Download Architecture of dorsal tissue aggregates from frog embryos.

ARCHITECTURE OF DORSAL TISSUE REAGGREGATES FROM XENOPUS LAEVIS EMBRYOS
Uma Balakrishnan, Joseph Shawky, and Lance Davidson
Department of Bioengineering, University of Pittsburgh
DFA, the epithelium is also peeled off and discarded, as this
INTRODUCTION
layer takes longer to dissociate in the solution, which would be
The central goal of tissue engineering is to recreate the
unhealthy for the rest of the cells.
form of clinically needed tissues together with their proper
The dissociated cells are placed in a specially designed
function. In tissue engineering today, 3D-printing is the newest
microcentrifuge tube with 30 µL of agarose in a micropipette
way to generate multicellular tissues for clinical use.[1] 3Dtip; the dissociation media is replaced with DFA. These cells
printing relies on mixing scaffolds of extracellular matrix or
are centrifuged for 10 minutes at 5 rpm (Fig 1c). This creates a
hydrogels with cells as a starting material. This method of
sheet of the non-differentiated cells to be cut into 3-4
building tissues can be useful today for tissues that have low
reaggregates (Fig 1d). In those experiments that require fixation
cell densities such as tendons or connective tissue; however, the
after a specific amount of time, the reaggregates are placed
majority of human tissues have a high ratio of cells to
under glass and allowed to develop further. For each
extracellular fluid.[2] Therefore, developing methods to engineer
experiment, at least two additional dorsal isolates were
tissues with high cell density is a critical problem that must be
microsurgically removed to serve as controls.
addressed for future clinical application.
One such method to develop engineered tissues with a high
cell density is to create tissue reaggregates.[2] In this method,
dissociated, non-differentiated cells from a tissue are packed
together using centrifugation, which mimics the forces present
during development. Dissociation is required to break down
any architecture present from the original tissue. Once
centrifuged, the packed-together cells are a brick, called a
reaggregate, of non-differentiated cells.
The African clawed frog Xenopus laevis embryo makes a
good model to develop this method to create scaffold-free
engineered tissues because it develops outside the mother and
can be microsurgically manipulated without damaging
individual cells. Specifically, the dorsal tissue of X. laevis
embryos between gastrulation and neurulation is a good tissue
Figure 1 Creating a tissue reaggregate. A frog embryo is (a)
to make aggregates because it contains all three germ layers
dissected to remove a dorsal tissue. This tissue is (b) dissociated and
(endoderm, mesoderm, and ectoderm) of undifferentiated
(c) centrifuged to yield a (d) tissue aggregate.
cells.[3] Having a model to make viable tissue aggregates is key
The Live/Dead Assay identified cells with a compromised
to developing high-density engineered tissues for clinical
cell membrane to determine the viability of the aggregated
application.
tissue. Cells were stained at room temperature for 2 hours.
For the other stains, the reaggregates were fixed in Dent’s
OBJECTIVE & HYPOTHESIS
Fix
at 4 ºC overnight and stained using immunochemistry.
The objective of this study is to evaluate various
Anti-rabbit
β-catenin primary antibody was used to tag
characteristics of tissue reaggregates from X. laevis embryos.
intercellular
junctions.
ZO-1 primary antibody was used to tag
For this study to be successful, staining and imaging the
tight
junctions
present
in the epithelial layer. The fluorescent
aggregates should give visual confirmation that the aggregates
TRITC
secondary
antibody
was used for all experiments.
survived the reaggregation process, reformed cell-to-cell
All
Live/Dead
and
β-catenin
staining occurred directly
contacts, and grown an epithelium.
after reaggregation. The ZO-1 staining happened at three
METHODS
distinct time points: (1) immediately after reaggregation, (2)
X. laevis embryos are harvested, fertilized, allowed to age
after six hours, and (3) after 24 hours.
to stage 12.5-15, at least until blastopore is closed. Dorsal tissue
All tissues were imaged using a confocal microscope.
