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Adhesion-Guided In Vitro Morphogenesis in Pure
and Mixed Cell Cultures
Department of Chemical Engineering and Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139
organ morphogenesis; adhesion; pure and mixed cell cultures
The ability to understand and control the morphogenesis of mammalian cells is a
fundamental objective of cell and developmental biology and tissue engineering research. Numerous
processes, both biochemical and biophysical in nature, have been studied in an attempt to elucidate
the mechanisms underlying this behavior. We focus here on the contributions of biophysical
phenomena to the morphogenetic behavior of pure and mixed cell populations on solid surfaces in
vitro. These principles are illustrated using characteristic liver tissue cells as a model system. The
studies discussed demonstrate that cell-substratum and cell-cell adhesive forces are critical
determinants of the ultimate morphology, cytoarchitecture, and organization achieved by these cells
in vitro. Microsc. Res. Tech. 43:379–384, 1998. r 1998 Wiley-Liss, Inc.
Morphogenesis of three dimensional tissue-like structures in vitro is of increasing interest for basic studies
of differentiated cell function as well as applications in
the growing field of therapeutic tissue engineering
(Langer and Vacanti, 1993). Although the roles of
specific signaling processes initiated by ligation of
growth factor or adhesion receptors are currently being
delineated, cell behavior in vivo ultimately results from
integration of many signals within the tissue environment, which are as yet unknown or unquantified.
Co-cultures or feeder layers are often used to provide
some of these missing signals, but failure to achieve cell
functions in vitro that are known to occur in vivo, such
as infection of human liver cultures by hepatitis B
virus, suggests that the hierarchichal structure of
tissue and the accompanying hierarchical delivery of
matrix and cytokine signals may also be an essential
factor to reproduce in vitro in order to maintain true
tissue function. Here, we focus on factors that influence
morphogenesis of tissue-like structures in pure and
mixed primary cell cultures, and emphasize a systematic approach to identifying culture parameters that
can predictably be manipulated to control such morphogenesis.
Enormous interest in the intrinsic cellular clues and
mechanisms guiding the morphogenetic process was
spawned by Townes and Holtfreter, who demonstrated
that the dissociated cells of vertebrate embryonic organs would not only re-aggregate but also reorganize in
an appropriate manner (Townes and Holtfreter, 1955).
It is generally agreed that relatively few mechanisms
are required to generate tissue organization, even if it
is not known exactly how they lead to the formation of
most structures. A variety of mechanisms have been
studied in terms of their contribution to various aspects
of morphogenesis including directed cell movement,
cytoskeletal contraction, cell proliferation, differences
in cell adhesion, and contact guidance (reviewed by
Bard, 1990).
Pioneering work by Steinberg suggests that the
sorting-out of intermixed embryonic cells, the spreading of one tissue over the surface of another, and the
specific inside/outside tissue stratification that arises
by either process are guided in large part by the
differential intercellular adhesivities of the cell types
involved (Steinberg, 1963, 1970, 1996). An important
implication of this analysis is that different molecular
configurations could lead to identical outcomes provided that they produce comparable adhesion forces. A
similar analysis involving cellular forces was perfomed
by Oster et al. (1983), who demonstrated that a variety
of intricate structural arrangments and patterns could
be predicted based strictly on biophysical force balances. Thus, while morphogenesis is a complex process
involving numerous signals and guidance clues that
can be both chemical and/or physical in nature (Gumbiner, 1996) the fundamental mechanisms responsible
for this process are very possibly biophysical in nature.
The findings discussed here detail investigations into
the role played by biophysical cell-substratum and
cell-cell forces in the morphogenesis and pattern formation of homogeneous and heterogeneous cell cultures.
Liver is used as the representative tissue system in
these studies based on the observed importance of
morphology on in vitro cellular behavior. The basic
functional unit of liver, the acinus, exhibits a wellcharacterized architecture consisting of continuous
plates or cords of a single layer of hepatocytes separated from the sinusoidal blood supply on both sides by
a layer of endothelial cells interspersed with stellate
cells and Kupffer cells; the plates continuously branch
*Correspondence to: Linda G. Griffith, Department of Chemical Engineering
and Center for Biomedical Engineering, Massachusetts Institute of Technology,
66–466, Cambridge, MA 02139. E-mail: [email protected]
Received 16 August 1998; accepted in revised form 25 August 1998
Contract grant sponsor: National Science Foundation; Contract grant numbers: BCS-9157321, BES-963–2714; Contract grant sponsor: National Institutes
of Health; Contract grant number: GM-50047; Contract grant sponsor: Harvard
Dental School/MIT.
