U.S. patent application number 10/200955 was filed with the patent office on 2003-01-02 for fabrication of vascularized tissue using microfabricated two-dimensional molds.
Invention is credited to Borenstein, Jeffrey T., Cunningham, Brian T., Pien, Homer, Vacanti, Joseph P..
Application Number | 20030003575 10/200955 |
Document ID | / |
Family ID | 26829934 |
Filed Date | 2003-01-02 |
United States Patent
Application |
20030003575 |
Kind Code |
A1 |
Vacanti, Joseph P. ; et
al. |
January 2, 2003 |
Fabrication of vascularized tissue using microfabricated
two-dimensional molds
Abstract
A method and materials to create complex vascularized living
tissue in three dimensions from a two-dimension microfabricated
mold has been developed. The method involved creating a two
dimensional surface having a branching structure etched into the
surface. The pattern begins with one or more large channels which
serially branch into a large array of channels as small as
individual capillaries, then converge to one or more large
channels. The etched surface serves a template within a mold formed
with the etched surface for the circulation of an individual tissue
or organ. Living vascular cells are then seeded onto the mold,
where they form living vascular channels based on the pattern
etched in the mold. Once formed and sustained by their own matrix,
the top of the mold is removed. The organ or tissue specific cells
are then added to the etched surface, where they attach and
proliferate to form a thin, vascularized sheet of tissue. The
tissue can then be gently lifted from the mold using techniques
such as fluid flow and other supporting material, as necessary. The
tissue can then be systematically folded and compacted into a
three-dimensional vascularized structure. This structure can then
be implanted into animals or paitents by directly connecting the
blood vessels to flow into and out of the device. Immediate
perfusion of oxygenated blood occurs, which allows survival and
function of the entire living mass.
Inventors: |
Vacanti, Joseph P.;
(Winchester, MA) ; Borenstein, Jeffrey T.;
(Holliston, MA) ; Pien, Homer; (Andover, MA)
; Cunningham, Brian T.; (Lexington, MA) |
Correspondence
Address: |
Thomas J. Kowalski
FROMMER LAWRENCE & HAUG LLP
745 Fifth Avenue
New York
NY
10151
US
|
Family ID: |
26829934 |
Appl. No.: |
10/200955 |
Filed: |
July 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10200955 |
Jul 22, 2002 |
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09560480 |
Apr 28, 2000 |
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6455311 |
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60131930 |
Apr 30, 1999 |
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60165329 |
Nov 12, 1999 |
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Current U.S.
Class: |
435/371 ;
435/299.1 |
Current CPC
Class: |
C12N 5/0697 20130101;
C12N 5/0671 20130101; A61K 35/44 20130101; C12N 2502/28 20130101;
A61K 35/12 20130101; A61L 27/3604 20130101; B33Y 70/00 20141201;
C12N 2502/14 20130101; A61L 27/3886 20130101; A61L 27/3895
20130101; A61L 27/38 20130101; C12N 2535/10 20130101 |
Class at
Publication: |
435/371 ;
435/299.1 |
International
Class: |
C12N 005/08; C12M
001/14; C12M 003/04 |
Goverment Interests
[0002] The United States government has certain rights in this
invention by virtue of grant number DAMD 17-99-2-9001 from the
Department of Defense.
Claims
We claim:
1. A system for creating thin tissue layers comprising: (a) a mold
comprising a support surface having a pattern of channels thereon,
suitable for attachment and culturing of cells to form lumens
within the channels, and suitable for attachment and culturing of a
different type of cells in the areas on the surface surrounding the
channels.
2. The mold of claim 1 wherein the system further comprises (b)
connections and means for circulation of culture fluid through the
mold for culturing of the cells attached thereto.
3. The system of claim 1 wherein the mold has branched channels
therein, beginning from one or more inlets, expanding into more
channels, and then converging back into one or more outlets.
4. The system of claim 1 further comprising pumping means for
circulating fluid through the mold.
