Dynamic Cell Patterning with Microparticle Self-Assembly

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DYNAMIC CELL PATTERNING WITH MICROPARTICLE SELF- ASSEMBLY W. Dai, K.N. Ren, Y.Z. Zheng and H.K. Wu* Department of Chemistry, The Hong Kong University of Science and Technology Clear Water Bay, Kowloon, Hong Kong

ABSTRACT In this paper, we introduce a method based on microparticle self-assembly for cell dynamic patterning. UV induced Norland Optical Adhesive (NOA) was used for the fabrication of microparticles with various shapes and sizes. The microparticles then were fixed in alginate with one surface exposed to the external free solution. After the cells attached to the exposed surfaces, the microparticles were released and finally assembled manually or automatically. This method could be used as a good platform for dynamic cell patterning. Also, it showed promising potential in large-scale co- patterning of multi types of cells at the same time.

KEYWORDS: Dynamic cell patterning, self-assembly, Norland Optical Adhesive (NOA) microparticle

INTRODUCTION Microfabrication techniques combined with surface chemistry and material science have provided important and use- ful tools to pattern cells at microscale to explore and remodel in vitro cell behaviors. Currently, many methods can achieve the micropatterning of cells on flat substrates by selectively modifying different surface areas, e.g., photolitho- graphy, microcontact printing [1, 2], and microfluidics[3]. However, it is non-trivial for them to form patterns of multiple types of cells, especially when cell type is more than two. Moreover, once cells are patterned, it is difficult to dynamically change the cell pattern as wanted on the substrate. Herein, we propose a new concept for cell micropatterning by dynamically assembling cell-attached microparticles. We fabricate cell-attached microparticles of various shapes and sizes that are defined by soft lithography, and assemble them in microwell arrays on a substrate, which have complemen- tary shapes and sizes. In this method, selective surface modifications are not required and multiple cells can be conveniently patterned with little more efforts than patterning one type of cells. Also, it is possible for this method to dynamically pattern cells by replacing the microparticles on the surface anytime during cell culture.

EXPERIMENTAL 1. Fabrication of microparticles The original master for fabrication of rounded bottom microparticles was made in positive photoresist AZ4903 (150 μm tall) by following the conventional photolithographic procedure. After development, the master was put into a 120 ℃ oven for about 20 min to melt the photoresist into a rounded profile. The pattern then was molded into PDMS to create masters of microwells with rounded bottom. The microparticles were made of NOA. The fabrication scheme is shown in Fig. 1 A. The PDMS wells were filled with NOA prepolymer and aligned to the corresponding photomask. After exposed to UV light to cure the NOA prepolymer, the formed microparticles were embedded in alginate gel. The flat surfaces of the NOA particles were exposed for subsequent cell attachment (Fig. 1 A). After cells were cultured on the NOA microparticles for two days, the microparticles were released with ethylene diamine tetraacetic acid (EDTA) (Fig. 2 C&D), which can chelate calcium ions and dissolve the alginate gel. Finally, the microparticles with cells attached were collected, and assembled together on a substrate with designed patterns of holes (Fig. 1 B). By changing the original photoresist pattern, the microparticles with various shapes and sizes could be generated (Fig. 2 A&B). In our experiment, we fabricated two types of microparticles. One was of pure NOA (Fig. 3) with sizes larger than 300 μm. The other type was magnetic NOA microparticles with sizes smaller than 300 μm. The fabrication processes for them were slightly different. Ferric magnetic nanoparticles were well dispersed in acetone to prevent aggregation. This nanoparticle-acetone solution was mixed with NOA prepolymer. After the acetone evaporated completely, the NOA prepolymer was exposed to UV light for curing. Other steps followed the procedure above.

2. Manipulation of the microparticles The microparticles were transferred, fixed in gel, seeded by cells and released for assembly. The procedure is shown in Fig. 1A and 1B. After cells firmly attached to the microparticles within a few hours, the particles were kept in the cell culture medium for two days before they were released from the alginate hydrogel. The released microparticles (with cell attached) were assembled into a substrate with patterned complimentary holes. The whole assembly process was operated as gently as possible to minimize any harm to the cells in a 37 ºC cell chamber .

