U.S. patent application number 15/064360 was filed with the patent office on 2016-09-08 for self-assembling tissue modules.
The applicant listed for this patent is Universiteit Maastricht. Invention is credited to Severine Le Gac, Nicolas Clement Rivron, Jeroen Rouwkema, Roman Truckenmuller, Clemens Antoni Van Blitterswijk, Erik Jacob Vrij.
Application Number | 20160257926 15/064360 |
Document ID | / |
Family ID | 39926404 |
Filed Date | 2016-09-08 |
United States Patent
Application |
20160257926 |
Kind Code |
A1 |
Rivron; Nicolas Clement ; et
al. |
September 8, 2016 |
SELF-ASSEMBLING TISSUE MODULES
Abstract
The disclosure relates to a new approach to constructing
cellular aggregates in vitro and their use in methods for producing
3D-tissue constructs in a modular way. In particular, the
disclosure is directed to a method for in vitro producing a tissue
construct comprising: a) combining living cells to form
supracellular aggregates using spatial confinement; b) combining
two or more of the supracellular aggregates in a mold or on a
biomaterial; c) applying conditions that induce self-assembly
within the combined supracellular aggregates to obtain the tissue
construct; and d) applying conditions that induce tissue
morphogenesis in the tissue construct.
Inventors: |
Rivron; Nicolas Clement;
(Juvisy sur orge, FR) ; Rouwkema; Jeroen; (Zwolle,
NL) ; Truckenmuller; Roman; (Flein, DE) ; Le
Gac; Severine; (Ormesson-sur-Marne, FR) ; Van
Blitterswijk; Clemens Antoni; (Ruigahuizen, NL) ;
Vrij; Erik Jacob; (Maastricht, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Universiteit Maastricht |
Maastricht |
|
NL |
|
|
Family ID: |
39926404 |
Appl. No.: |
15/064360 |
Filed: |
March 8, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12999841 |
Mar 14, 2011 |
9303245 |
|
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PCT/NL09/50368 |
Jun 22, 2009 |
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15064360 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2533/52 20130101;
C12N 5/0663 20130101; C12N 5/0062 20130101; C12N 5/069 20130101;
C12N 2513/00 20130101; C12N 2533/54 20130101; C12N 5/0691
20130101 |
International
Class: |
C12N 5/00 20060101
C12N005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 20, 2008 |
EP |
08158652.1 |
Claims
1. A method for in vitro producing a tissue construct comprising:
a) combining living cells to form supracellular aggregates using
spatial confinement; b) combining two or more of the supracellular
aggregates in a mold or on a biomaterial; c) applying conditions
that induce self-assembly within the combined supracellular
aggregates to obtain the tissue construct; and d) applying
conditions that induce tissue morphogenesis in the tissue
construct.
2. A method according to claim 1, wherein said tissue morphogenesis
comprises migration and/or differentiation of cells.
3. A method according to claim 1, wherein said spatial confinement
comprises one selected from an array of microwells, Hanging drop
method, or microfluidic channels.
4. A method according to claim 3, wherein said spatial confinement
comprises an array of microwells, said microwells having an
enveloping diameter in the range of 50-500 .mu.m and a depth in the
range of 100-1,000 .mu.m.
5. A method according to claim 3, wherein the microwells have a
shape that is different from a cylinder.
6. A method according to claim 5, wherein the shape of at least
some of the microwells is such that the resulting aggregates can
self-assemble according to the lock-and-key principle.
7. A method according to claim 1, wherein 2-500,000 cells per
microwell are combined to form a supracellular aggregate,
preferably 10-100,000 cells per microwell, more preferably
10-10,000 cells per microwell.
8. A method according to claim 1, wherein the living cells of the
same or different cell type are combined to form the supracellular
aggregates.
9. A method according to claim 1, wherein the cells are selected
from the group consisting of endothelial cells, smooth muscle
cells, striated muscle cells, neural cells, connective tissue
cells, osteoblasts, osteoclasts, chondrocytes, hepatocytes,
cardiomyocytes, myocytes, Schwann cells, urothelial cells,
parenchymal cells, epithelial cells, exocrine secretory epithelial
cells, epithelial absorptive cells, keratinizing epithelial cells,
extracellular matrix secretion cells, or undifferentiated cells,
such as embryonic cells, progenitor cells, (mesenchymal) stem
cells, bone marrow cells, satellite cells, fibroblasts, and other
precursor cells.
10. A method according to claim 1, wherein the supracellular
aggregates have a mean particle size of 20-400 .mu.m as measured by
light microscopy.
11. A method according to claim 1, wherein the biomaterial is
selected from the group consisting of ceramics, (bio)glasses,
polymeric materials (biodegradable or non-biodegradable), and
metals.
12. A method according to claim 3, wherein the array of microwells
is prepared by microchip technology, hot embossing, selective laser
sintering, solid free-form fabrication, and phase separation
micro-molding.
13. A method according to claim 3, wherein the array of microwells
comprises at least two microwells having a substantially different
size and/or shape.
14. A method according to claim 3, wherein the microwells are made
of agarose, PEG (polyethyleneglycol) or PDMS.
15. A method according to claim 3, wherein the microwell surface is
coated with one or more compounds capable of reducing and/or
preventing cellular adhesion, such as PEG, BSA, collagen and/or
fibronectin.
16. A method according to claim 1, wherein the living cells are
combined in the presence of fibronectin and/or collagen.
17. A method according to claim 1, wherein the surface properties,
the magnetic charge, and/or the electrical charge of the
supracellular aggregates are modified before combining two or more
of the supracellular aggregates.
18. A method according to claim 1, wherein the supracellular
aggregates are combined in a moving liquid, for instance, a
microfluidic chamber and channel.
