Advanced Materials Research Vol 1112 (2015) pp 441-444 © (2015) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/AMR.1112.441
Submitted: 2014-08-31 Revised: 2015-02-13 Accepted: 2015-02-13
Wax-Impregnated Cotton Fabrics as Cell Culture Platform Norsamsiah Muhamad Wahab1, Syazwani Abdul Jamil1, Dwi Gustiono Riban2, Fadzilah Adibah Abdul Majid3, Mohamed Rafiq Abdul Kadir1 and Dedy Hermawan Bagus Wicaksono1,a 1
Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310, Johor Bahru, Johor, Malaysia 2
Pusat Teknologi Material, Badan Pengkajian dan Penerapan Teknologi (BPPT), Puspitek Serpong, Banten 15310, Indonesia
3
Department of Bioprocess Engineering, Faculty of Chemicals Engineering, Universiti Teknologi Malaysia, 81310, Johor Bahru, Johor, Malaysia a
[email protected]
Keywords: Wax, Cotton Fabrics, Cell Culture, Cell-based Assay.
Abstract. Microwell plates are widely used in various cell-based assay and drug screening. Usually these plates are made from non-biodegradable materials such as polystyrene or polyethylene. In this report, we propose the use of wax-impregnated cotton fabrics as an alternative microwell plate that is easy to fabricate, simple to use, sustainable and environmentally friendlier. Several researchers recently used soft lithography and photolithography technique to fabricate various cell culture platforms. Our proposed method consists instead of simple dipping and drying process. The platform is made of a series of fabrics being stacked and held together with various wax formulations. With the exception of the base layer, each fabric layer has a circular hole opening with increasing diameter towards the top layer; thereby forming a well where cells are cultured at its flat bottom. We characterized the chemical and physical properties of the platform surface which affect cells attachment and proliferation. These properties include the surface chemistry, hydrophobicity and roughness. We cultured human skin fibroblast (HSF 1184 Cell Line) on the platform as our preliminary proof of concept. We observed proliferation of the cells after 24 hours. The result indicates the potential use of the platform for future cell-based assay applications. Introduction The standard microwell plates are applied for pharmaceutical and cell culture applications. Conventionally, the microwell plates for cell platform are made from polystyrene and polyethylene [1, 2]. Recently, some researchers have conducted a series of studies on modifying the existing materials of microwells for cell-based assay [2-4]. They have fabricated microwell plates using materials that are less expensive [5], to reduce the cost of fabrication, as well as to simplify the fabrication procedures [6]. They have been using substrate materials such as glass [7, 8] polystyrene [2] and paper [9] for cell culture. Besides that, there is a group of researchers who used photolithography on polyester with automatic laser micromachining as an alternative fabrications technique [6]. These methods, however, still require special equipment and trained operators. The fabrication cost is therefore still considerably high. This study introduces a low cost, environmental friendly and simple technique for fabrication of wax impregnated cotton fabric formulations. We discovered a feature that allows wax to be used as a coating agent for cotton fabric and induce cell-material interactions. Besides, cotton fabrics has a high absorption rate [10] to mold with wax and is more flexible, making it resistant to wax cracking. In order to ensure that the cells are cultured optimally, we fabricated wax-impregnated platform with intermediate water contact angle range [11, 12] and optimum surface roughness.