5. The system of claim 1 wherein the mold is formed from a material
selected from the group consisting of silicon, glass, natural cell
substrates like hydroxyapatite, and polymer.
6. The system of claim 1 further comprising a coating on the
channels which promotes adhesion and lifting of cells as intact
sheets from the mold.
7. A method for making a mold comprising a support surface having a
pattern of channels thereon, suitable for attachment and culturing
of cells to form lumens within the channels, and suitable for
attachment and culturing of a different type of cells in the areas
on the surface surrounding the channels, comprising: (a) selecting
a material for forming the support surface from the group
consisting of silicon, metals, polymers, and natural cell
substrates like hydroxyapatite, (b) patterning channels in the
surface of the material to create lumens which can be seeded with
cells to form tubular structures for fluid flow, and (c) forming
the mold so that cells can be seeded within the mold to form a thin
layer of tissue surrounding the tubular structures.
8. The method of claim 7 wherein the patterning is done by a
process selected from the group consisting of micromachining,
lithography, etching, or molding.
9. A method for forming complex tissues comprising (a) selecting a
mold having a complex pattern of channels formed into at least one
surface into which cells can be seeded; (b) seeding cells into the
channels to form blood vessels or other lumens; (c) culturing the
cells under conditions until they form vessels or lumens; (d)
seeding other type(s) of cells onto or into the mold so that a
tissue is formed incorporating the vessels or lumens; and (e)
removing the tissue layer.
10. The method of claim 9 further comprising (f) assembling
multiple layers of the tissue layers until the desired complex
structure is formed.
11. The method of claim 10 further comprising (g) implanting into a
body the complex structure and anastomizing the vessles with the
blood supply or lumens in other organs or tissues within the
body.
12. The method of claim 11 further comprising applying to the
channel a coating on the channels which promotes adhesion and
lifting of cells as intact sheets from the mold.
13. A complex structure comprising multiple layers of tissue and
vasculature formed by a method comprising (a) selecting a mold
having a complex pattern of channels formed into at least one
surface into which cells can be seeded; (b) seeding cells into the
channels to form blood vessels or other lumens; (c) culturing the
cells under conditions until they form vessels or lumens; (d)
seeding other type(s) of cells onto or into the mold so that a
tissue is formed incorporating the vessels or lumens; and (e)
removing the tissue layer.
14. The structure of claim 13 wherein the vasculature comprises
endothelial cells.
15. The structure of claim 14 wherein the other cells are selected
from the group consisting of parenchymal cells, cells forming
cartilage or bone, muscle cells, and nerve cells.
16. The structure of claim 15 wherein the parenchymal cells are
derived from organs selected from the group consisting of heart,
liver, pancreas, intestine, brain, kidney, reproductive tissues and
lung.
Description
[0001] This application claims priority to U.S.S.No. 60/131,930
filed Apr. 30, 1999, and U.S.S.No. 60/165,329 filed Nov. 12,
1999.
BACKGROUND OF THE INVENTION
[0003] The present invention generally relates to the fields of
organ transplantation and reconstructive surgery, and to the new
field of Tissue Engineering. It more specifically is a new method
and materials for generating tissues requiring a blood vessel
supply and other complex components such as a nerve supply,
drainage system and lymphatic system.
[0004] Organ transplantation, as currently practices, has become a
major lifesaving therapy for patients afflicted with disease which
destroy vital organs including the heart, liver, lungs, kidney and
intestine. However, the shortage of organs needed for
transplantation has become critical and continues to worsen.
Likewise, every major field of reconstructive surgery reaches the
same barrier of tissue shortage. Orthopedic surgery, vascular
surgery, cardiac surgery, general surgery, neurosurgery, and the
others all share this fundamental problem. Therefore, countless
patients suffer as a result.