19. A method according to claim 1, wherein the supracellular
aggregates are combined in a well having an enveloping diameter of
at least 500 .mu.m.
20. A method according to claim 1, wherein the conditions in step
c) comprise one or more selected from mechanical constraints,
compression, shaking, electrical fields, magnetic fields, and
gradients of morphogens or growth factors.
21. A method according to claim 1, wherein step d) comprises
compaction of the cellular aggregates, preferably by applying
geometrical constraints to the tissue construct.
22. A method according to claim 1, wherein in step a) or b) the
living cells or the supracellular aggregate is combined with an
object and/or wherein in step c) or d) the tissue construct is
combined with an object, preferably a biodegradable object and/or a
metallic object, to induce a local response.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/999,841, filed Mar. 14, 2011, pending,
which is a national phase entry under 35 U.S.C. .sctn.371 of
International Patent Application PCT/NL2009/050368 filed Jun. 22,
2009, designating the United States of America and published in
English as International Patent Publication WO 2009/154466 on Dec.
23, 2009, which claims the benefit under Article 8 of the Patent
Cooperation Treaty to European Patent Application Serial No.
08158652.1 filed Jun. 20, 2008, the disclosure of each of which is
hereby incorporated herein in its entirety by this reference.
TECHNICAL FIELD
[0002] The disclosure relates to a new approach to constructing
cellular aggregates of defined sizes and shapes in vitro and their
use in methods for producing 3D-tissue constructs in a modular
way.
BACKGROUND
[0003] Most tissues consist of multiple cell types organized with
specific microscale heterogeneity. Typically, one cubic centimeter
can hold up to 300 million cells. These cells form different
structures within the tissue, including blood capillaries and a
neural network, which are crucial for nutrition, innervation and
homeostasis of the tissue. Cells organize and interact in a
multitude of architectures and synthesize a variety of biologically
active molecules for mechanical support and cellular instruction.
Therefore, living tissues are highly complex.
[0004] Tissue engineering is a term used for attempts to produce
living tissue in vitro from individual or groups of cells. It aims
at repairing or replacing portions of or whole tissues and provides
solutions to shortage of organ donation or to the use of
experimental animals for testing new therapies.
[0005] Due to the high complexity of living tissue, efforts to
produce or mimic living tissues in vitro have been in vain to date
and new methods and technologies to assemble cells into tissue
structures are needed. This is currently a main limitation in
disciplines like regenerative medicine, pharmaceutics, oncology and
developmental biology in which 2D culture and crude small 3D cell
aggregates (see, e.g., WO-A-00/75286) are still standards. As a
result, biological in vitro experiments do not even come close to
complex biological reality, research progress is severely
inhibited, and experimental animals have to be used as an
unsatisfactory experimental alternative instead.
[0006] With the recent developments in both adult and embryonic
stem cell biology, it is becoming truly feasible to induce cells in
culture into more and more of the individual cell types that are
found in the human body and in spectacular high numbers.
[0007] Unfortunately, a satisfactory technology to go from a large
pool of cells including different cell types to a tissue mimic with
a complex architecture has not yet been developed. Several possible
strategies, such as organ printing (see, e.g., WO-A-2005/081971)
and cell sheet technology, are currently being explored. These
strategies rely heavily on the possibility of positioning (pools
of) cells in a predefined organization. These strategies are
encountering obstacles that prevent the translation of a complex
architecture to an actual centimeter scale tissue (i.e., remodeling
of the tissue construct over time due to physical shrinkage or cell
migration). Furthermore, the rationale behind organ printing is
still beyond reach of contemporary science as it simply requires
too many (10.sup.8) single steps. Even at a currently unattainable
speed of depositing one thousand individual cells per second at the
correct three-dimensional location with micrometer accuracy, it
would take close to four days to build a single cubic centimeter of
tissue. These approaches and related technologies result in a
metastable multicellular construct: the construct is not stable but
will remodel according to complex biological principles. This means
that with these strategies, a designed structure and complexity is
not translated to the objective tissue. Promoting the self-assembly
and self-organization of pools of cells is thus a more powerful
approach. In this approach, cells are assembled into a construct
prone to a predictable remodeling over time. Under appropriate
boundary conditions, the construct leads to a final organized
tissue. This is achieved by using a bottom-up approach to
sequentially assemble cells into, subsequently, supracellular
aggregates and tissues and by promoting the self-organization of
the tissue using boundary conditions.
[0008] Several attempts to assemble cells into tissues using a
bottom-up approach are already described, which are different from
the presented disclosure. McGuigan and Sefton (PNAS 2006, 103 (31),
11461-11466) have undertaken an attempt to overcome these practical
difficulties by starting from microscale modular components
consisting of submillimeter-sized collagen gel rods seeded with
endothelial cells into a micro-vascularized tissue. These modules
were manually assembled into a larger tube and perfused by medium
or blood. However, their approach requires the use of a gel, in
this case a collagen gel, to obtain the modules and retain their
structural integrity during the subsequent manual assembly into
larger structures. Although the use of a gel can be advantageous in
some cases to control, for instance, cell density, the entrapment
of cells within a gel will decrease the plasticity of the modules
and prevent fusion between modules. Eliminating the necessity to
use gels for the formation of tissue modules allows for more
plasticity and physiological remodeling of the tissue during the
self-assembly process. Sodunke et al. (Biomaterials 2007, 28 (27),
4006-4016) describe a similar approach based on a biomatrix
hydrogel. Gels have the disadvantages in that the interface is not
available and in that the cells have low movability.
[0009] An early attempt to generate gel-free cellular aggregates
for use as building blocks to construct bigger tissues has been
described by Kelm et al. (Tissue Eng. 2006, 12 (9), 2541-2553).
