Directing the assembly of spatially organized multicomponent tissues from the bottom up

Abstract

The complexity of the human body derives from numerous modular building blocks assembled hierarchically across multiple length scales. These building blocks, spanning sizes ranging from single cells to organs, interact to regulate development and normal organismal function but become disorganized during disease. Here, we review methods for the bottom-up and directed assembly of modular, multicellular, and tissue-like constructs in vitro. These engineered tissues will help refine our understanding of the relationship between form and function in the human body, provide new models for the breakdown in tissue architecture that accompanies disease, and serve as building blocks for the field of regenerative medicine.

Figure 1

Modular functional units in mammalian organs. (a) Human skeletal muscle cross section (b) Human mammary terminal ductal lobular unit (TDLU) cross section (c) Pig liver cross section showing repeating lobules (d) Mouse embryonic kidney. Reproduced from [79], Pathpedia, Werning, S., 2007 (http://calphotos.berkeley.edu/cgi/img_query?seq_num=223971&one=T), and [80] with permission.

Figure 2

DNA-programmed assembly. (a) Fluorescent epithelial cells labeled with complementary ssDNA (or other interacting molecules) are brought together through molecular recognition. Mixing cell populations 1-to-50 results in discrete multicellular aggregates that can be purified using fluorescence activated cell sorting. Assembled aggregates are then cultured in laminin-rich ECM to form polarized microtissues. Genetically distinct input cells can be incorporated to build mosaic aggregates. (b) Mosaic microtissues assembled from single H2B-GFP-expressing MCF10AT cells, which express low levels of H-Ras^V12, and wild-type MCF10A neighbors display emergent behaviors that are not observed in homogeneous assemblies. (scale bar, 10 μm). (c) Quantification of the emergent behaviors in homogeneous and mosaic aggregates. Mean values of great than 500 observations are displayed, with error bars representing the standard deviation of the mean. Reproduced from [21] with permission.

Figure 3

Directed assembly of cell aggregates. (a) Pre-formed, spheroidal cell aggregates (100 μm in diameter) assembled in trough-shaped wells fused into rod-shaped structures over 24 hours. (b) Pre-cultured heterogeneous spheroids with a core of human fibroblasts (red) and surrounding rat hepatoma (green) cells cultured in wells for 24 hours. (c) Confocal image shows that the assembled spheroids retained the inner fibroblast core during spheroid fusion (scale bars, 200 μm). (d) Different combinations of three cell sheet layers are stacked to make heterogeneous tissues of endothelial (green) and fibroblast (pink) cells. (e) After 3 days, endothelial sheets formed vessels (outlined with indirect CD31 staining in green) when stacked within or under fibroblast sheets (actin staining in red in merged images; scale bars, 20 μm). Reproduced from [39] and [50] with permission.

Figure 4

Directed assembly of cell-laden hydrogels. (a) Donut-shaped hydrogels with an inner hydrogel ring loaded with endothelial cells (green) surrounded by an outer ring loaded with smooth muscle cells (red) were made using sequential photolithography steps. (b) Side view of tubular structure formed after sequential assembly of hydrogel units from a (scale bars, 100 μm). (c, d) Lock-and-key (cross- and rod-shaped) hydrogels stained with fluorescent dextran were made using photolithography. (e, f) Lock-and-key hydrogels loaded with fluorescent murine fibroblast cells assembled through self-association in a hydrophobic medium (scale bars, 200 μm). (g) 3D volume reconstruction of DNA-directed hydrogel assemblies. Spherical hydrogels bearing green fluorescent tracking beads and labeled with ssDNA were bound to a microarray template. A second layer of hydrogels loaded with red beads and labeled with complementary DNA was assembled onto the first population (Scale bar, 100 μm). Reproduced from [59], [60], and [62] with permission.

Box Figure I

The modular and hierarchical organization of the human mammary gland. (a) Individual glandular epithelial cells exchange signals with each other and the basement membrane. (b) Epithelial cells of the ducts and acini also exchange signals with the surrounding lobular stroma. (c) Ducts and acini are organized into terminal ductal lobular units (TDLUs) that are embedded in a second type of collageneous ECM and that also contains many adipocytes. (d) The entire organ is integrated with the rest of the body to mediate its function in delivering milk during breast-feeding.