Microfluidic Cell Culture Systems

Christopher Bettinger, Jeffrey T Borenstein, Sarah L Tao, Christopher Bettinger, Jeffrey T Borenstein, Sarah L Tao

• Publisher: William Andrew
• Year: 2012
• ISBN:
  9781437734607
• Topic: Biological Sciences
• Subtopic: Biotechnology
• Index: Biological Sciences

The fields of microfluidics and BioMEMS are significantly impacting cell biology research and applications through the application of engineering solutions to human disease and health problems. The dimensions of microfluidic channels are well suited to the physical scale of biological cells, and the many advantages of microfluidics make it an attractive platform for new techniques in biology.

This new professional reference applies the techniques of microsystems to cell culture applications. The authors provide a thoroughly practical guide to the principles of microfluidic device design and operation and their application to cell culture techniques. The resulting book is crammed with strategies and techniques that can be immediately deployed in the lab. Equally, the insights into cell culture applications will provide those involved in traditional microfluidics and BioMEMS with an understanding of the specific demands and opportunities presented by biological applications.

The goal is to guide new and interested researchers and technology developers to the important areas and state-of-the-practice strategies that will enhance the efficiency and value of their technologies, devices and biomedical products.

• Provides insights into the design and development of microfluidic systems with a specific focus on cell culture applications
• Focuses on strategies and techniques for the design and fabrication of microfluidic systems and devices for cell culture
• Provides balanced coverage of microsystems engineering and bioengineering

Part 1

Materials and Fabrication Methods

Chapter 1 Microfluidic Cell Culture Platforms with Embedded Nanoscale Features

Chapter 2 Microvascular Networks for Tissue Engineering

Chapter 3 Microfluidics for Engineering 3D Tissues and Cellular Microenvironments

Chapter 4 Fabrication of Advanced Microcontainer Arrays for Perfused 3D Cell Culture in Microfluidic Bioreactors

Chapter 5 Mechanobiological Approaches for the Control of Cell Motility

Chapter 6 Transport Models for Three-Dimensional Cell Culture Systems

Chapter 1

Microfluidic Cell Culture Platforms with Embedded Nanoscale Features

1.1 Introduction

In most living tissues, cells typically reside in a microenvironment where cells interact with the extracellular matrix (ECM) as well as the neighboring cells. The ECM is composed of diverse biomacromolecules including glycosaminoglycans, fibrous proteins such as collagen, elastin, and fibronectin, and nonfibrous proteins such as growth factors and cytokines, with size ranging from several to hundreds of nanometers. For example, collagens show a hierarchical structure of collagen fibrils of 10–300 nm in diameter to collagen fibers up to several microns in diameter [1]. The ECM constructed from these biomacromolecules often includes significant topography at the nanoscale. Basement membrane, for example, is a ubiquitous component of ECM that plays an important role in tissue development and organization. Basement membranes manifest a complex mixture of pores, ridges, and fibers with sizes in the nanometer range [2–6]. In an ECM of connective tissues, the fibrous proteins penetrate through a hydrophilic matrix composed of proteoglycans and interstitial fluids to form a 3-D network with nonfibrous proteins dispersed inside [7]. Although the interstitial fluid flows, driven by dynamic stresses or/and pressure gradients, can be extremely slow (between 0.1 and 4.0 µm/s [8]), they nevertheless play an important role in nutrient transport, tissue maintenance, and remodeling, as well as establishment of the microenvironment [9,10]. Together, the ECM serves as a structural support for cells and provides, in concert with the spatiotemporally arranged signaling molecules and external stimuli, topographical and mechanical cues that serve to regulate the phenotypes and function of mammalian cells.

