Using Microfabrication and Electrostatic Layer-by-layer (LbL) Self-Assembly Technologies to Improve the Growth and Alignment of Smooth Muscle Cells 1
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Mengyan Li , Hua Ai , David K. Mills , Yuri M. Lvov , Michael J. McShane1, Bruce K Gale Institute for Micromanufacturing, Department of Biomedical Engineering, College of Engineering and Science 2 School of Biological Sciences, College of Applied and Natural Sciences 3Department of Mechanical Engineering, University of Utah Louisiana Tech University, Ruston LA 71272, USA Phone: (801)585-5944, Fax: (801)585-9826; e-mail:
[email protected]
ABSTRACT: Smooth muscle cells (SMCs) were cultured on polydimethylsiloxane (PDMS) based cell culture substrates. Two types experiments were performed to address the cell behaviors on these substrates. One was culturing smooth muscle cells on bare PDMS flat surfaces and gelatin coated PDMS flat surfaces deposited using electrostatic layer-by-layer self-assembly technology. The other was culturing smooth muscle cells on two microstructured PDMS microchannel substrates and PDMS flat surface substrates. The microchannels are 5-µm channels (line width = 5 µm, spacing width = 5 µm, depth = 5 µm) and 100-µm channels (line width = 100 µm, spacing width = 100 µm, depth = 50 µm) respectively. All substrates were coated with multilayers (50 nm in thickness) of gelatin using electrostatic layer-by-layer self-assembly technology in order to improve the attachment of the cells. We concluded that surface treatment, such as gelatin coating, is able to help smooth muscle cells attach on PDMS substrates. Accordingly, it will increase the potential growth of cells on the engineered PDMS substrates. Second, smooth muscle cells showed a clear preference of alignment long the channel sidewall on the 100-µm channel substrate as compared to that on the flat surface substrate. Microchannels are able to align the growth of smooth muscle cells, and the ability of controlling the alignment depends on the dimension of the microstructures, as well as the surface treatment for increasing cell attachment. Microfabrication and electrostatic layer-by-layer self-assembly technologies have significant potential for application in the field of tissue engineering.
I. INTRODUCTION Cells play a major role in building and maintaining tissue functions in their innate environment. However, after cells are removed from their innate environment to in vitro environment, they lose their in vivo normal behavior. Therefore, a principal objective of tissue engineering is to reach a fundamental understanding of the factors in the microenvironment surrounding cells, which can induce and affect the basic functions of cells. Using microfabrication technologies[1,2,3], we may design and fabricate desired substrates for cell culture, and together with electrostatic layer-by-layer (LbL) technology[4,5,6], we may make a treatment of the substrate surface to improve the growth and alignment of smooth muscle cells (SMCs).
II. METHODOLOGY A. Substrate Fabrication PDMS flat surface substrates and microchannel substrates were prepared for the experiment. Later on, we coated multilayers of gelatin in the PDMS substrate surface with electrostatic layer-by-layer self-assembly technology to evaluate the efficiency of the gelatin surface treatment for the growth of smooth muscle cells and the function of microchannels for the alignment of smooth muscle cells. 1) Photolithography In our experiments, we applied AZ 1813, a popular positive photoresist to fabricate the photoresist pattern as the mask for later ICP etching. The photoresist pattern was fabricated using a standard recipe as following: first, silicon substrate was prebaked at 115 °C for 2 minutes, then spun AZ 1813 at 1000 RPM for 50 seconds after 10 second ramp to achieve the 2 µm thickness. After spining, the substrate was soft baked for 90 seconds at 115 °C, then exposed to 20 mW/cm2 of UV light for 6 seconds followed by development in AZ 300 developer for 1 minute to get the complete photoresist pattern. 2) ICP etching Inductively-coupled plasma (ICP) etching is widely used for etching materials in micromachining applications. In ICP systems, very high aspect ratio etching has been demonstrated. The recipe we used in our experiment to obtain the 5-50 µm deep microchannels was indicated in Table 1. 3) Soft lithography Soft lithography represents an alternative set of techniques for fabricating micro- and nanostructures. Polydimethylsiloxane (PDMS) elastomer replication has been utilized mostly in BioMEMS. PDMS is durable, optically transparent, and inexpensive. In our experiment, microchannel patterned PDMS substrates are the major scaffolds for the cell culture study. Using silicon patterns obtained previously, PDMS microchannels were fabricated with a typical molding procedure. By
Table 1 Recipe of ICP etching for fabrication of silicon mold
Source Power SF6 flow rate / time C4F8 flow rate / time Pressure (%) Temperature Bias Power Process time
180 W 300 sccm / 7s 150 sccm / 3s 20 % 20 °C 30 W Depends on etching depth