This attempt is based on the so-called "hanging drop"-method,
wherein cells in an inverted drop of tissue culture medium
precipitate and aggregate. However, this method cannot generate
sufficiently large numbers of cellular aggregates in a short enough
time. Conventional methods for producing multicellular models (such
as the hanging drop method or micro-mass culture) suffer from a
number of limitations including (i) a poor control of size and
shape of the aggregates, and reproducibility, (ii) tedious and
time-consuming manipulations, and (iii) low production yield of
micro-tissues. Napolitano et al. (Tissue Engineering 2007, 13 (8),
2087-2095) describe a method to form cellular aggregates by
self-assembly on micro-molded non-adhesive hydrogels. This document
does not describe the formation of pre-condensed cellular
aggregates in a first step and subsequent self-assembly of the
cellular aggregates as building blocks in a second step. This
method thus induces intense and non-predictable remodeling (e.g.,
shrinking) of the tissue construct.
BRIEF SUMMARY
[0010] The disclosure aims at overcoming one or more of these
problems by producing supracellular aggregates of cells of any cell
type using spatial confinement. These aggregates are used as
building blocks and combined using boundary conditions promoting
their self-assembly and self-organization to create complex
multicellular architectures.
[0011] In a first aspect, the disclosure relates to a method for in
vitro producing a tissue construct comprising: [0012] a) combining
living cells to form supracellular aggregates using spatial
confinement; [0013] b) combining two or more of the supracellular
aggregates in a mold or on a biomaterial; [0014] c) applying
conditions that induce self-assembly within the combined
supracellular aggregates to obtain the tissue construct; and [0015]
d) applying conditions that induce tissue morphogenesis in the
tissue construct.
[0016] The disclosure provides various advantages over prior art
methods, including the use of simple tools that can be handled in
most biology labs, the ability to generate a very large amount of
aggregates in quick and simple procedures (e.g., 220,000 aggregates
per conventional 12-well plate), and the absence of a hydrogel as
supporting material.
[0017] The spatial confinement can be achieved in various manners.
A well-known and often applied way is by using arrays of
microwells. Other ways of imposing spatial confinement include
using air-liquid interfaces like the Hanging drop method or
microfluidic channels. Any biocompatible, processable material can
be used for the spatial confinement applied for assembling the
cells into supracellular aggregates.
[0018] The term "microwell" as used in this application is meant to
refer to an array of numerous cup-like structures formed in a
substantially uniform layer of material by photolithographic
patterning, molding, embossing or other manufacturing processes.
Each microwell thus includes a lower wall (which may be formed by a
substrate on which the microwell material is deposited) and one or
more peripheral side walls (e.g., a single circular wall, or three
or more contiguous substantially straight walls) that extend upward
from the bottom wall and surround a predefined lower wall area,
with upper edges of the peripheral side walls defining an open end
of the microwell. Typically, microwells having an enveloping
diameter of 50-500 .mu.m can be used. The depth of the microwells
is normally in the range of 100-1000 .mu.m. For seeding, it is
advantageous that individual microwells are close to each other in
order to prevent cells staying on the spaces between the
microwells. Thus, the maximum distance between two individual
neighboring microwells on the array can be, for example, 300 .mu.m
or less, preferably 200 .mu.m or less, more preferably 100 .mu.m or
less, such as about 50 .mu.m. The number of microwells in the array
can vary depending on the size of the microwells and the distance
between individual microwells. One array can suitably have
50-20,000 wells, such as 50-5000, or 100-2000 wells.
[0019] The term "self-assembly" as used in this application is
meant to refer to the creation of tissue units (or small building
units) by association of individual cells or cellular aggregates.
The individual cells or cellular aggregates adhere together in
specific arrangements to give one-dimensional, two-dimensional or
three-dimensional superstructures. The aggregation may be
spontaneous without human intervention, or may be as a result of
changing local environmental conditions, e.g., temperature,
concentration of cells, physical boundaries (such as specific shape
or dimension of the microwells and/or mold), etc.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1. Top: illustrative scheme of the disclosure. Bottom:
tools that can be used to bring the disclosure into practice.
[0021] FIG. 2. SEM picture of a microwell array (diameter 100
.mu.m, depth 350 .mu.m) for the spontaneous formation of
micro-tissues. In insert, enlarged view of wells molded in
PDMS.
[0022] FIG. 3. Spheroids prepared from HUVECs cells in a PDMS
microsystem (10:0.5) coated with 35,000 MW PEG at 50 mg/ml.
Microwells are of 200 .mu.m diameter and 350 .mu.m depth.
[0023] FIGS. 4A and 4B. Spheroids prepared from human bone
marrow-derived mesenchymal progenitor cells in a PDMS microsystem
(10:0.5) coated with 35,000 MW PEG at 50 mg/ml. Microwells are of
200 .mu.m diameter and 350 .mu.m depth. FIG. 4A picture is taken
just after seeding and picture of FIG. 4B after two days of
culture.
[0024] FIG. 5. Stainless steel mold (left) used as a master to
replicate an agarose chamber (right) used to assemble
aggregates.
[0025] FIG. 6. Aggregates of HUVEC and hMSC assembled into the
agarose chamber just after seeding after three days of culture. The
width of the chamber is 1 mm; the depth of the chamber is 1 mm; and
the length of the chamber is 1 cm.
[0026] FIGS. 7A and 7B. Self-assembly of aggregates of different
cell types. Cross-section of cylinder described previously after
three days (FIG. 7A) and six days (FIG. 7B). On day 3, one can
observe the segregation of cell types with the HUVEC, in red,
forming an exterior layer. After six days, the angiogenesis
processes took place and capillaries are formed in the tissue
construct.
