Design and Application of Multiscale Double-layered Microfluidic Device for Multi-cellular Co-culture

Abstract

In this thesis, we propose about designing new microfluidic device to culture microvessel in three-dimensional space and suggest methods to analyze microvessel behavior to external condition. The device have cell culture region both in horizontal and vertical direction providing in vivo like culture condition among cells. Mainly the devices are made in flexible polymer PDMS (polydimethylsiloxane) and composed with two-layered microchannel with thin porous membrane in between. Considering the pore size of the membrane is important because it can vary the aim of the experiment. The biggest advantage of two-layered microchannel with porous membrane is that the device can provide both isolated region and shared region in cell culture. Cells can be cultured independently in the isolated culture region while they can also share their secreting factors and even their exclusive functions through shared region. For these reasons, there are many researches and publishes dealing cell responses using this type of microfluidic device. However, there are also limitations in fabricating methodology to provide various applications so far. Here, we have designed two types of two-layered microfluidic culture platform which can be considered invaluable system for further experiment in the field of cell culture. Each platform have an individual aim respectively

(1) Platform #1: we have established self-assembled microvessel network and analyzed microvessel damage by treating with toxic chemical

(2) Platform #2: we have observed microvessel behavior by co-culturing with cancer spheroid. Both platforms provide chemical diffusion through porous membrane at the intersecting region of upper and lower microchannel. Cells in this region have more opportunity to be nourished with fresh media which have direct influence on cell viability. The platform #1, we have precisely aligned 0.4 μm porous polyester membrane in between the layered microchannel. Upper part microchannel is designed to culture and differentiate endothelial cells into microvessel. Once the microvessel is assembled and stabilized, toxic chemical – in this case low concentration of SDS (sodium dodecyl sulfate) – is introduced into lower part of the microchannel. As SDS molecule is tiny enough to penetrate through the porous membrane, the external side of the microvessel is exposed to the chemical instantly. According to the toxicity of the chemical, microvessel can be maintained or damaged gradually. By mixing fluorescent molecule with the chemical, we were able to observe the fluorescent molecule penetrate through microvessel wall which can be an indicator of considering vessel wall damage. The platform #2, we have observed microvessel behavior by co-culturing with cancer spheroid. The strong point of this platform is that we were able to culture μm sized microvessel and mm sized cancer spheroid at the same time while observing the blood vessel sprouts in vertical direction as well. In the paper, we present method steps to fabricate 200 μm sized pores in 75 μm thick PDMS membrane. This micro-pore can connect upper part and lower part and also guide microvessel to sprout towards the upper part. Upper part of the device is Ø6 mm open reservoir and lower part of the device is microchannel design to culture and differentiate endothelial cells into microvessel. During microvessel development, cancer spheroid is co-cultured in the upper part of the same device. As cancer spheroid secret plenty of growth factors to induce formation of the microvessel, we were able to observe thicker microvessel growth in the presence of cancer spheroid. With the platform #2, we also tested selective flow by flowing two types of fluorescent dye through upper and lower part of the device. As lower part is the region where the microvessel is assembled, dye introduced in lower part can flow through microvessel lumen while dye introduced in upper part can access through micropore to external side of the vessel. In this way, microvessel can experience different types of media supply at inner-and external side of the blood vessel barrier, which is usual condition for blood vessels in vivo. This condition realized in vitro is meaningful because selecting media is very important element to co-culture two types of different cells in one system. To conclude, in order to fully develop the in vitro culture condition close to in vivo, we need to consider not only the cell types but also the spatial relevance among them. Reminding that our designed platform provide horizontal and vertical co-culture, most of the three-dimensional culture condition can be demonstrated in the device. Our development enables to overcome the difficulties experienced so far with existing devices and offers opportunity to design various experimental concepts in the field of microfluidic bioengineering.