A number of recent findings underscore the phenomenon that mammalian cells have the capacity to respond to environmental features at the nanoscale on synthetic surfaces [11–15]. In addition to inducing pronounced changes to cell morphology, and consequently gene expression, nanotopographical cues could potentially help induce the differentiation of stem cells into certain lineages [11,13,15]. For instance, by manipulating levels of nanoscale order of nanopits (120 nm in diameter, 100 in. depth), Dalby et al. [11] have shown that the near-square topography stimulates human mesenchymal stem cells (hMSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements at levels similar to the cells cultured with osteogenic media; highly ordered nanotopography permits prolonged retention of multipotency of hMSCs [12]. Another study shows that hMSCs cultured on small (~30 nm diameter) TiO₂ nanotubes exhibit promoted adhesions without noticeable levels of differentiation, whereas larger (~70–100 nm) nanotubes elicit a dramatic cell elongation (~10-fold increase compared with the flat control), which induces cytoskeletal stress and selective differentiation into osteoblast-like cells [13]. In addition, fluid shear stress can remodel cytoskeletal organization, alter gene and protein expression, and influence cell proliferation [16]. For instance, hMSCs seeded on silicate-substituted tricalcium phosphate scaffolds demonstrate improved proliferation and osteogenesis in the flow perfusion culture compared with the static cell culture. Flow perfusion culture also facilitates homogenous distribution of cells and ECM proteins throughout the entire scaffold, whereas only a peripheral layer is obtained after static culture [17]. Intriguingly, it has been reported that shear stress promoted the differentiation of smooth muscle cells and embryonic stem cells into endothelial cells [18,19].

It is thus vital to integrate these external cues and engineer a dynamic cell culture platform with embedded nanoscale features for cellular studies [20]. Microfluidic systems, in particular the ones based on poly(dimethylsiloxane) (PDMS), have been extensively applied to the studies of cell stimulation and selection [21,22], cell lysis and biochemical analysis [23], cell manipulation [24,25], and cell culture [26–30]. In cell culture, the microfluidic setup can work as a circulatory system, enhance mass transfer of nutrients, gases, and metabolites, provide a spatiotemporal control of delivery of signaling molecules, and create mechanical strain in the physiological range. However, it is challenging to integrate nanoscale features into a microfluidic platform [31].

The two main challenges of fabricating a microfluidic platform with embedded nanoscale features are creating a large area of nanopatterned surface for cell culture and preserving the fidelity of the nanotopography after assembly. In this chapter, we first review techniques for engineering nanoscale features. Particular emphasis is placed on generating a large area of nanopatterned surface. The assembly techniques suitable for PDMS-based microfluidic platforms are then discussed. Several studies that engineer microfluidic platforms with embedded nanoscale features for cancer detection and stem cell research are discussed.

1.2 Engineering of nanoscale features

Nanotechnology is referred to a length scale of 1–100 nm in the physical realm [32]. However, considering that cellular structures are built from biomolecules, it is appropriate to extend its length scale beyond 100 nm and upward to the submicrometer range. Here, we review the conventional techniques for fabricating irregular and regular nanoscale features, and generating large area of nanopatterned surfaces.

1.2.1 Fabrication of irregular nanoscale features

Chemical etching, in addition to modifying the surface chemistry of cell culture substrates [33,34], may generate irregular nanoscale features [34]. By combining silicon etching and silver deposition, Peng et al. [35] synthesized large silicon nanowire arrays. The diameters of the nanowires range from 30 to 150 nm and their lengths are up to 50 µm. These nanowire arrays provide numerous surface area and have been utilized to capture circulating tumor cells (CTCs) [36].

Self-assembly offers a simple and low-cost process to make large-area periodic nanostructures. It requires only the mixing of components such as colloidal nanoparticles and polymers, which can be spontaneously assembled into a nanotextured structure. Colloidal lithography forms nanopatterns based on the colloid–colloid and colloid–substrate interactions. The resulting nanopatterns can either be used in the current form or as masks. Lipski et al. [37] aminosilated silica nanoparticles of 50 and 100 nm and self-assembled them onto metal substrates, thus demonstrating a single-step fabrication of nanoscale surface with controllable surface characteristics of charge, roughness, and chemistry. Some polymer blends spontaneously undergo phase separation during spin coating. This approach, often called polymer demixing, can produce different topographies such as pits, islands, or ribbons of varying height. The ratio of the polymers used varies with the topography, and the concentration of polymer solution changes the feature sizes [38,39]. Demixing the polystyrene (PS) and poly(4-bromostyrene) (PS/PBrS) system can form nanoislands with a variety of sizes, and PS segregates to the surface upon annealing [40–42]. Another example is PS–poly(n-butylmethacrylate) (PS/PnBMA), in which the PnBMA segregates spontaneously to cover the surface at ambient temperature without annealing [43,44]. Both systems can provide the nanotopography with a single chemistry at the top surface.

Self-assembly as a stand-alone method for nanofabrication is presently unable to produce structures with precise spatial positioning and arbitrary shapes [45]. Higher density of defects compared to conventional nanofabrication techniques is also a drawback. Templated self-assembly, which uses top-down lithographic approaches to prov...