[0027] FIGS. 8A-8F. Cellular aggregates of hMSC can be assembled
into tissue constructs of different shapes to build tissue units
that can then be assembled into bigger constructs. FIGS. 8A and 8B:
15 minutes after seeding the aggregates. FIGS. 8C and 8D: 5 hours
after seeding the aggregates. FIGS. 8E and 8F: tissue constructs
were released from the wells 24 hours after seeding the
aggregates.
[0028] FIG. 9A. The size of the building blocks depends on the
number of cells seeded and on the size of the microwells.
[0029] FIG. 9B. Different compositions of culturing media induce
different levels of compaction of the spheroids over time.
[0030] FIG. 9C. Different cell types show different plasticity and
maintenance of the shape over time.
[0031] FIG. 10. Remodeling of the tissue construct into a desired
geometry by using compensated shapes.
[0032] FIG. 11A. Local compaction of the tissue depends on the
geometrical shape.
[0033] FIG. 11B. Local stress of the tissue depends on the
geometrical shape.
[0034] FIG. 12. Tissues can further be assembled into
centimeter-scale tissues.
DETAILED DESCRIPTION
[0035] In a first step, a method according to the disclosure
comprises producing a supracellular aggregate of cells. These cells
may be of the same ("homocellular") or different ("heterocellular")
type within one aggregate. It is preferred, however, that one
aggregate is formed of cells of one cell type. Diversification of
the tissue construct to be produced may be achieved by combining
aggregates of different cell types.
[0036] Many cell types may be used to form the cell aggregates. In
general, the choice of cell type will vary depending on the type of
three-dimensional construct to be built. For example, for a blood
vessel type three-dimensional structure, the cell aggregates will
advantageously comprise a cell type or types typically found in
vascular tissue (e.g., endothelial cells, smooth muscle cells,
etc.). In contrast, the composition of the cell aggregates may vary
if a different type of construct is to be produced (e.g.,
intestine, liver, kidney, etc.). One skilled in the art will thus
readily be able to choose an appropriate cell type for the
aggregates, based on the objective type of three-dimensional
construct. Non-limiting examples of suitable cell types include
contractile or muscle cells (e.g., striated muscle cells and smooth
muscle cells), neural cells, connective tissue (including bone,
cartilage, osteoblasts, osteoclasts, cells differentiating into
bone-forming cells and chondrocytes, and lymph tissues),
hepatocytes, cardiomyocytes, myocytes, Schwann cells, urothelial
cells, parenchymal cells, epithelial cells (including endothelial
cells that form linings in cavities and vessels or channels,
exocrine secretory epithelial cells, epithelial absorptive cells,
keratinizing epithelial cells, and extracellular matrix secretion
cells), and undifferentiated cells (such as embryonic cells,
progenitor cells, (mesenchymal) stem cells, bone marrow cells,
satellite cells, fibroblasts, and other precursor cells), among
others. In addition, plant cells and algae may suitably be
used.
[0037] The aggregates may vary in both size and shape. They may,
for example, be in the form of a sphere, a cylinder (preferably
with equal height and diameter), a rod, a cube, or the like.
Although other shaped aggregates may be used, it is generally
preferable that the cell aggregates are spherical, cylindrical
(with equal height and diameter), or cuboidal (i.e., cubes), as
aggregates of these shapes may be easier to manipulate. The shape
of the cellular aggregates can play an important role in promoting
self-assembly. Different shapes of aggregates can generate
different arrangements by stacking. The shapes of the cellular
aggregates can, for instance, promote close proximity between
cellular aggregates (e.g., key-lock system), or create free space
at their interfaces. Aggregates are substantially uniform in size
and substantially uniform in shape when they are combined but
different shapes and sizes can be assembled to generate different
heterogeneous structures.
[0038] Although the exact number of cells per aggregate is not
critical, it will be recognized by those skilled in the art that
the size of each aggregate (and thus the number of cells per
aggregate) is limited by the capacity of nutrients to diffuse to
the central cells, and that this number may vary depending on cell
type. Cell aggregates may comprise a minimal number of cells (e.g.,
two or three cells) per aggregate, or may comprise many hundreds or
thousands of cells per aggregate. Typically, cell aggregates
comprise hundreds to hundreds of thousands of cells per
aggregate.
[0039] The number of cells in one aggregate can be controlled by
the applied spatial confinement. For instance, the number of cells
in one aggregate can be dependent on the number of cells that are
seeded in a microwell and the size of the well. There is, however,
no 1:1 ratio, because cell death and proliferation may occur during
formation of the aggregate. In a suitable embodiment, the number of
cells that is provided per microwell is 2-500,000, such as
100-100,000, or 100-50,000. Furthermore, the number of cells
applied, e.g., per microwell, also depends on the desired aggregate
size.
[0040] For purposes of the present disclosure, the cellular
aggregates are typically from about 100 microns to about 600
microns in size, such as from about 200 to about 400 microns,
although the size may be greater or less than this range, depending
on cell type. In one embodiment, the cell aggregates are from about
250 microns to about 400 microns in size. In another embodiment,
the cell aggregates are about 250 microns in size. For example,
spherical cell aggregates are preferably from about 20 microns to
about 600 microns in diameter (such as from about 100 microns to
about 600 microns), cylindrical cell aggregates are preferably from
about 100 microns to about 600 microns in diameter and height, and
the sides of cuboidal cell aggregates are preferably from about 100
microns to about 600 microns in length. Aggregates of other shapes
will typically be of similar size. The size of the aggregates can
be measured using standard light microscopy techniques.