[0005] Over the last twelve years, the new field of tissue
engineering has arisen to meet this need. The field brings the
expertise of physicians, life scientists and engineers together to
solve problems of generating new tissues for transplantation and
surgical reconstruction. The initial approaches to this problem
were described in the 1980's. Yannas and Burke (Bell, et al.,
Science 221,1052 (1981); Burke, et al., Ann Surg 194, 413
(1981)(described methods to generate new tissues in vivo by
implanting non-living materials such as modified collagens which
are seeded with cells to promote guided regeneration of tissue such
as skin. Vacanti and Langer (Langer and Vacanti Science 260, 920
(1993); Vacanti, et al., Materials Research Society 252,367 (1992))
described synthetic fibrous matrices to which tissue specific cells
were added in vitro. The matrices are highly porous and allow mass
transfer to the cells in vitro and after implantation in vivo.
After implantation, new blood vessels grow into the devices to
generate a new vascularized tissue. However, the relatively long
time course for angiogenesis limits the size of the newly formed
tissue.
[0006] The field of Tissue Engineering is now maturing and
undergoing explosive growth. See, for example, Vacanti and Langer,
Lancet 354, 32 (1999); Langer and Vacanti Science 260, 920 (1993);
Rennie, J. ed. Special report: The promise of tissue engineering.
Scientific American 280, 37 (1999); and Lysaght, et al., Tissue Eng
4, 231 (1998). Virtually every tissue and organ of the body has
been studied. Many tissue-engineering technologies are becoming
available for human use. See, Lysaght, et al. Tissue Eng 4, 231
(1998); Bell, et al., Science 221,1052 (1981); Burke, et al., Ann
Surg 194, 413 (1981); Compton, et al., Laboratory Investigation 60,
600 (1989); Parenteau, et al., Journal of Cellular Biochemistry 45,
24 (1991); Parenteau, et al., Biotechnology and Bioengineering 52,
3 (1996); Purdue, et al., J. Burn Care Rehab 18, 52 (1997);
Hansbrough and Franco, Clinical Plastic Surg 25, 407 (1998);
Vacanti, et al., Materials Research Society 252,367 (1992).
[0007] Over time, several techniques to engineer new living tissue
have been studied. Technologies include the use of growth factors
to stimulate wound repair and regeneration, techniques of guided
tissue regeneration using non-living matrices to guide new tissue
development, cell transplantation, and cell transplantation on
matrices. More recently, new understanding in stem cell biology has
led to studies of populations of primordial cells, stem cells, or
embryonic stem cells to use in tissue engineering approaches.
[0008] To date, all approaches in tissue engineering have relied on
the in-growth of blood vessels into tissue-engineered devices to
achieve permanent vascularization. This strategy has worked well
for many tissues. However, it falls short for thick, complex
tissues such as large vital organs, including liver, kidney, and
heart. Techniques using three-dimensional printing technology to
achieve ordered arrays of channels have been described to begin to
solve this problem. See, for example, Griffith, et al., Ann NY Acad
Sci 831, 382 (1997); Langer and Vacanti J P Sci Am 280, 62
(1999).
[0009] In parallel to these advances, the rapidly emerging field of
MicroElectroMechanical Systems (MEMS) has penetrated a wide array
of applications, in areas as diverse as automotives, inertial
guidance and navigation, microoptics, chemical and biological
sensing, and, most recently, biomedical engineering, Langer and
Vacanti Sci Am 280, 62 (1999); McWhorter, et al. "Micromachining
and Trends for the Twenty-First Century", in Handbook of
Microlithography, Micromachining and Microfabrication, ed. P.
Rai-Choudhury, (Bellingham, Wash.: SPIE Press, 1997).