A B

Figure 1: Scheme of fabrication and manipulation of NOA microparticles. (A) the NOA microparticles are molded and transferred to a silver coated glass substrate. Then they are fixed by hydrogel and seeded by cells.(B) the microparticles of different types are assembled together on a predesigned complimentary substrate.

RESULTS AND DISCUSSION Current approaches of cell patterning are generally based on two techniques: surface modification and microfluidics. In these methods, once cells have been patterned onto surfaces, it is difficult to remove them or replace them with other cells. It is desired in many applications to be able to dynamically change cell patterns during the study. Furthermore, arranging cells of multiple types in arbitrary patterns is still an arduous task. Our strategy of assembling cell-attached microparticles is aimed to provide a different tool for both dynamical and multiple cell patterning. In our method, each microparticle with cells attached acts as the unit for assembly; the microparticle is the carrier for the cells that facilitates both the manipulation and the assembly of the cells. We chose NOA as the material for the fabrication of our microparticles because its prepolymer can be conveniently cured with UV light and the cured polymer is transparent, soft and biocompatible for cell culture. All microparticles had one flat surface and a rounded bottom. This particular shape is important in our method in that (1) the microparticles are easier to find corresponding holes and to assemble into the holes on the substrate and (2) after the assembly, all cells are patterned on a flat surface. To facilitate the assembly process, magnetic nanoparticles were added to the NOA prepolymer during the fabrication to produce magnetic microparticles. A magnetic field of a magnet placed nearby was used to guide the microparticles to assemble into the correct holes on the substrate. Also, because of their high density, the magnetic nanoparticles settled down at the bottom of the microparticles during the fabrication of the NOA particles. The heavy bottom of the NOA microparticles forced them to orient correctly (flat surface facing up) in solution, which made it easier for the assembly and minimized the harm to the cells on the flate surface during the assembly process. .

Figure 3. Fluorescent image of manual manipulated microparticles. (A)&(B) the NOA microparticles of cir- cle, square and triangle in shape were manually ar- ranged to form a simple pattern.

Figure 2: Image of microparticles transferred and with cell attachment. (A)&(B) the NOA microparticles in 100μm and 200μm in diameter had been transferred to a silver coated glass slide. (C) after two days culture, the cells grew well on fixed NOA microparticles. (D) the microparticles were released from gel and collected.

164 Figure 4: The cell-attached magnetic microparticles as- (A) phase contrast image; (B) fluorescent image. sembled into corresponding holes on a flat substrate. We also found that the alginate hydrogel can affect the cells that grow on it. For the gelation of the alginate solution, low calcium concentrations made hydrogel so soft that it was difficult to handle and high calcium concentrations could significantly affect the cells that were seeded on the microparticles. Our experimental results show that 0.1 M calcium chloride was the optimal concentration for alginate gelation. Also, during the dissolution of the alginate hydrogel with EDTA, we normally treated the hydrogel with 5mM EDTA for ~20-30 min to keep any effects from the EDTA on the cells minimal. To facilitate the assembly, the size of the holes on the substrate was normally designed to be slightly larger than (~10 μm larger in lateral size) that of their corresponding microparticles. The assembly of larger NOA microparticles and smaller magnetic microparticles are shown in Fig. 3 and Fig. 4, respectively.

CONCLUSION We demonstrated a novel method of patterning biological cells with a convenient fabrication process at a low cost. It shows potentials for large-scale co-patterning of different kinds of cells simultaneously and for dynamical cell patterning.

ACKNOWLEDGEMENTS Thanks for the financial support from DuPont Young Professor Award, Hong Kong RGC (604509 and N_HKUST617109), and the NSNT program at HKUST.

REFERENCES [1] D. Falconnet, M. Textor, “Surface engineering approaches to micropattern surfaces for cell-based assays,” Biomaterials, 27, 3044 (2006). [2] C.M. Nelson, C.S. Chen, “Degradation of micropatterned surfaces by cell-dependent and -independent processes,” Langmuir, 19,1493 (2003). [3] D.T. Chiu, G.M. Whitesides, “Patterned deposition of cells and proteins onto surfaces by using three-dimensional microfluidic systems,” PNAS, 97, 2408 (2000).

CONTACT H. K. Wu, tel:852-23587246; [email protected]

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