[0041] The size of the cellular aggregates can be controlled by the
spatial confinement, such as by size of the microwells, as well as
by the number of cells that is used, such as the number of cells
seeded to the microwells. Importantly, the size of the aggregates
depends more on the number of cells than on the enveloping diameter
of the spatial confinement. The size and/or the shape of the
spatial confinement can be roughly adjusted to facilitate proper
aggregate formation. If the spatial confinement is too large, the
cells will not find each other and will not aggregate. If the
spatial confinement is too small, then not all cells will fit in
the well. For example, for aggregates having a size between 0 and
90 .mu.m, circular microwells with a diameter of 100 microns are
suitable; for aggregates having a size between 90 and 150 .mu.m,
circular microwells with a diameter of 200 .mu.m are suitable; for
aggregates between 150 and 350 .mu.m, circular microwells with a
diameter of 400 microns are suitable.
[0042] As mentioned above, with a suitable embodiment of the
disclosure, aggregates of cells are produced using arrays of
microwells that can be produced with technologies that include, but
are not limited to: microchip technology, hot embossing, selective
laser sintering, solid free-form fabrication, and phase separation
micro-molding. With these technologies, arrays of microwells can be
produced in sheets of different materials including, but not
limited to: PDMS (polydimethylsiloxane), collagen, gelatin,
hydrogels, and the like. An important advantage of the
above-mentioned technologies is that they can produce microwells
with different size and shape.
[0043] The disclosure considers both the use of spatial confinement
with single morphology (such as arrays containing single-microwell
morphology) and spatial confinement with two or more morphologies
(such as arrays containing two or more microwell morphologies). In
an embodiment, cell aggregates are formed by applying a cell
suspension on top of the microwell array. Typically, the cell
concentration in the cell seed suspension is in the range of
500,000 cells per ml to 5,000,000 cells per ml. Cells either settle
in the microwells spontaneously due to gravitational forces, or are
forced in the microwells using, for instance, centrifugal,
capillary forces or microfluidic devices.
[0044] Upon spatial confinement, the cells will aggregate
spontaneously by adhesion between the cells. The adhesion between
the same cell types is not necessarily better than between
different cell types, although this may be the case for some
specific cell types. The adhesion between the different cells
differs from cell type to cell type. For instance, human
mesenchymal stem cells will form spheroids that condense a lot due
to strong adhesion between the cells, HUVEC will form spheroids
that hardly condense due to moderate adhesion between the cells,
and Chinese hamster ovary cells will instead form plates of
spheroids due to low adhesion between the cells. Assembly of the
cells into supracellular aggregates may be assisted by various
tools known in the art, such as microfluidic tools, moving liquids,
confining chambers with modular properties (adherent/non-adherent
surfaces), using surfaces with topographies, or using surfaces with
coatings.
[0045] It is important to note in this aspect that in order for
aggregates to form, the adhesion between cells and the surrounding
material (such as the material of the microwell) is preferably
lower than the adhesion between the cells themselves. This can, for
example, be achieved by using microwells of materials that display
low cellular adhesion, such as PEG (polyethyleneglycol), PDMS or
the like, or by coating the microwell surface with molecules that
prevent cellular adhesion, such as PEG and BSA (bovine serum
albumin). Moreover, it is important to note that the formation of
aggregates depends on the cellular adhesion of the cell type used.
When a certain cell type is unable to form cellular aggregates
spontaneously, aggregation may be initiated using compounds such as
fibronectin or collagen that can be added to the cell
suspension.
[0046] The shape of the aggregates can be manipulated by altering
the spatial confinement shape. The size of the aggregates can be
manipulated by altering the size of the spatial confinement, the
cell concentration of the cell suspension used, and/or the
composition of the culture medium that is used during cellular
aggregation.
[0047] An advantage of using microwells when compared to classical
methods to produce cellular aggregates, like the hanging-drop
method, is that in a single "step," one can make thousands of
aggregates at the same time, instead of merely one aggregate. This
enables the fabrication of the vast quantities of aggregates that
are needed for this bottom-up approach. For instance, in comparison
with the spontaneous aggregation in a cell suspension, the
microwells allow a precise control and reproducibility of the
shape, size, and surface properties of the aggregates.
[0048] The disclosure is further illustrated in FIG. 1. The top
scheme describes the technical steps to assemble cells into tissues
with geometric steps in a bottom-up approach. Cells are assembled
into spheroids that are used as building blocks to build tissues.
These tissues are shaped, e.g., to promote self-remodeling and can
be influenced to self-organize. For example, sharp tips of a
triangular tissue promote compaction of the construct inducing
further developmental mechanisms. The bottom picture shows some of
the tools that can be used to bring the disclosure into practice.
Polymeric stamps can be used to replicate structures into agarose.
Agarose chips can be inserted into a conventional microwell plate
and used for cell and tissue culture.
[0049] The cell suspension can suitably be added to a container
(for instance, 12-well plate) in which an array of microwells has
been placed on the bottom. The cells can then sink into the wells
by gravitational or centrifugational forces. In principle, the
values of temperature and pH do not have to vary from the values
that are used during standard culture of the respective cell types.
However, there are applications foreseeable where, for instance, a
change in temperature can be used to initiate cell aggregation. The
basis for the cell suspension is normally a culture medium
supplemented with standard nutrients (not different from normal
cell culture). Aggregation usually takes place in a standard
incubator (humidified, 37.degree. C., 5% CO.sub.2). If different
cell types are used, they can be mixed in one cell suspension, or
they can be applied separately, depending on the initial situation
one wants to create. If both cells are mixed in one cell
suspension, the different cell types will typically be regularly
mixed in the resulting aggregate. If the different cell types are
applied in different cell suspensions one after the other, the
resulting aggregate will typically consist of two (or more)
distinct regions containing the two (or more) different cell
types.