Microfabrication methods for MEMS represent an extension of
semiconductor wafer process technology originally developed for the
integrated circuit (IC) industry. Control of features down to the
submicron level is routinely achieved in IC processing of
electrical circuit elements; MEMS technology translates this level
of control into mechanical structures at length scales stretching
from less than 1 micron to greater than 1 cm. Standard bulk
micromachining enables patterns of arbitrary geometry to be
imprinted into wafers using a series of subtractive etching
methods. Three-dimensional structures can be realized by
superposition of these process steps using precise alignment
techniques. Several groups (Kourepenis, et al., "Performance of
MEMS Inertial Sensors," Proc. AIAA GN&C Conference, Boston,
Mass., 1998; Griffith, et al., Annals of Biomed. Eng., 26 (1998);
Folch, et al., Biotechnology Progress, 14, 388 (1998)) have used
these highly precise silicon arrays to control cell behavior and
study gene expression and cell surface interactions. However, this
approach is essentially a two-dimensional technology and it has not
been apparent that it might be adapted to the generation of thick,
three-dimensional tissues.
[0010] PCT US96/09344 by Massachusetts Institute of Technology
describe a three-dimensional printing process, a form of solid free
form fabrication, which builds three-dimensional objects as a
series of layers. This process uses polymer powders in layers bound
by polymer binders whose geometry is dictated by computer assisted
design and manufacture. This technique allows defined internal
architectures which could include branching arrays of channels
mimicking a vascular supply. However, this technique is limited by
the characteristics and chemistry of the particular polymers. Also,
it severely limits the types of tissue to be fabricated. Polymer
walls do not allow the plasma exchange that is needed at the
alveolar capillary wall of the lung.
[0011] The object of the present invention is to provide a method
and materials for creating complex, living vascularized tissues for
organ and tissue replacement, especially complex and/or thick
structures, such as liver tissue.
SUMMARY OF THE INVENTION
[0012] A method and materials to create complex vascularized living
tissue in three dimensions from a two-dimension microfabricated
mold has been developed. The method involved creating a two
dimensional surface having a branching structure etched into the
surface. The pattern begins with one or more large channels which
serially branch into a large array of channels as small as
individual capillaries, then converge to one or more large
channels. The etched surface serves a template within a mold formed
with the etched surface for the circulation of an individual tissue
or organ. Living vascular cells are then seeded onto the mold,
where they form living vascular channels based on the pattern
etched in the mold. Once formed and sustained by their own matrix,
the top of the mold is removed. The organ or tissue specific cells
are then added to the etched surface, where they attach and
proliferate to form a thin, vascularized sheet of tissue. The
tissue can then be gently lifted from the mold using techniques
such as fluid flow and other supporting material, as necessary. The
tissue can then be systematically folded and compacted into a
three-dimensional vascularized structure. This structure can then
be implanted into animals or paitents by directly connecting the
blood vessels to flow into and out of the device. Immediate
perfusion of oxygenated blood occurs, which allows survival and
function of the entire living mass.
[0013] The design of the branching channels can be constructed by a
number of means, such as fractal mathematics which can be converted
by computers into two-dimensional arrays of branches and then
etched onto wafers. Also, computers can model from live or
preserved organ or tissue specimens three dimensional vascular
channels, convert to two-dimensional patterns and then help in the
reconversion to a three-dimensional living vascularized structure.
Techniques for producing the molds include techniques for
fabrication of computer chips and microfabrication technologies.
Other technologies include laser techniques. The two-dimensional
surface of the mold can also be varied to aid in the folding and
compacting process. For example, the surface can be changed from
planar to folded accordian like. It can be stacked into multiple
coverging plates. It could be curvilinear or have multiple
projections.
[0014] Different types of tissue, or multiple layers of the same
type of tissue, can be placed adjacent to each other prior to
folding and compacting, to create more complex or larger
structures. For example, a tubular system can be layered onto a
vascular system to fabricate glomerular tissue and collecting
tubules for kidneys. Bile duct tubes can be onlaid over
vascularized liver or hepatocyte tissue, to generate a bile duct
drainage system. Alveolar or airway tissue can be placed on lung
capillaries to make new lung tissue. Nerves or lymphatics can be
added using variations of these same general techniques. The
two-dimensional surface of the mold can also be varied to aid in
the folding and compacting process. For example, the surface can be
changed from planar to folded accordian like. It can be stacked
into multiple coverging plates. It could be curvilinear or have
multiple projections.
