MATERIALS AND METHODS

Cell lines and culture conditions: In this study, HeLa cells were used. HeLa cells are cultured in standard culture flask in Rosewell Park Memorial Institute (RPMI) 1640 media (Sigma) with 10% fetal bovine serum until 90% confluence. The cell was maintained in the atmosphere of 5% CO₂ at 37°C and were split once reaching confluence, usually every 5-6 days⁸ (Fig. 1).

Microcontact printing method: Figure 2 shows the concept of the fabricating high resolution patterns of protein using a μCP method with a PDMS stamp start⁹ at A and B.

Fig. 1:	HeLa cell with 50% confluence

Fig. 2:	Process flow schematic of microcontact printing method

Where, A show step of inking the PDMS stamp with protein solution and B shows the transferring the proteins to a substrate area or contact or it called step of stamping PDMS with protein at the new substrate by applying a small amount of force and C shows a patterned surface after remove the stamp from the stamping step, the ink is patterned on the glass substrate¹⁰.

Electroporation technique: For the electroporation technique, Fig. 3 and 4 shows the main components of the experimental setup.

Fig. 3(a-c):	(a) High voltage pulsed system (ECM® 830), (b) Chamlide TC stage and (c) Microchamber slide (Chamlide EC)

Fig. 4:	Nikon inverted microscope (Ti series)

A nikon inverted research microscope (Ti series) offers improved speed, increased flexibility and efficient multi-mode microscopy as part of fully integrated microscope system for cell live imaging. In order to expose biological cells to high voltage pulse electric field, two main subsystems are needed: (1) Imaging chamber system (Chamlide EC) which is connected with the main components, dedicated to the applications of field stimulation, which supplied with a pair of platinum electrodes¹¹ and (2) High voltage pulsed system (ECM® 830) which allow to deliver 10-600 μsec duration of pulses with the adjusted amplitude upto 3 kV¹².

RESULTS AND DISCUSSION

Patterning test with Fetal Bovine Serum (FBS) proteins: Fetal Bovine Serum (FBS) is used initially to test μCP functionality as in protein patterning. Figure 5 shows that the protein patterning outcome by using microcontact printing method with variety size such as 100, 50, 25 and10 μm. The variety size of stamp used to identify which pattern size will be suitable in microcontact printing method to analyze the cell alignment activity close to 100%.

Electroporation effect on HeLa cell cultured on protein surface and empty surface: With the availability of the HeLa cancer cell, this test was conducted in order to see the effect of the electroporation technique on HeLa cancer cell with the protein solution, which is in this experiment we used fibronectin and the electroporation technique we used 2000 kV with pulse length of 30 µsec in order to achieve high electrical field intensity.

Fig. 5(a-d):	Patterns of proteins by microcontact printing method with variety of size, (a) 75 µm, (b) 250 µm, (c) 300 µm and (d) 30 µm

Fig. 6:	Electroporation effect on HeLa cell within 24 h

Fig. 7:	Graph shows a confluence rate of HeLa cell in this test

Where in this experiment, in one well, it divides into two sections of the substrate. Which is the left side is the empty surface while the right side is the protein surface (Fig. 6).

It had been left for 24 h to see the effect of the electroporation technique on HeLa with the protein solution. After 24 h, the result as Fig. 7 shows that the proliferation rate of HeLa cell with EP is increased dramatically at 24 h compared with HeLa cell without EP and also most of HeLa cell attracts with protein surface with the ratio confluence 3:7. This shows that HeLa cell can be used in the fabricating process on the next objective of this study.

CONCLUSION

In conclusion, the preliminary result shown in the previous section, confirm the cell adhesion and proliferation occurs mainly on the fibronectin coated area. By comparing the previous research on the microcontact printing and electroporation, there is minor study on the combination of these two method. The basic concepts and techniques of electroporation and microcontact printing were highlighted. Both EP and μCP were found to be related to wound healing process, depending on the level of their threshold and application (gene therapy, electrochemotherapy and wound healing or tissue ablation). Thus, investigations of EP and μCP showed that the two methods can be combined in in vitro to see the cell response to μCP with the PEF applied to it. Important parameters (pulse number and pulse duration) should be considered along with MCP method/technique, which include the cell type and system configuration. We strongly believe that the result of this study can lead to the development of wound healing and cancer treatment.

ACKNOWLEDGMENTS

The researchers wish to acknowledge the financial support by UTHM GIPS grant sponsor. We would also thank to individual who have helped in this study.