Fig. 1. Comparison of rat hepatocyte cellsubstratum adhesion strengths with cell traction forces. Primary rat hepatocytes were cultured on Matrigel substrata which ultimately
lead to the formation of monolayers (䊏) or
spheroids (䊊, ⴛ). The shear force required to
detach these cells was determined after 24
hours. in culture. Shaded areas represent cell
traction force values for fibroblasts (F) (Oliver
et al., 1994), keratocytes (K) (Lee et al., 1994),
and the estimated range for hepatocytes (H).
Ref. Powers et al. (1997).
and interconnect between the portal tract and the
central vein. Three distinct epithelial domains are
present in the hepatic plates: an apical domain forming
the bile canaliculi, a lateral domain consisting of epithelial junctions that interconnect the hepatocytes, and a
basal (sinusoidal) domain that is adjacent to the sinusoidal endothelial cells and other non-parenchymal cell
types. At least some semblance of this acinar cytoarchitecture and organization seems to be essential for the
expression of liver-specific function in hepatocellular
cultures (Clayton et al., 1985; Dunn et al., 1991; Moghe
et al., 1996). Investigations into the morphogenetic
behavior of pure cultures of hepatocytes and mixed
cultures of hepatocytes and endothelial cells— the two
predominant cell types defining the architecture of
liver— therefore provide a useful paradigm with which
to study the morphogenetic behavior of dispersed tissue
cells as they re-form into native tissue structures.
Aggregation of primary hepatocytes has been observed on numerous surfaces comprised of different
architectures and/or molecular compositions (Koide et
al., 1989, 1990; Landry et al., 1985; Parsons-Wingerter
and Saltzman, 1993; Peshwa et al., 1994; Saltzman et
al., 1991). These substrata have been shown to play an
important role in the ultimate morphology achieved by
these cell aggregates (Koide et al., 1989; Landry et al.,
1985). Since a substratum is capable of imparting both
biochemical and biophysical signals to cells, it is essential to delineate the contributions of each to the morphogenetic process. We have shown previously (Powers et
al., 1997) that hepatocytes are capable of forming both
monolayered and spheroidal aggregates on a diverse
array of rigid extracellular matrix substrata, including
laminin, fibronectin, collagen I, and Matrigel. Hepatocytes attach to each of these matrices through different
sets of cell-surface receptors (Clement et al., 1989;
Forsberg et al., 1990; Gullberg et al., 1992; Pujades et
al., 1992; Stamatoglou et al., 1990a,b), suggesting that
differences in biochemical signalling, if present on the
different substrata, do not significantly affect the resulting aggregate morphologies on these surfaces. Rather,
the morphology of the hepatocyte aggregates correlates
strongly with the ligand density present on each surface. For each type of matrix studied, high surface
ligand concentrations leads almost exclusively to the
formation of monolayered aggregates. Conversely, low
matrix concentrations induce the formation of spheroidal aggregates. Quantitation of cell-substratum adhesion strength on these surfaces indicates that it is the
biophysical forces present in the system that provide a
basis for the observed morphological results: substrata
on which monolayers form promote higher levels of
cell-substratum adhesion than do those surfaces on
which spheroids form.
On a general level these analyses provide a basis for
simple assessment of the relative affinity of cells for
these different substrata (i.e., high or low) based on the
morphology of the resulting aggregates. Investigations
into the mechanisms responsible for the observed aggregation and morphological behavior provide further
clues as to the impact of other cell-based forces in this
Hepatocytes on rigid substrata aggregate through a
mechanism involving active cell membrane protrusion
leading to cell-cell attachment and followed by what
appears to be a discrete contraction event which often
causes a rapid retraction of the peripheral edges of the
aggregating cells (Powers and Griffith-Cima, 1996).
The membrane extension or spreading of one cell on top
of another was not observed to occur (i.e., cells did not
form directly into multilayered aggregates). These observations suggest that cell-generated contractile forces,
which have been identified as key determinants in the
morphogenesis of other cell systems (Bard, 1990; Oster
et al., 1983), are critical in the determination of hepatocyte aggregate morphology. Further evidence for this
supposition is provided by studies of hepatocyte aggregation on biochemically identical gel substrata of varying deformabilities. On such substrata, spheroids form
on malleable gels that are easily deformed by the
cytoskeletal forces that the aggregating cells generate;
when the gels are present in a rigid, non-deformable
state monolayers result (Coger et al., 1997; Linblad et
al., 1991).
