Transcript imaging.bioen.illinois.edu

Kenneth M. Yamada & Edna Cukierman
Introduction
2D cell cultures
Animal models
2D cell cultures
• Studies in standard cell culture
have produced many important
conceptual advances
• Nevertheless, cells grown on flat
2D tissue culture substrates can
differ considerably from
physiological 3D environments
Animal models
• Animal models provide definitive
tests of the importance of specific
molecules and processes
• There can, however, be puzzling
discrepancies between conclusions
from gene ablation studies
In Vitro 3D Models
In vitro 3D tissue models provide a third approach that bridges the gap between
traditional cell culture and animal models
5 types of In Vitro 3D Models:
 Organotypic explant cultures
 Stationary or rotating microcarrier cultures
 Micromass cultures
 Free cells in rotating vessel
 Gel-based cultures
In Vitro 3D Models
The morphologies of fibroblasts, including cytoskeletal organization and types of cell
adhesions, are also more similar to their in vivo behavior
Planar fibronectin
Cell-derived 3D matrix
In Vitro 3D Models
3D models are closer to physiological conditions than routine 2D cell culture, but they
also have their weaknesses, such as their lack of vasculature and ability to progress
Key Strengths and Weaknesses of 3D Models
Advantages
• Cell morphology and signaling are often more physiological than routine 2D cell culture
• Permit rapid experimental manipulations and testing of hypotheses
• Permit much better real-time and/or fixed imaging by microscopy than in animals
Disadvantages
• Vary in their ability to mimic in vivo tissue conditions
• Currently lack vasculature and normal transport of small molecules
• Generally mimic static or short-term conditions, whereas in vivo systems often progress
In Vitro 3D Models
Comparisons of 2D and 3D models reveal that the latter are better, but not exact, models
of in vivo tissues
3D-Dependent Cell Behavior and Signaling
Function
2D versus 3D
Regulatory Mechanisms
Cell Shape
Loss of epithelial cell polarity and altered
epithelial and fibroblast shape in 2D
Growth factor receptors and
pathways; cell-adhesion signals
Gene Expression
Cells in 2D versus 3D often have different
patterns of gene expression
ECM, hormones, and adhesion
molecules
Growth
3D matrix-dependent regulation of cell
growth
Adhesion and growth factor-related
pathways, apoptotic genes
Morphogenesis
3D matrix-induced vessel sprouting and
gland branching
ECM, adhesion, growth factorrelated pathways, apoptotic genes
Motility
Altered single and collective cell motility
patterns in 3D matrices
ECM and its regulators; adhesions
and growth factor-related pathways
Differentiation
3D matrix-induced cell differentiation
ECM and growth factors; motor
molecules
Model Choice Affects Outcome
In any 3D model system, the specific cellular and matrix microenvironment provided to
cells can substantially influence experimental outcome
Model Choice Affects Outcome
Physical properties such as matrix stiffness and cell polarity in 3D models can also play
surprisingly important roles
 Matrix stiffness
 Stiffness (compliance) of the extracellular matrix can be sensed
by cells through bidirectional interaction between the cell surface
integrin receptors and the contractile cytoskeleton
 Cell and tissue polarity
 Polarity in vivo depends both on the cell type and the cellular
microenvironment, and it is important for tissue organization and
direction secretion of products
Matrix Stiffness
The stiffness of a matrix can affect the distribution of cell surface integrin receptors,
types of cell adhesions, cytoskeletal structures, cell proliferation, and other factors
Cell and Tissue Polarity
Cells explanted into routine tissue cultures often flatten and lose differentiation markers;
they generally regain their correct polarity when placed back in 3D culture conditions
Edna Cukierman, et al.
Introduction
Our current knowledge about the
roles of cell-matrix adhesions in cell
adhesion, migration, signaling, and
cytoskeletal function is derived
primarily from studies on planar 2D
tissue culture substrates
There are two types of cell-matrix
adhesions:
 Focal adhesions
 Fibrillar adhesions
Focal Vs. Fibrillar Adhesions
Focal adhesions
Fibrillar adhesions
 Located at cell periphery
 Elongated/beaded structures
 Vinculin
 Located centrally

 Tensin
 Focal adhesion kinase (FAK)
 Integrin αVβ3
 Associated with fibronectin
fibrils
2D Vs. 3D Cell Cultures
Relatively little is known about the cell-matrix adhesive structures formed in
3D matrices in living tissues
 Fibroblastic cells have been studied mainly in 2D cell cultures
 In contrast to 3D matrices, fibroblasts on 2D substrates:
 Induce an artificial polarity
 Differ in their morphology and migration
“3D-Matrix Adhesions”
FOCAL ADHESIONS
 Located at cell
periphery
 Vinculin
FIBRILLAR ADHESIONS
 Elongated/beaded
structures
 Located centrally
 Tensin
 Focal adhesion
kinase (FAK)
 Integrin αVβ3
3D-MATRIX ADHESIONS
 Associated with
fibronectin fibrils
 Associated with
fibronectin fibrils
Cell Response to 3D-Matrix Adhesions
#1: CELL ATTACHMENT
The cell-derived 3D matrix was more
effective (by a factor of over 6) in
mediating cell adhesion than was 2D
substrates
Cell Response to 3D-Matrix Adhesions
#2: CELL MORPHOLOGY
Fibroblasts in 3D matrices achieved their
final, elongated morphology by 5 hours.
Fibroblasts on cell-derived 2D matrices
eventually achieved the same
morphology, but it took over 18 hours.
Cell Response to 3D-Matrix Adhesions
#3: MIGRATION
Cells showed enhanced migration rates
(by a factor of 1.5) in 3D matrices relative
to individual protein-coated surfaces.
Cell Response to 3D-Matrix Adhesions
#4: PROLIFERATION
Proliferation rates of fibroblasts in the
cell-derived 3D matrices were more than
double fibronectin-coated 2D substrates.
Three-Dimensionality Vs. Composition

The properties of 3D-matrix
adhesions could be due either to
the three-dimensionality of the
matrix or to its composition
 A 3D-matrix was flattened by
mechanical compression to form a
virtually 2D matrix with the same
composition as its 3D counterpart
 The cell-derived 2D matrix did not
show the same triple
colocalization of α5 integrin,
paxillin, and fibronectin as the cellderived 3D matrix
 The authors concluded that threedimensionality was important to
achieving “3D-matrix adhesions”
Conclusions
 Cukierman et al. speculated that focal and fibrillar adhesions studied
in vitro represent exaggerated precursors of in vivo 3D-matrix
adhesions
 Fibroblasts initially require culture for days at high cell density to
generate 3D matrices and evolve 3D-matrix adhesions, yet when
added back to cell-free 3D matrices, they begin regenerating matrix
adhesions within 5 minutes
 Requirements for 3D-matrix adhesions include three-dimensionality,
integrin α5β1, fibronectin, other matrix components, and pliability
(data not discussed in this presentation)