapture2.0 software and select the magnification of the objective used in capturing the image. 3. Select the line measurement from the measurement tools as shown below. 4. Select a unit of measurement from software as shown below. 5. Left click on the first point and drag the line measurement tool to the end of the point and left click again to finish the measurement. 6. Repeat step four for all the lengths measurement. 7. Click on MPW icon on the toolbar and export the data to excel by clicking the export to excel icon on the MPW. A sample of a line measurement is shown in Figure 1 with the red line and blue writing. click again to finish the measurement. 6. Repeat step four for all the angles measurement. 7. Click on MPW icon on the toolbar and export the data to excel by clicking the export to excel icon on the MPW. A sample of an angle measurement is shown in Figure 2 with the red line and blue writing.

Statistical analysis
All experiments in this research were repeated three times and presented as the mean plus or minus the standard deviation (+/-SD). This is because triplicate is the minimum number to have for standard deviation and the number of replicates often depends on other factors such as cost of running the experiment. Data obtained in this research were tested for normality using a Kolmogorov-Smirnov test for normality. Data that showed a normal distribution with P-value greater than 0.05 were analysed through one-way analysis of variance (ANOVA) followed by post Hoc Turkey SHD test using SPSS software. For data that were not normally distributed, the Serial Mann-Whitney test was used. A Pvalue of less than 0.05 would be taken as data indicating a statistically significant difference. Figure 3 depicts the images of the PDMS stamps of different width (25, 50 and 100µm) under microscope. The PDMS stamps were used for the creation of the fibronectin and collagen patterns on the glass coverslips. The various ECM protein pattern was successfully produced as shown in Figure  4. The result of angular alignment is interpreted as the alignment angle of forty-five degrees (45 o ) or greater suggesting that orientation of cell to the pattern is random, that is, no alignment [5], [17]. Whereas, an alignment angle of less than 45 o implies that cell aligns to the pattern, where zero degrees designate complete alignment (100%) to the pattern [5], [19], [17].   10 Table 2 contains the data obtained from quantitative analysis of the mean angle of HT29 cell alignment on all the ECM protein used on different patterned substrates (25, 50 and 100µm) and the control substrate. The quantitative analysis of the data obtained revealed that the HT29 cells showed no alignment on control coverslip with mean angle of 54.  In all experimental data obtained with respect to cells angle of alignment, the data were found to be normally distributed (P > 0.05) and therefore one-way analysis of variance (ANOVA) followed by Post Hoc Turkey HSD test was used in analysing the data for statistically significant difference. The results showed that between fibronectin and collagen protein patterns there is a significant difference in angle of alignment on the entire stamp width pattern used (P < 0.05). This implies that alignment is better on fibronectin protein irrespective of the stamp size. It could therefore be stated that the HT29 binding to the surface is mostly via fibronectin specific integrin.

Measurement of Cell Alignment
The statistical results further revealed that there is no significant difference in alignment angle between 50 and 100µm width pattern of fibronectin protein (P > 0.05). Whereas, there was significant difference in alignment angle between 50 and 100µm width pattern of collagen protein (P < 0.05). Moreover, there was a significant difference in the angle of alignment between 50µm width pattern and the other pattern width (25µm and control substrates) of the two ECM protein utilised (P < 0.05 in all case). Similarly, there is a significant difference in the angle of alignment between 100µm width pattern and the other patterns used (25µm and control substrate) of the two ECM protein used (P < 0.05 in all case). Additionally, Figure 6 shows a graphical representation of quantitative data obtained for cell angular alignment. Small angle of alignment was signifying that the cells aligned better on that particular pattern.