[0050] It is an advantage of the disclosure that aggregates may be
formed that do not contain anything but living cells. In
particular, the use of a gel is not necessary. This way, aggregates
of particular high cell density may be formed. In some cases, this
can lead to a better contact between the different cells for
exchange of compounds, since some cells rely on direct cell contact
for cellular communication. In addition, the absence of a gel
allows for the cells to better produce their own extracellular
matrix in a physiological way. Furthermore, the addition of a gel
from xenogenous origin may impose a complication for clinical
applications. By only using autologous cells, the product is
completely patient-owned.
[0051] Step a) allows the condensation of cells into building
blocks (supracellular aggregates). This condensation process that
is occurring over time is essential, since shaped micro-tissues
cannot be produced on a large (mm) scale by seeding the cells in
large wells. Condensation of the small aggregates minimizes the
condensation of the bigger shapes in a later stage. If the step of
forming the supracellular aggregates through condensation would be
skipped, then the shape of a subsequent seeding surface (such as
macro-wells) will not be translated to the desired construct. After
seeding, the tissue will condense toward a spheroid, regardless of
the shape of the seeding surface. Apart from that, the inventors
found that pre-condensation (i.e., formation of supracellular
aggregates) allows seeding a larger number of cells as aggregates
(e.g., spheroids), compared to seeding a cell suspension. It is,
therefore, necessary to first condense cells into dense building
blocks (supracellular aggregates) that thereafter can be used and
assembled into bigger constructs.
[0052] Thereafter, the cellular aggregates are combined to obtain
larger tissue constructs. This can be described as a two-stage
process.
[0053] The first stage is the self-assembly of cellular aggregates
into a bigger tissue construct. In a suitable embodiment, the
aggregates are removed from a microwell array by flushing (culture)
medium over the surface of the microwell. Another possibility is to
invert a chip with microwells onto the surface to be seeded.
Aggregates are then released by gravitational or centrifugational
forces and transferred to the seeding surface (e.g., biomaterial,
scaffold, macro-well). For self-assembly, the aggregates can, for
instance, be transferred into wells having an enveloping diameter
of at least 500 .mu.m. Any biocompatible, processable material can
be used for the spatial confinement applied for assembling the
supracellular aggregates into tissue constructs.
[0054] Self-assembly of the cellular aggregates will be governed by
imposed "boundary conditions" of the cellular aggregates, such as
supracellular aggregate size, supracellular aggregate shape,
supracellular aggregate surface properties (for instance,
hydrophilicity/hydrophobicity or a coating with bioactive molecules
that results in specific interactions between the cellular
aggregates), supracellular aggregate electrical charge,
supracellular aggregate magnetic charge, and of "boundary
conditions" of the chamber used to assemble the cellular
aggregates, such as adherent or non-adherent surfaces of the
chamber, topographies of the surface of the chamber, protein
deposition and patterning at the surface of the chamber and the use
of microfluidic to promote the arrangement and assembly of the
cellular aggregates.
[0055] Preferably, the "boundary conditions" are imposed on the
aggregates before they are released from the initial spatial
confinement. Depending on the type of boundary condition, this may
or may not require an extra active step. For instance, the boundary
condition "supracellular aggregate size" is already imposed by the
spatial confinement and the seeding density. The boundary condition
"supracellular aggregate surface properties" can be adjusted, for
instance, by coating the aggregates before releasing them from the
spatial confinement. The boundary condition "supracellular
aggregate magnetic charge" can be imposed during seeding (e.g., by
including magnetic particles) or by coating the aggregates before
releasing them from the spatial confinement.
[0056] After incorporating these boundary conditions to the
cellular aggregates or the chamber used for their self-assembly,
self-assembly of the cellular aggregates can be guided, e.g., in a
chamber or in a moving liquid by applying, for instance, mechanical
constraints, shear stress using a liquid, compression, shaking,
electrical fields, magnetic fields, or gradients of morphogens
and/or growth factors. The shape, size and cell type(s) of the
supracellular aggregates is important in the early stage of the
assembly to promote mesoscale organization and create the
heterogeneous structure of interest. Self-assembly of the
aggregates normally takes several hours. Typically, it takes at
most one day. The structure of interest can include the simple
assembly of spherical aggregates into the shape of a cylinder or
the more complex assembly of spherical aggregates into blocks
(cubes, triangles, etc.) that can then be assembled into bigger
constructs. For example, using the plastic properties of cells,
chambers with compensated shapes can be designed, which result in
the desired tissue construct shapes. The design and structures of
those constructs should promote the creation of local environment
leading to further remodeling and tissue development.
[0057] Some illustrative examples of conditions that can be used to
promote self-assembly of the cellular aggregates into a tissue
construct include the cell type, the medium used to culture the
tissue, and the time of incubation on, e.g., the microwell array
before the transfer to the final chamber.
[0058] The second stage involves the remodeling and/or
reorganization of the cells and/or tissue in the construct. In this
stage, conditions are applied that induce tissue morphogenesis in
the tissue construct. The term "morphogenesis" in this application
is meant to refer to a coordinated series of molecular and cellular
events that shape the structure of the tissue construct. Tissue
morphogenesis and can be governed by migration of cells, physical
traction of cells, differentiation of cells, local production of
soluble or insoluble (extra-cellular matrix) biological factors, or
combinations thereof. Remodeling and/or reorganization can further
involve compaction of the cells and/or tissue in the tissue
construct. This second stage can be characterized as further
development of the tissue construct and can again be guided by
applying artificial parameters such as mechanical constraints,
compression, shaking, electrical fields, magnetic fields, the
action of objects embedded into the construct and may or may not be
subjected to external forces, or gradients of morphogens and/or
growth factors. Typically, the combination of cellular aggregates
of different sizes in a stirred liquid promotes the formation of
patterned arrangements. The combination of cellular aggregates of
complementary shapes promotes the formation of tissues with
repetitive units.