[0015] Examples of tissues and organs which can be fabricated using
these methods include, but are not restricted to, organs currently
transplanted such as heart, liver, lung, kidney and intestine.
Other tissues such as muscle, bone and breast tissue could also be
engineered.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1a is a schematic of the process for making a branching
pattern ono a silicon substrate. FIG. 1b is a more detaild
schematic showing the process to make a more complex structure,
with channels of varying depths.
[0017] FIG. 2a is a schematic of an etched surface showing a
branching structure which branches out from a single inlet and then
converges back into a single outlet. FIGS. 2b, c, and d are
schematics of a cross-sectional view of different etched channels
in the surface of FIG. 2a.
[0018] FIGS. 3a-g are schematics of multiple tissue layers
assembled to form three dimensional structures.
[0019] FIGS. 4a, b and c are schematics of the process for making a
tissue layer.
[0020] FIG. 5 is a schematic of an assembled complex tissue or
organ formed by the process of FIGS. 4a-c.
[0021] FIG. 6 shows how the organ of FIG. 5 can be connected to a
fluid by anastomosis of the inlet and outlet.
[0022] FIG. 7 is a schematic of the pattern etched using an
inductively-coupled (IPC) system.
[0023] FIG. 8 is a schematic of the process for fabricating
U-shaped trenches in silicon wafers.
[0024] FIG. 9a shows the vascular branching network pattern used
for silicon and pyrex wafer micromachining (FIG. 9a), FIG. 9b shows
the optical micrograph or portion of the capillary network etched
into the silicon wafer using the process shown in FIG. 7, and FIG.
9c is a scanning electron micrograph of the anisotrophic etching
process used to form angled sidewall trenches.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Due to the difficulties of the prior art, an approach to
provide a complete vascular system to the engineered structure
before implantation was developed using microfabrication techniques
such as three dimensional printing to provide an ordered array of
branching channels in a substrate formed of a material such as
silicon or a biocompatible polymer, which are then seeded with
cells. A complete branching vascular circulation is made in two
dimensions on the surface of silicon using microfabrication. The
vascular circulation is then lifted from the silicon mold and
folded or rolled into a compact three dimensional structure.
[0026] Microfabrication technology has been used in important
studies in cell and developmental biology to understand complex
biologic signaling events occurring at the cell membrane-surface
interface, as described, for example, by Kane, et al., Biomaterials
20, 2363 (1999). It has also been used in tissue engineering to
guide cell behavior and the formation of small units of tissue, as
described by Griffith, et al., Annals of Biomed. Eng., 26 (1998).
As demonstrated by Example 1, a coherent structure over a broad
range of scale has now been made which demonstrates the efficacy of
this method for tissue engineering for construction of complex
and/or thick structures such as liver. The device constructed in
the example includes channels that begin as a single inlet channel
with a diameter of 500 microns, branch through four generations
following a geometric scaling law which halves the channel width
for each successive generation, form an array of capillary channels
10 microns in diameter, and then sequentially branch back to a
single outflow vein. Living endothelial cells seeded into these
channels and provided with flow of appropriate nutrients and gases
will line the channels to form blood vessels. In Example 2, it has
been demonstrated that cells seeded onto surfaces of silicon and
pyrex will lay down matrix and form sheets of tissue of the cell
type of origin, either hepatic or endothelial. These sheets can be
peeled from the surface and formed into three dimensional units of
tissue. In effect, the wafer of silicon or pyrex has acted as a
mold for the formation of tissue.