Measurement of Cell Elongation or Cell Length
The result of cell elongation is interpreted as follows: the longer the cell length the better the alignment of the cell to the pattern [5], [17]. The result of cells elongations agrees/reflects well with the result of cell alignment, i.e. cells showing best alignment revealed the highest elongation. Table 3 shows the data obtained from quantitative analysis of the mean cell length of all the ECM protein used on different patterned substrates (25, 50 and 100µm), and control substrate. The mean cell length on the control substrate was 39.6µm ± 5.90. In the case of fibronectin, the mean cell lengths were 53.0µm ± 8.3, 88.8µm ± 13.3 and 71.0µm ± 11.8, respectively, on the 25, 50 and 100µm width patterned substrates. Furthermore, the mean cell lengths on the 25, 50 and 100µm width patterned substrates of collagen were 42.7µm ± 7.8, 58.5µm ± 6.2 and 50.5µm ± 7.7, respectively. The results showed that between fibronectin and collagen protein patterns, there is statistically significant difference in cell elongation in all the stamp sizes used with P < 0.05 in all cases. The 12 statistical results revealed that there is significant difference in the cell elongation between all other stamp sizes of the two ECM proteins used (P < 0.05). Except for the case of 25µm of collagen and control which showed no significant difference. Figure 7 shows the graphical representation of quantitative data obtained for the cell elongation. Longer cell elongation was signifying that the cells aligned better on that particular pattern. The result of the study showed that HT29 is most readily aligned to the 50 and 100µm stamp size. This could be connected to the cell size, whereby for smaller stamp size (relative to the average cell length of the HT29 cell line), the cell could not distinguish between the patterns protein lines and unpattern protein lines, in which case the cell could overlap [16]. Whereas for stamp size greater than the average cell length of the HT29 cell line, as in the case of 50 and 100µm stamp size, the cells could easily distinguish between the protein pattern lines and the protein un-pattern lines. Hence, they could easily follow the guidance cues [17], [19]. The results obtained in this research (results without PEF treatment) are qualitatively in agreement to that of Khaghani et al., (2008), Berend, (2012) and Sefat (2013) that also demonstrated that Chondrocyte, Keratinocyte cells and MG63 osteoblast bone cells, respectively, were most readily aligned to 50 and 100µm [16], [19], [20]. In order to have a better understanding, we further investigated the cell alignment and elongation with application of PEF.

Influence of PEF on Cell Alignment and Cell Elongation
Cell alignment on 50µm stamp pattern of fibronectin and collagen were the best for the respective protein patterns. Hence, these stamp sizes were used to investigate the influence of PEF on the HT29 cell line alignment on MCP surface. The cells were electrically treated before plating on the patterned substrates of 50µm for fibronectin and collagen. The cells were cultured for 48 hours with the same number of cells as in the case of the untreated cells and under the same physiological environment. Figure 8 shows the images of HT29 cell line after 48 hours of seeding on 50µm stamp pattern of fibronectin and collagen. The results of the HT29 cell alignment and elongation after exposure to PEF on the 50µm pattern of fibronectin and collagen have shown significant improvement. The angular alignments of HT29 cells were 4.0 o ± 1.2 and 11.2 o ± 3.5 for the 50µm pattern of fibronectin and collagen, respectively, under PEF treatment as shown in Table 4. On the other hand, the cell lengths of HT29 cells were 101.4µm ± 13.6 and 86.5µm ± 21.2 for the 50µm pattern of fibronectin and collagen, respectively, under PEF treatment as shown in Table 5. Moreover, Figures 9 and 10 show the graphical representation of the cell angular alignment and elongation of the HT29 cell line in the PEF treated and control group.   The data obtained from PEF treatment revealed that there was an improvement on the alignment of HT29 cell line on 50µm pattern of fibronectin and collagen. The angles of alignment decreased from 5.0 o ± 1.3 to 4.0 o ± 1.2 and from 16.0 o ± 3.4 to 11.2 o ± 3.5, respectively, for the fibronectin and collagen under PEF treatment. The results are also statistically significance (P = 0.003, i.e. P < 0.05) with a mean difference of 1.0 o and (P = 0.0001, i.e. P < 0.05) and mean difference of 4.8 o on the 50µm pattern of the fibronectin and collagen, respectively, under PEF exposure. Similarly, the cell elongation under PEF on the 50µm patterned of fibronectin and collagen increased from 88.8µm ± 13.3 to 101.4µm ± 13.6 and from 58.5µm ± 6.2 to 86.5µm ± 21.2, respectively. The results are also statistically significance (P = 0.001, i.e. P< 0.05) in both cases with a mean difference of 12.6µm and 28.0µm, on the 50µm pattern of fibronectin and collagen, respectively, under PEF exposure.
The result has shown that PEF has great influence on the HT29 cell line alignment and elongation. This could be due to the fact that PEF stimulates the integrin and cadherin which are the molecules responsible for cell-cell and cell-ECM adhesion [21], [22]. In addition, it could be that the PEF