[0059] Remodeling and/or reorganization can, for instance, involve
applying geometrical constraints (such as using a chamber with
specific geometry) to the tissue construct. This can induce
self-organization into tissues (such as local compaction and local
growth of capillaries).
[0060] The geometrical shape of the tissue in itself can induce
local remodeling and/or reorganization of the cells, including
compaction of the cells, local stress, and local sprouting of
endothelial cells into blood capillaries.
[0061] The cells can be assembled on chips made of biocompatible
materials including agarose, PDMS or PLLA cast on etched silicon
wafers by conventional lithography process or replicated by
hot-embossing. Polymers can be further functionalized to modify
their interaction with cells by using coatings with polymers (e.g.,
PEG) or proteins (e.g., BSA), or patterns of adhesive proteins
promoting local adhesion of the tissue construct or nanometer and
micrometer topographies. Chips are in the order of centimeter scale
and fit in classical cell-culture well-plates. Wells in the order
of 100 to 1500 .mu.m (such as in the order of 500 to 1500 .mu.m)
are generated in which the aggregates can self-assemble.
[0062] Depending on the methods that are used for the self-assembly
of the cells and/or cellular aggregates, a wide variety of
construct shapes can be designed and prepared using the method of
the disclosure. For instance, when using wells in which the
aggregates are combined to constructs, the shape of the wells will
be translated to the shape of the construct.
[0063] In addition, the construct size may vary widely. However,
the maximum size may be limited by the diffusion distance of oxygen
and nutrients. A way to overcome this is, for instance, by using
perfusion or superfusion systems. The constructs will normally have
a size of at least 500 .mu.m, or at least 1 mm. The upper limit of
the size can, for instance, be 4 mm or 1.5 cm.
[0064] When combining the cell aggregates to obtain a tissue
construct, self-assembly may be assisted using a biomaterial, e.g.,
to form a scaffold and provide mechanical support or to assist in
achieving a particular desired shape. In addition, biomaterials or
bio-active factors may be included that guide the development or
organization of the tissue construct. Types of biomaterials that
can be incorporated include, but are not limited to: ceramics,
bioglasses, polymeric materials (biodegradable or
non-biodegradable), metals, and gels. Types of bio-active factors
that can be incorporated include, but are not limited to: enzymes,
receptors, neurotransmitters, hormones, cytokines, cell response
modifiers such as growth factors and chemotactic factors,
antibodies, vaccines, haptens, toxins, interferons, ribozymes,
anti-sense agents, plasmids, DNA, and RNA. Biodegradable objects
and/or metallic objects are preferred. It is possible to combine
the object with living cells, to combine the object with
supracellular aggregates, and/or to combine the object with tissue
constructs. The object can thus be introduced in steps a) or b)
and/or in steps c) or d). Metallic objects can be used to modify
the cellular aggregate or tissue by using an electrical or magnetic
field.
[0065] An important aspect of the disclosure is that the
aggregates, after having been combined, will self-assemble into
biological tissues, which may vary in complexity. To this end,
aggregates of different cell types are preferably combined.
Aggregates of the cell types that make up a tissue may be combined
to replicate the tissue. Features to incorporate in tissues may
include, but are not limited to, a vascular network (endothelial
cells and smooth muscle cells/pericytes), a neural network (neural
cells), and a lymphatic network (lymphatic endothelial cells). For
instance, for skeletal muscle tissue, aggregates of skeletal muscle
cells, neural cells, endothelial cells, smooth muscle
cells/pericytes, and lymphatic endothelial cells may be
combined.
[0066] Without wishing to be bound by theory, it is postulated that
the self-assembly of the aggregates into tissue structures (also
referred to as tissue morphogenesis) can be caused by migration of
cells, physical traction or compaction of cells, local production
of soluble or insoluble (extra-cellular matrix) biological factors,
differentiation of cells, or combinations thereof.
[0067] The obtained tissue constructs can be used for various
applications. They can, for instance, serve as a platform for
creating constructs for tissue repair, or as a platform for
studying tissue development (as a scientific tool), as an in vitro
test model for compound testing in pharmacology or cosmetics, etc.
The disclosure will now be further elucidated by way of the
following, non-restrictive examples.
Examples
[0068] The micro-device shown in FIG. 2, consisting of an array of
microwells of various dimensions (well diameter, spacing and depth)
fabricated in PDMS, was used for the spontaneous and simultaneous
formation of a number of microscale spheroids in a fast, controlled
and reproducible way (see FIG. 3). Aggregate formation is
straightforward and requires reduced amounts of cells and
biological factors. The size of the micro-tissues is tunable
(.about.25 to 100,000 cells) and more suitable for imaging
purposes. First, the optimal properties of the material were
studied, i.e., giving little or no cellular adherence and strong
cellular aggregation for the preparation of spheroids based on
hMSCs (human Mesenchymal Stem Cells) or HUVECs (Human Umbilical
Vein Endothelial Cells), and the PDMS composition (curing
agent:base ratio) in combination with various coatings was notably
investigated. Both parameters greatly influence cellular adherence
and aggregation. The results range from strong to no adherence on
the surface, and cellular assembly from isolate cell "suspension"
to extensive cell aggregation. Best efficiency in the formation of
spheroids is observed with a coating of 35,000 MW PEG and a 10:0.5
PDMS composition. PDMS 10:0.5 gives the smallest cellular adherence
and 35,000 MW PEG at a concentration of 50 mg/ml promotes cellular
aggregation (see Table 1). The resulting spheroids exhibit a size
in the hundreds of micron range depending on the size of the
microwells and the cell seeding density, see FIGS. 4A and 4B.