[0027] These examples demonstrate that microfabrication technology
can be adapted to suit the needs of forming living tissue. The
power of the technique lies in its control of form over extremely
small distances. The resolution is on the order of 0.1 microns from
point to point. This level of precision adds new levels of control
in the ability to design and guide new tissue formation. For
instance, surfaces can be imprinted with submicron grooves or
scallops, and corners can be made rounded, angled or sharp with
this same level of submicron precision. Geometric control at this
scale can have a powerful impact on cell adhesion through
mechanisms such as contact guidance, as described by Den Braber, et
al. J. Biomed. Mater. Res. 40, 291 (1998). This technology
overcomes the problems with the prior art which was limited to very
thin structures because designs were constrained to the surface of
the silicon wafers. Using the wafer as a temporary mold, then
lifting the tissue from it and folding the tissue into
three-dimensional space, overcomes this limitation.
[0028] As described herein, complex tissues are formed by
laminating layers of thin vascularized tissues to form thicker
tissue structures or more complex organ equivalents. The thin
vascularized tissue layers are formed by:
[0029] (1) designing a mold having a complex pattern of channels
formed into at least one surface into which cells can be
seeded;
[0030] (2) seeding vascular (endothelial) cells into the
channels;
[0031] (3) culturing the vascular cells under conditions until they
form vasculature;
[0032] (4) seeding other type(s) of cells onto or into the mold so
that a tissue is formed incorporating the vasculature;
[0033] (5) removing the vascularized tissue layer; and
[0034] (6) assembling multiple layers of the vascularized tissue
layers until the desired complex structure (or organ equivalent) is
formed.
[0035] This structure can then be implanted and the vasculature, if
properly designed, anastomized into the existing vasculature to
provide an immediate blood supply for the implanted organ
equivalent.
[0036] Molds for Manufacturing Thin Vascularized Tissue Layers
[0037] Materials for Forming Molds
[0038] Fabrication of the wafer molds begins by selection of an
appropriate substrate. Any of a variety of materials can be used to
form the surfaces on which the branching structures can be molded
or etched. These include "inert" materials such as silicone,
polymers such as polyethylene vinyl acetate, polycarbonate, and
polypropylene, and materials such as a ceramic or material such as
hydroxyapatite. In particular, the mold can be constructed from
metals, ceramics, semiconductors, organics, polymers, and
composites. Representative metals and semiconductors include
pharmaceutical grade stainless steel, gold, titanium, nickel, iron,
gold, tin, chromium, copper, alloys of these or other metals,
silicon, silicon dioxide.
[0039] Typically, micromachining is performed on standard bulk
single crystal silicon wafers of a diameter ranging between 50 and
300 millimeters, and of thickness ranging between 200 and 1200
microns. These wafers can be obtained from a large number of
vendors of standard semiconductors material, and are sawn and
polished to provide precise dimensions, uniform crystallographic
orientation, and highly polished, optically flat surfaces. Wafers
made from pyrex borosilicate or other glasses can also be procured
and inserted into micromachining processes, with alternative
processes used to etch the glassy materials.
[0040] The choice of a substrate material is guided by many
considerations, including the requirements placed on the
fabrication process by the desired mold dimensions, the desired
size of the ultimate template, and the surface properties of the
wafer and their interaction with the various cell types,
extracellular matrix ("ECM") and polymeric backbone. Cost may also
be a consideration, depending upon the overall size requirements of
the tissue mold.
[0041] Unless otherwise specified, the term "polymer" includes
polymers and monomers which can be polymerized or adhered to form
an integral unit. The polymer can be non-biodegradable or
biodegradable, typically via hydrolysis or enzymatic cleavage,
although biodegradable matrices are not typically preferred since
the molds are not implanted and are preferably reusable.
Non-polymeric materials which can also be used include organic and
inorganic materials such as hydoxyapatite, calcium carbonate, which
are solidified by application of adhesive rather than solvent. In a
preferred embodiment, polymers are selected based on the ability of
the polymer to elicit the appropriate biological response from
cells, for example, attachment, migration, proliferation and gene
expression. As noted above, many non-biodegradable plastics are
known which are currently in use in cell culture, including
polystyrene, polycarbonate, polypropylene, polyvinylacetate, as
well as biodegradable polymers such as polyhydroxy acids and
polyhydroxyalkanoates. Photopolymerizable, biocompatible
water-soluble polymers include polyethylene glycol tetraacrylate
(Ms 18,500) which can be photopolymerized with an argon laser under
biologically compatible conditions using an initiator such as
triethanolamine, N-vinylpyrrolidone, and eosin Y. Other suitable
polymers can be obtained by reference to The Polymer Handbook, 3rd
edition (Wiley, N.Y., 1989).
