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Cell encapsulation in sub-mm sized gel modules using replica molding.

McGuigan AP, Bruzewicz DA, Glavan A, Butte MJ, Butte M, Whitesides GM - PLoS ONE (2008)

Bottom Line: 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 (10(8)-10(9) cells/cm(3)).

View Article: PubMed Central - PubMed

Affiliation: Harvard University, Cambridge, Massachusetts, United States of America.

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 microm, cell densities of 10(5)-10(8) cells/cm(3), and total volumes between 1x10(-7) and 8x10(-4) cm(3). 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 (10(8)-10(9) cells/cm(3)). 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.

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Related in: MedlinePlus

Confocal microscopy through a module after 2 days in culture.Series of images at increasing depth in a 500-μm wide module, 2 days after fabrication. The series of images indicates that cells are evenly distributed throughout the module. The scale bar for each image is 50-μm wide. DAPI (blue) indicates cell nuclei, CFSE (Green) indicates cell cytoplasm where esterase activity is present, and phalloidin (red) indicates actin filaments.
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pone-0002258-g003: Confocal microscopy through a module after 2 days in culture.Series of images at increasing depth in a 500-μm wide module, 2 days after fabrication. The series of images indicates that cells are evenly distributed throughout the module. The scale bar for each image is 50-μm wide. DAPI (blue) indicates cell nuclei, CFSE (Green) indicates cell cytoplasm where esterase activity is present, and phalloidin (red) indicates actin filaments.

Mentions: Light microscopy images suggested that cells were distributed evenly throughout a module after fabrication (Figure 2B, 2C and 2F), as long as the cells were evenly distributed in the collagen gel initially during fabrication of the modules. Mixing cellular aggregates instead of individual cells into the collagen during fabrication resulted in modules with uneven cell distributions, which may find use in some applications. Confocal microscopy was used to demonstrate even distributions of cells in modules formed using a well mixed suspension of cells. Figure 3 shows the even distribution of cells in successive layers, from the surface of the module to approximately 20 μm into the module. The focal length of the microscope was sufficient to enable visualization of the module to a depth equivalent to the thickness of approximately three cells. Beyond this depth, the laser did not penetrate the dense tissue sufficiently to produce a detectable signal.


Cell encapsulation in sub-mm sized gel modules using replica molding.

McGuigan AP, Bruzewicz DA, Glavan A, Butte MJ, Butte M, Whitesides GM - PLoS ONE (2008)

Confocal microscopy through a module after 2 days in culture.Series of images at increasing depth in a 500-μm wide module, 2 days after fabrication. The series of images indicates that cells are evenly distributed throughout the module. The scale bar for each image is 50-μm wide. DAPI (blue) indicates cell nuclei, CFSE (Green) indicates cell cytoplasm where esterase activity is present, and phalloidin (red) indicates actin filaments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376064&req=5

pone-0002258-g003: Confocal microscopy through a module after 2 days in culture.Series of images at increasing depth in a 500-μm wide module, 2 days after fabrication. The series of images indicates that cells are evenly distributed throughout the module. The scale bar for each image is 50-μm wide. DAPI (blue) indicates cell nuclei, CFSE (Green) indicates cell cytoplasm where esterase activity is present, and phalloidin (red) indicates actin filaments.
Mentions: Light microscopy images suggested that cells were distributed evenly throughout a module after fabrication (Figure 2B, 2C and 2F), as long as the cells were evenly distributed in the collagen gel initially during fabrication of the modules. Mixing cellular aggregates instead of individual cells into the collagen during fabrication resulted in modules with uneven cell distributions, which may find use in some applications. Confocal microscopy was used to demonstrate even distributions of cells in modules formed using a well mixed suspension of cells. Figure 3 shows the even distribution of cells in successive layers, from the surface of the module to approximately 20 μm into the module. The focal length of the microscope was sufficient to enable visualization of the module to a depth equivalent to the thickness of approximately three cells. Beyond this depth, the laser did not penetrate the dense tissue sufficiently to produce a detectable signal.

Bottom Line: 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 (10(8)-10(9) cells/cm(3)).

View Article: PubMed Central - PubMed

Affiliation: Harvard University, Cambridge, Massachusetts, United States of America.

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 microm, cell densities of 10(5)-10(8) cells/cm(3), and total volumes between 1x10(-7) and 8x10(-4) cm(3). 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 (10(8)-10(9) cells/cm(3)). 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.

Show MeSH
Related in: MedlinePlus