[0069] Cellular aggregates can then be harvested and assembled into
different shapes, and different cell types can be combined. Here,
the case of the assembly of hMSC and HUVEC aggregates into an
agarose mold was presented. The mold is made by replication of
agarose on a stainless steel master (1.times.1.times.10 mm), see
FIG. 5. Five thousand cellular aggregates of each cell type were
combined into this mold. They self-assembled into a stratified tube
with a layer of HUVEC surrounding a core of hMSC (FIGS. 7A and 7B).
This self-assembly process is due to the differential surface
tension of the two types of aggregates promoting segregation. Over
time, the construct will remodel according to biological processes
of angiogenesis and lead to a vascularized cylinder of dense
tissue.
TABLE-US-00001 TABLE 1 Preparation of micro-tissues in coated
PDMS-based microwells: Cellular aggregation and adherence on the
surface depending on the PDMS composition and the coating nature.
Coating PDMS BSA BSA PEG 300 PEG 35,000 composition O Fibronectin
10 mg/ml 50 mg/ml 10 mg/ml 50 mg/ml Agarose 10:0.5 Adherence:
Adherence: Adherence: Adherence: Adherence: Adherence: Adherence: +
+ --- +++ - No No Aggregation: Aggregation: Aggregation:
Aggregation: Aggregation: Aggregation: Aggregation: + ++ --- ++ +
+++ --- 10:1 Adherence: Adherence: Adherence: Adherence: Adherence:
Adherence: Adherence: -- - --- +++ --- + --- Aggregation:
Aggregation: Aggregation: Aggregation: Aggregation: Aggregation:
Aggregation: -- +++ -- +++ - +++ --- 10:3 Adherence: Adherence:
Adherence: Adherence: Adherence: Adherence: Adherence: +++ +++ -
+++ + + ++ Aggregation: Aggregation: Aggregation: Aggregation:
Aggregation: Aggregation: Aggregation: ++ + +++ + + + ++
[0070] In FIGS. 7A and 7B, human mesenchymal stem cell from bone
marrow and human umbilical vein endothelial cells were separately
cultured and aggregated onto chips. The chips are made of PDMS
coated with 50 mg/ml BSA. The microwells on the chip are 200
microns diameter and 300 microns deep. Cells were allowed to
aggregate into spheroids over 24 hours. hMSC are cultured in
DMEM+glutamax, 100 nM dexamethasone (Sigma), 1% Pen/Strep (100
U/100 .mu.g/ml, GIBCO), 50 mg/ml ITS-plus Premix (BD), 50 .mu.g/ml
ascorbic acid (Sigma), 40 .mu.g/ml proline (Sigma), 100 .mu.g/ml
sodium pyruvate (Sigma). HUVEC are grown and aggregated in EGM2
medium (Lonza).
[0071] Five thousand spheroids of each cell type (10,000 spheroids
total) were transferred to an agarose chip with one trench (1 mm
width, 1 mm depth and 1 cm long). This agarose (4%) is molded on a
stainless steel mold.
[0072] The 10,000 spheroids quickly aggregated and formed a
cylindrical tissue construct. This construct was cultured for six
days and sectioned and immunostained at days 3 and 6 for CD31 and
Dapi.
[0073] A self-assembly of the two cell types in two concentric
layers was observed at day 3 where the HUVEC are forming an
external layer and the hMSC an internal core. This was followed by
an invasion of the HUVEC into the center of the construct on day 6
and the formation of a primitive capillary network.
[0074] In FIGS. 8A-8F, spheroids of hMSC were produced as described
above. Fifteen thousand spheroids of 100 microns diameter were
transferred onto an agarose chip with wells of different shapes
(i.e., squares, triangles and circles). The agarose chip (4%
agarose) is molded on a PDMS mold. The wells have a total surface
area of 0.64 mm.sup.2 and a depth of 1 mm. The spheroids were
seeded onto the chip and formed mesoscale tissue of defined size
and shape. Those mesoscale tissues were harvested after 24 hours
and can be combined and used to build tissue models or tissue
implants.
[0075] FIG. 9A shows that the size of the building blocks depends
on the number of cells seeded and on the size of the microwells.
Using human mesenchymal stem cells and two different sizes of
microwells (200 .mu.m and 400 .mu.m), building blocks from 30 .mu.m
to 150 .mu.m were assembled. In FIG. 9B, it is also shown that
different culturing media can induce different levels of
compaction. Furthermore, as shown in FIG. 9C, different cell types
show different plasticity and maintenance of the shape over time.
This plasticity decreased with longer incubation on the microwells
array. For each cell type and each culturing medium, a time of
incubation on the microwells array has to be adjusted.
[0076] FIG. 10 shows that compaction of the tissue construct is not
uniform for all shapes. Corners are regions of greater compaction.
Compensated shapes can be designed to promote remodeling of the
tissue into a desired geometry.
[0077] In FIGS. 11A and 11B, it is shown that both the local
compaction and the local stress of the tissue depend on the
geometrical shape. FIG. 11A shows a nuclear staining of 7 .mu.m
cuts. A local compaction of the cells (nuclei are closer to each
other) was observed on the outside of tissues compared to the
inside in discs (left pictures) and tip effects with local cell
compaction in the tips of triangular tissues (right pictures). FIG.
11B shows a cytoskeleton staining of 7 .mu.m cuts. Regions of more
intense F-actin populations of cells were observed. The shape of
the tissues created local microenvironments of stress.
[0078] As can be seen from FIG. 12, further assembling into tissues
of clinically relevant size (such as centimeter-scale) is possible.
Tissues spontaneously fuse and can be manipulated, thus achieving
clinical relevance.
* * * * *