These results can be interpreted by considering the
contributions of cytoskeletal contraction to hepatocyte
aggregate morphology. It has been demonstrated that
the balance between cell-generated traction forces,
which represent that part of the total contractile force
transmitted to the substratum (Lauffenburger and
Horwitz, 1996), and cell-substratum adhesive forces
play an important role in the determination of hepatocyte aggregate morphology (Powers et al., 1997). Figure
1 illustrates the data that support this hypothesis,
showing that the estimated hepatocyte traction forces
are lower than the expected cell-substratum adhesive
forces of a majority of cells attached to the ‘‘monolayerpromoting’’ substrata but greater than most of the
cell-substratum adhesive forces present on the ‘‘spheroid-promoting’’ surfaces. In other words, spheroids form
when cytoskeletal contractile forces can act to ‘‘pull’’ the
cells together, while monolayers result when the cellsubstratum adhesion strength is insurmountable.
While this analysis provides convincing evidence
that the interplay between cell-substratum adhesion
and cellular contraction is critical to hepatocyte aggregate morphogenesis, cell-cell adhesion may also play a
role in this process. Martz et al. (1974) outline a model
system by which variation in cell-cell and cell-substratum adhesive interactions alone could account for multiple morphological states. In this system, aggregate
morphology evolves through a thermodynamic free
energy minimization process. As cells aggregate, physical remodeling results from small perturbations in the
local molecular (i.e., receptor-based) equilibrium as the
cell aggregate attempts to increase high adhesion (either cell-cell or cell-substratum) contact regions. This
behavior is analagous to that observed by water droplets interacting with surfaces: the water molecules will
bead on hydrophobic surfaces and spread on a hydrophilic surfaces. The model therefore predicts that, when
cell-substratum attachment sites are sparse, surfacefree energy is minimized by maximizing the more
highly adhesive intercellular adhesions, and spheroids
result. Conversely, when cell-substratum attachment
sites are plentiful, surface-free energy is minimized by
the maximizing the number of cell-substratum adhesion bonds, resulting in monolayers. Active, directional
activation of the cytoskeletal machinery does not play a
role in this model. In this sense, morphogenesis would
occur through a passive mechanism with the relative
strengths of cell-cell vs. cell-substratum adhesive interactions ultimately determining the outcome.
In our system, previously cited observations (i.e., lack
of direct multilayering of coupled cells, the occurrence
of a discrete contractile event, cell behavior on gel
substrata) suggest that contractile force is the defining
event in the initiation of morphogenesis; however, the
maturation of aggregate morphology into multi-layer
spheroids is likely to be at least partially due to the the
free-energy minimization phenomena described above.
It is important to stress that both parameters are likely
to contribute to the ultimate morphological state of the
aggregates, with the contractile behavior of the cells
allowing the morphogenesis to occur on a kinetically
favorable time scale.
Fig. 2. Predicted morphological outcomes for cells sorting on
surfaces based on the adhesive properties of the system. Examples are
shown for cells that exhibit different levels of cell-cell and cellsubstratum affinity. When cell-cell forces are dominant, spheroidal
aggregates would be predicted to form with the high affinity cells
sorting internally to the low affinity cells. When cell-surface forces are
dominant, monolayers would be expected, with the cells sorting from
one another only within this morphological framework. Two intermediate cases are also shown.
These analyses indicate that the morphological state
of a cellular aggregate provides valuable clues as to the
biophysical ‘‘hierarchy’’ present in the system. When
monolayers are present, the cell-substratum forces are
dominant over cell traction forces and/or cell-cell adhesion forces,while the formation of spheroids indicates
that the opposite is true.
Due to the observed importance of biophysical properties in the morphogenesis of homogeneous cell cultures,
Fig. 3. Phase contrast images of primary rat hepatocytes and bovine aortic endothelial cells coated on
high (left), intermediate (middle), and low (right) concentrations of Type I collagen. Images were
recorded 24 hours (top) or 4 days (bottom) post-seeding. Examples of cellular morphology are labeled H
(hepatocytes) or E (endothelial cells) where discernible.
we now focus on the role played by these phenomena in
the sorting of mixed cocultures on rigid substrata. Since
most tissues are comprised of multiple cell types, these
studies provide a more tissue-like system with which to
investigate these phenomena. For the purposes of this
analysis, we again focus on the liver. Because of the
importance of hepatocytes and endothelial cells in
acinar architecture, we have chosen to study interactions between these two cell types. The role played by
biphysical cell-cell and cell-surface phenomena in the
sorting of these two cell types from one another therefore presents an intriguing case study. In considering
our approach to these analyses, it should be noted that
the nature of interaction between hepatocytes and
endothelial cells in vitro is largely unknown. In vivo
interactions between hepatocytes and endothelial cells
in the postnatal, nonregenerating liver are typically
modulated through the extracellular matrix present in
the Space of Disse. In vitro, cells are devoid of endogenous matrix at the time of initial plating, but matrix
deposited during culture may ultimately control hepatocyte-endothelial cell interactions in vitro. Hepatocytes
are indeed capable of synthesizing and secreting extensive arrays of matrix proteins in vitro (Stamatoglou et
al., 1987; Sudhakaran et al., 1986; Tamkun and Hynes,
1983) with which endothelial cells are capable of associating. Direct hepatocyte-endothelial adhesion may also
be possible through cadherin-based adhesion. Although
these cell types express different cadherin receptors
—E-cadherin in hepatocytes (Vestweber et al., 1985)
and VE- and N- cadherin in endothelial cells (Navarro
et al., 1998) — it has been postulated that nonidentical
cadherin systems on different cell types could mediate
intercellular adhesion (Brackenbury et al., 1981; Mur-
phy-Erdosh et al., 1995; Volk et al., 1987). But regardless of the nature of hepatocyte-endothelial interaction,
these associations are likely to be more temporal and
less able to resist cytoskeletal forces than the stronger,
more irreversible epithelial adhesion complexes present in aggregated hepatocytes (Farquhar and Palade,
1963; Miettinen et al., 1978; Stamatoglou and Hughes,
1994), leading us to focus on the role played by heterotypic cell-cell adhesion in this system.