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Experimental White plain cotton cloth fabrics (Mirota Batik, Surabaya, Indonesia) were used as raw materials in this experiment. In these experiments, Beeswax (Jadi Batek Gallery, Kuala Lumpur), Bleached Beeswax (cat. no. 243221 sigma Aldrich), Candelila (cat. no. 432288, sigma Aldrich), Carnauba (cat. no. 243213 sigma Aldrich) and Paraffin (cat. no. 327204 sigma Aldrich) were used for wax impregnated cotton fabrics; all wax formulations were with a uniform ratio 3:2:1 with 3 different combinations, exceptions to formulation (S2) 5:1 in sample 2 (S2). Samples compositions are as follows: sample 1 (S1) beeswax (Jadi Batek), candalila, carnauba, sample 2 (S2) bleached beeswax, paraffin, sample 3 (S3) beeswax (Jadi Batik), bleach beeswax, paraffin, and sample 4 (S4) beeswax (Jadi Batik), bleach beeswax and candalilla. Layers of wax-impregnated cotton fabrics with microwells were accordion-folded, followed by a subsequent layer without microwells at the bottom to create a stack of wax-impregnated cotton fabrics that resembles the shape of 96-well plates with an enclosed bottom part. The holes were made by the used of fiber etches (Silkpaint, Waldron) that functionalized to removes the fiber. The stack of wax-impregnated cotton fabrics was then immersed again in melted wax to ensure that all layers of the stack have been fully impregnated with wax. The completed wax-impregnated cotton fabric microwell plate was then left to dry. Results and Discussion From the spectrum, the hydrophilic groups were identified as –(C-O)- and -(C=O)-. 150
(S1)
arbitrary unit (a.u)
Surface chemistry. As can be seen in the FTIR spectrum Fig. 1 there are no significant differences in IR spectrum between different wax formulations. The presence of C-H stretching bonds indicates the presence of saturated carbons as observed at segment 2927.92 cm-1 and 2852.32 cm-1. Besides that, segment 1757.92 cm-1 corresponds to stretching C=O bonds, and indicates carbonyl function in all types of wax formulations. Aside from that, the presence of bonds at segment 1472.8 cm-1 corresponds to interactions of O-C-H. Segment 1173.11 cm-1 represents C-O stretching bonds corresponding to esters. Also, functional C-H bonds are apparent at segment 719.87 cm-1.
(S2)
100
(S3) 50
(S4)
0
4000
3500
3000
2500
2000
1500
1000
500
-1
wavelength (cm )
Fig. 1 The FTIR spectra of difference wax formulations.
Hydrophobicity. Wettability is an important surface attribute for in vitro studies; hence it was investigated on the platforms with different wax formulations by determining the contact angle. The contact angles of platforms S1, S2, S3 and S4 are 98.10o ± 0.39, 87.29o ± 2.66, 81.99o ± 0.69, and 93.69o ± 0.50 respectively. S1 was the most hydrophobic while S3 was the least. Many studies have shown that surface wettability is one of the most important physicochemical properties of biomaterials that regulate cell behavior [13]. Aside from that, there have been some reports that cells adhere and proliferate best on hydrophobic surfaces or surfaces with an intermediate contact angle of around 70º [12]. Surface Roughness. Surface roughness of the platforms with different wax formations were obtained by observation via optical profiler system. Surface roughnesses ranging from 3.642 µm to 15.552 µm were obtained. The surface roughness of platforms with S1, S2, S3 and S4 are 3.986 µm, 15.552 µm , 6.807 µm and 3.642 µm respectively. Of the four, platforms with S2 composition had the roughest surface with an average roughness of 15.552 µm. Surface roughness of platforms that have undergone certain treatment are known to be different than that of commercial origin. In one report, the surface roughness of a treated platform was found to be 18.7 nm, more than 3 times
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compared to the surface roughness of commercial platforms (5.36 nm) [12]. Our study agrees with this; wax-impregnated cotton fabrics with wax formulation S2 is rougher (15.5 µm) and it may allow more cells to attached the platform.
3.0
R elative O ptical D ensity (% O D )
MTT Assays for Cell proliferation. The MTT assay was used to quantify the viability of cell on different platforms. The relative optical density was measured to determine the cell proliferation at a given time; to assess the effects of different wax formulations on cell growth. From Fig. 2, it is clear that cell proliferation in all four platforms far exceed that of commercial 96-well plates. The higher surface roughness of the wax-impregnated platforms contributes to their higher surface area, while keeping the microwell diameter to be consistent with commercial microwell plates. A platform with S2 composition promotes better cell proliferation, followed by S3, S1 and S4. Cell viability also varies amongst them. The use of wax-impregnated cotton fabrics for culturing HSF 1184 cells seems to positively correlate with cell viability (p