reversing the silicon patterns, we obtained the microchannles with the same shape and spacing given for the silicon substrates in PDMS. The PDMS (Sylgard 184, Dow Corning, Midland, MI) used was supplied as twopart liquid component kit comprised of a base and a curing agent. The liquid components were thoroughly mixed in the ratio of 1 part curing agent to 10 parts silicone elastomer. The molding process was performed with the assistance of a vacuum pump to insure a quality reproduction of the silicon microchannels. The PDMS film was then peeled from the silicon substrate to obtain the reversed PDMS substrate. The final dimensions of PDMS microchannel are 5 µm and 100 µm. Most of our experimental results are based on this kind of material. 4) Electrostatic layer-by-layer self-assembly As the experiment was designed, we applied the electrostatic layer-by-layer assembly technique and procedures on the PDMS cell culture substrate using the standard assembly procedure as following: a) Take aqueous solutions of polyions - Poly styrenesulfonate (PSS), Poly dimethyldiallylammonium chloride (PDDS) and gelatin at a concentration of 1-3 mg/mL and at pH 7.4. PSS and gelatin are negatively charged. PDDA is positively charged. b) Take a PDMS substrate carrrying a negative surface charge. c) Carry out alternate immersion of the substrate in polyion and gelatin solution for 10-15 minutes with 1 minute intermediate water rinse. d) Dry the sample using a stream of nitrogen. e) Repeat step a) to step d). In our experiment, five double-layered PDDA/PSS were coated on the bare PDMS substrate as the precursor, and then another five double-layered biocompatible PDDA/Gelatin were coated as the outmost surface for cell culture. The overall thickness is about 50 nm. B. Monolayer Cell Culture Generally, in our experiments, smooth muscle cells (rat aortic smooth muscle cells) were obtained from Louisiana State University Medical Center at Shreveport. RPMI culture media, consisting of 5.2 g RPMI powder (HyClone, HyQRPMI-1640 Medium cell culture reagents), 1 g Sodium Bicarbonate powder, 450 mL type I water, 50 mL Fetal Bovine Serum (FBS) and 1 mL 10x antibiotic/antimicotic (ABAM), was used for the culture
of all cells. The substrates were sterilized in ethanol for at least 10 minutes on each side before being transferred to cell culture dishes containing the RPMI media. The cells were observed daily using an optical phase contrast microscope for a period of up to two weeks for each culture system. The RPMI media was changed every other day over the course of the culture. C. Fluorescent Staining Cells After observing the growth of the cultured cells and taking pictures using optical microscopy during the first two weeks of culture, we fixed and stained the cells in order to compare the results obtained from the optical images and further prove our experimental results and conclusions. We used a nuclear marker to stain the nuclei of smooth muscle cells. This nuclear stain is a very useful method to count the cell numbers even in situations in which we can not clearly see the cells using a general phase contrast optical microscope. As a nuclear stain, we used HOECHST 33342 (Bisbenzimide H33342 (MW: 615.99), Nr.15091, Serva, Heidelberg, Germany, Nr.B-2261, Sigma, St.Louis, USA). The working solution is 1:1,000 dilution of stock solution in PBS. Following is the procedure that we used to fix and stain smooth muscle cells in our experiment: a) Remove PRMI media from the cell culture dish. b) Rinse the substrate with PBS twice. c) Immerse the substrate in ethanol to fix the cells for 10 min at room temperature. d) Rinse the substrate with PBS twice. e) Immerse the substrate in Hoechst 33342 dye and keep in the incubator at 37°C for staining for 20 min. f) Rinse the substrate with PBS twice. g) Observe with fluorescent microscope (Nikon, ECLIPSE TS100/TS100-F inverted microscope). D. Data Measurement and Statistical Analysis In our experiments, we compared the number of cells growing on the bare PDMS flat surface and that on the gelatin coated PDMS flat surface. We also measured the alignment angles on the flat PDMS surface substrate and the 100-µm channel PDMS substrate. From these collected data, we investigated the attachment and spreading of smooth muscle cells on the microfabricated substrates as well as the biocompatibility of the materials after surface modifications. Both of the methods are based on optical microscopy images collected during cell culture. 1) Data measurement The easiest way to count the cell numbers is to take several pictures of the cells cultured on the bare PDMS flat surface and on the gelatin coated PDMS flat surface during the cell culture. Then, the cell numbers were manually counted and the average cell numbers were calculated. The alignment angle is defined as the absolute value of the angle between the long axis of smooth muscle cells and a defined reference line. After
measuring the alignment angle of each cell on the images, the alignment angles were classified into four categories. 2) Statistical analysis Based on the observations and data collected, we are able to use χ2 (Chi square test) for parameter estimation and hypothesis testing, and to draw a statistical conclusion from the final cell culture experiments. If there is a significant difference (P