The hypothesis that intercellular adhesion phenomena provide a basis for cell sorting behavior was first
proposed by Steinberg (1963), who theoretically and
experimentally demonstrated that basic sorting behavior could be predicted through phase ordering principles (Steinberg, 1963, 1970, 1996; Steinberg and
Takeichi, 1994). Given a culture system comprised of
two cell types in which a cell-cell affinity hierarchy is
established (i.e., cell type A is more ‘‘cohesive’’ than cell
type B), it was shown that the cells will sort from one
another so as to minimize the free energy of the system.
That is, the high affinity (or high surface ‘‘free energy’’)
cells form a spherical core, surrounded by a shell of the
low affinity (low surface ‘‘free energy’’) cells. This is
analogous to the behavior seen when two immiscible
liquids of different surface tensions are mixed.
The principles set forth by Steinberg can now be
combined with the previously described model of Martz
et al. (1974) to allow for cell sorting on a surface. This
analysis adds another level of complexity to the system
as cell-surface interactions are included in the aforementioned cell affinity hierarchy (Fig. 2). Using the principles of free energy minimization, sorting outcomes for
cells on high, intermediate, and low surface free energy
cases can be predicted. As seen in these examples of
the formation of spheroidal aggregates with hepatocytes sorting internally to a ‘‘shell’’ of endothelial cells.
This architecture has also been observed to occur in
other hepatocyte co-culture systems (Landry et al.,
1985; Takezawa et al., 1992). According to our model
these results suggest that homotypic interactions between hepatocytes are of a higher affinity than homotypic interactions between endothelial cells, not a surprising result given the epithelial nature of hepatocyte
intercellular adhesions. Surfaces with intermediate
ligand densities lead to the attachment of endothelial
cells on top of monolayered hepatocyte aggregates,
indicating that the adhesion of endothelial cells to
hepatocytes is still thermodynamically preferred over
endothelial attachment to a surface. Both hepatocytes
and endothelial cells attach as monolayers to high
ligand concentration surfaces, a conformation in which
interaction with the surface is maximized and interaction with each other is minimized.
These results suggest that the morphology and architecture of heterologous cellular structures are guided
by biophysical phenomena. Furthermore, this behavior
can be predicted based simply on the principles outlined here, potentially providing a simple and effective
alternative to more costly and time-consuming approaches (e.g., staged/directed cell seeding; surface
patterning) for achieving histotypical architectures in
vitro. Extensions of this analytic approach to evolution
of structure in more complex three-dimensional systems may ultimately enable in vitro organogenesis of
functional tissue (Griffith et al., 1997).
Fig. 4. Vertical histological sections (3 µm) of hepatocyte (H)endothelial cell (E) cultures as in Figure 3. High (top), intermediate
(middle), and low (bottom) ligand density cultures were fixed on day
4, embedded in glycol methacrylate resin, sectioned, and stained with
Hematoxylin & Eosin. Note similarities to predicted outcomes from
Figure 2.
Figure 2, a wide variety of outcomes could result simply
by changing the affinity of cell-substratum interactions.
In order to test these predictions of cell sorting on
surfaces, mixtures of primary rat hepatocytes and
bovine aortic endothelial cells were cultured on substrata of high, intermediate, and low extracellular
matrix ligand densities (Fig. 3). Vertical histological
sections of these cultures indicate the formation of
cellular structures with intercellular organizations
closely resembling those predicted from biophysical
principles (Fig. 4). Furthermore, inspection of the specimens from intermediate and low ligand density surfaces reveals structures reminiscent of the histotypic
arrangement of cells in the acinar architecture. Low
surface ligand concentrations appear to promote a
situation where cell-cell forces are dominant, leading to
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