is microsurgically removed from the embryo using hair tools
Images for the Live/Dead Assay and β-catenin staining are
under a dissection microscope in Danilchik’s For Amy (DFA)
cross-sections of the tissues. Images for ZO-1 staining are from
solution with 0.1% bovine serum albumin (Fig 1a). In each
the surface of the dorsal isolates and tissue aggregates.
experiment, 10-12 dorsal isolates are explanted. Each explant is
RESULTS
dissociated in a Ca2+/Mg2+ free solution for 5-7 minutes to
The Live/Dead Assay shows qualitatively that the majority
cause cells to endocytose the intercellular junctions.
of
the
cells are alive toward the core of the tissue (Fig 2a). On
Additionally, the tissue is mechanically dissociated by
the
edges
of the aggregate, there are many shriveled, dead cells
chopping the tissue into individual cells (Fig 1b). Also while in
1
surrounding the aggregate (Fig 2b). However these cells do not
appear as if they are attached to the actual tissue.
Figure 2 Live/Dead Assay. Images from (a) the center and on (b) the
periphery of a tissue aggregate with live (green) and dead (red) cells.
Scale = 50 µm (n=7)
The control tissue stained for β-catenin shows the highly
specific and organized structure of cells in the dorsal tissue (Fig
3a). In the tissue aggregate, this architecture is completely
disrupted, however there are clearly intercellular junctions
present (Fig 3b).
Figure 4 ZO-1 staining. Epithelium in (a) dorsal isolate and evidence
of epithelial cells (white arrows) in tissue aggregates after (b) 0 hours
(n=2), (c) 6 hours (n=3) and (d) 24 hours (n=2). Scale = 50 µm
Figure 3 β-catenin staining. Intercellular junctions present in both the
(a) dorsal isolate and (b) tissue aggregate (n=6). Scale bar = 50 µm
ZO-1 staining revealed an overall lack of growth of
epithelial cells on the surface of the tissue aggregates.
Immediately after microsurgery, there is absolutely no
fluorescent signal for ZO-1 on the surface of the aggregate as
compared to the control epithelium on a dorsal isolate (Fig
4a,b). After 6 and 24 hours, some epithelium was present;
however, it did not cover a significant portion of the tissue and
its surface area did not seem to increase with time.
DISCUSSION
The qualitative data of this study strongly support that the
tissue aggregates both survive the dissociation and
reaggregation process and reform intercellular junctions with
their new cellular neighbors. Although there are dead cells on
the periphery of the tissue aggregates, the new tissue itself is
viable and useful for future research.
This study does not provide conclusive evidence that
epithelial cells grow on the surface of the aggregate. The small
numbers of cells present and lack of ZO-1 signal increase over
time may suggest that these cells are simply left over from the
original tissue, a result of incomplete discarding of the
epithelial layer during dissociation. However, if more
epithelium is shown to grow on the aggregate it may be
evidence of trans-differentiation occurring at the surface where
cells are exposed to different surface tensions than the cells on
the inside of the tissue.
This study could be further developed by comparing the
volumes of dead and live cells to the tissue volume, quantifying
disorganization in a tissue, and calculating the relative surface
area which the epithelial cells cover on the aggregate surface.
In the future, these aggregates can be induced to various
factors to cause them to differentiate into various cell types.
Multiple aggregates of differing cell types and shapes may be
used to build larger structures, much like building a house using
individual bricks. To use this aggregation method for clinical
applications, aggregates must be made from human stem cells.
However, first understanding and developing this method in a
cheaper and easier to manipulate model is important to the
eventual usefulness of tissue aggregates in tissue engineering.
ACKNOWLEDGMENTS
Thank you to the whole Davidson Mechanics and
Morphogenesis Lab for their support on this project.
REFERENCES
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in the shape of a human ear. Plastic and reconstructive
surgery, 100(2), 297-302.
2. Miller, J. S. (2014). The billion cell construct: will threedimensional printing get us there? PLoS Biol, 12(6),
e1001882.
3. Wilson, P. A., Oster, G., & Keller, R. (1989). Cell
rearrangement and segmentation in Xenopus: direct
observation of cultured explants. Development, 105(1), 155166.
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