Abstract
For many types of cells, behavior in two-dimensional (2D) culture differs from that in three-dimensional (3D) culture. Among biologists, 2D culture on treated plastic surfaces is currently the most popular method for cell culture. In 3D, no analogous standard method—one that is similarly convenient, flexible, and reproducible—exists. This paper describes a soft-lithographic method to encapsulate cells in 3D gel objects (modules) in a variety of simple shapes (cylinders, crosses, rectangular prisms) with lateral dimensions between 40 and 1000 μm, cell densities of 105 – 108 cells/cm3, and total volumes between 1×10−7 and 8×10−4 cm3. By varying (i) the initial density of cells at seeding, and (ii) the dimensions of the modules, the number of cells per module ranged from 1 to 2500 cells. Modules were formed from a range of standard biopolymers, including collagen, Matrigel™, and agarose, without the complex equipment often used in encapsulation. The small dimensions of the modules allowed rapid transport of nutrients by diffusion to cells at any location in the module, and therefore allowed generation of modules with cell densities near to those of dense tissues (108 – 109 cells/cm3). This modular method is based on soft lithography and requires little special equipment; the method is therefore accessible, flexible, and well suited to (i) understanding the behavior of cells in 3D environments at high densities of cells, as in dense tissues, and (ii) developing applications in tissue engineering.
Highlights
Growing cells on rigid 2D substrates is a standard and convenient method for cell culture
Module fabrication technique We formed poly(dimethyl siloxane) (PDMS) membranes to serve as templates for gel structures using replica molding [5]. (Figure 1 is a schematic of process)
Spin-casting PDMS around the posts generated a membrane that bore an array of holes in a variety of shapes with precise dimensions
Summary
Growing cells on rigid 2D substrates is a standard and convenient method for cell culture. To study the behavior of cells in 3D environments, there is a need for 3D culture systems that (i) accurately mimic the environment of in vivo tissues; (ii) conveniently integrate with current tools of biology (such as optical microscopy and highthroughput screening); and (iii) provide flexibility similar to that of current 2D culture, in terms of the ability to control experimental variables, to study different cell types, and to ask a wide range of biological questions Such systems for 3D culture would be useful for studying 3D biological phenomena, such as the organization and pathology of tissues, or the metabolism and distribution of drugs. This system is simple to use and versatile, and it encapsulates cells in a highly reproducible form that is compatible with standard biological tools for characterization and highthroughput screening
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