[0042] Solvents for most of the thermoplastic polymers are known,
for example, methylene chloride or other organic solvents. Organic
and aqueous solvents for protein and polysaccharide polymers are
also known. The binder can be the same material as is used in
conventional powder processing methods or may be designed to
ultimately yield the same binder through chemical or physical
changes that occur as a result of heating, photopolymerization, or
catalysis.
[0043] These can be coated with a material enhancing cell adhesion.
In some embodiments, attachment of the cells to the polymer is
enhanced by coating the substrate with compounds such as basement
membrane components, agar, agarose, gelatin, gum arabic, collagens
types I, II, III, IV, and V, fibronectin, laminin,
glycosaminoglycans, mixtures thereof, and other materials known to
those skilled in the art of cell culture.
[0044] Properties of the mold surface can also be manipulated
through the inclusion of materials on or in the mold material which
alters porosity, cell attachment (for example, by altering the
surface charge or structure), flexibility or rgidity (which may be
desirable to facilitate removal of tissue constructs).
Methods for Designing Mold Surfaces
[0045] Once the substrate material has been selected, the process
sequence for mold generation must be defined. The geometry of the
mold, in particular the number of different feature depths
required, is the major factor determining the specific process
sequence. The simplest case is that of a single depth dimension for
the mold. Specifically, for a silicon substrate, the process
sequence (shown in FIG. 1a) is as follows: First, the silicon wafer
is cleaned, and a layer of photosensitive material is applied to
the surface. Typically, the layer is spun on at a high revolution
rate to obtain a coating of uniform thickness. The photoresist is
baked, and the wafer is then exposed to ultraviolet or other
short-wavelength light though a semitransparent mask. This step can
be accomplished using any one of several masking techniques,
depending on the desired image resolution. The resist is then
developed in an appropriate developer chemistry, and the wafer is
then hard-baked to remove excess solvent from the resist. Once the
lithographic process has been completed, the wafer can be etched in
a plasma reactor using one of several possible chemistries. Etching
serves to transfer the two-dimensional pattern into the third
dimension: a specified depth into the wafer. Plasma parameters are
determined by the desired shape of the resulting trench
(semicircular, straight-walled profile, angled sidewall), as well
as by the selectivity of the etchant for silicon over the masking
photoresist. Once the etching has been completed, the photoresist
can be removed and the wafer prepared for use in the tissue molding
process.
[0046] Glass and polymeric wafer molds can be fabricated using a
similar sequence, but the actual process may be modified by the
addition of an intervening masking layer, since etchants for these
materials may attack photoresist as well. Such intervening
materials simply function to transfer the pattern from the
photoresist to interlayer and then on to the wafer below. For
silicon etched in one of several wet chemistries, an intervening
layer may also be necessary.
[0047] Increased flexibility in the geometry of wafer mold may be
obtained by inserting additional cycles of masking and etching, as
shown in FIG. 1b. Here, a second step in which a masking layer has
been applied and open areas etched is shown. This modification
provides the opportunity to machine channels of varying depths into
the wafer mold. For vascular branches with different diameters,
this increased flexibility becomes very important. The techniques
may be extended to provide as many additional layers and different
depths as is desired.
[0048] The mold surface is configured as required to create tissue
sections having the desired vascular or other tubular structures.
In the case where a naturally occurring structure is mimicked,
channels are etched in the same two-dimensional pattern that occurs
in the natural tissue. A difference is that the natural tissue will
not be limited to a two-dimensional structure, but instead would
include vasculature that extends three dimensionally. This differs
from the thin tissue or organ sections created as described
herein.
[0049] The design of the branching channels can be constructed by a
number of means, such as fractal mathematics which can be converted
by computers into two-dimensional arrays of branches and then
etched onto wafers. Also, computers can model from live or
preserved organ or tissue specimensthree dimensional vascular
channels, convert to two-dimensional patterns and then help in the
reconversion to a three-dimensional living vascularized structure.
CAD-CAM type software programs would typically be most useful for
design of these structures.
Methods for Configuring the Molds
[0050] The selection of the material which is used as the support
determines how the surface of the mold is configured to form the
branching structure. Materials can be configured by molding,
particularly in the case of polymers, etching using techniques such
as lasers, plasma etching, or chemical etching, photolithography,
or solid free form techniques including three dimensional printing
(3DP), stereolithography (SLA), selective laser sintering (SLS),
ballistic particle manufacturing (BPM) and fusion deposition
modeling (FDM), micromachining, or combinations thereof.
Conventional Polymer Processing
[0051] Polymers can be configured using standard techniques such as
solvent casting or extrusion molding into a pre-fabricated mold,
shaped using one of the solid free form techniques or configured
after shaping, using chemical etching, micromachining, lasers, or
other methods described herein. These methods can also be used to
form the molds from materials other than polymers.
Micromachining and Chemical Processing of Silicon Materials
[0052] In one embodiment, the mold devices are made by
microfabrication processes, by creating small mechanical structures
in silicon, metal, polymer, and other materials. These
microfabrication processes are based on well-established methods
used to make integrated circuits and other microelectronic devices,
augmented by additional methods developed by workers in the field
of micromachining.
[0053] Microfabrication processes that may be used in making the
molds disclosed herein include lithography; etching techniques,
such as wet chemical, dry, and photoresist removal; thermal
oxidation of silicon; electroplating and electroless plating;
diffusion processes, such as boron, phosphorus, arsenic, and
antimony diffusion; ion implantation; film deposition, such as
evaporation (filament, electron beam, flash, and shadowing and step
coverage), sputtering, chemical vapor deposition (CVD), epitaxy
(vapor phase, liquid phase, and molecular beam), electroplating,
screen printing, and lamination. See generally Jaeger, Introduction
to Microelectronic Fabrication (Addison-Wesley Publishing Co.,
Reading Mass. 1988); Runyan, et al., Semiconductor Integrated
Circuit Processing Technology (Addison-Wesley Publishing Co.,
Reading Mass. 1990); Proceedings of the IEEE Micro Electro
Mechanical Systems Conference 1987-1998; Rai-Choudhury, ed.,
Handbook of Microlithography, Micromachining & Microfabrication
(SPIE Optical Engineering Press, Bellingham, Wash. 1997). The
following methods are preferred for making molds.
[0054] Electrolytic anodization of silicon in aqueous hydrofluoric
acid, potentially in combination with light, can be used to etch
channels into the silicon. By varying the doping concentration of
the silicon wafer to be etched, the electrolytic potential during
etching, the incident light intensity, and the electrolyte
concentration, control over the ultimate pore structure can be
achieved.
[0055] This process uses deep plasma etching of silicon to create
molds with diameters on the order of 0.1 .mu.m or larger. Needles
are patterned directly using photolithography, rather than
indirectly by controlling the voltage (as in electrochemical
etching), thus providing greater control over the final mold
geometry.
[0056] In this process, an appropriate masking material (e.g.,
metal) is deposited onto a silicon wafer substrate and patterned
into dots having the diameter of the desired molds. The wafer is
then subjected to a carefully controlled plasma based on
fluorine/oxygen chemistries to etch very deep, high aspect ratio
trenches into the silicon. See, e.g., Jansen, et al., "The Black
Silicon Method IV: The Fabrication of Three-Dimensional Structures
in Silicon with High Aspect Ratios for Scanning Probe Microscopy
and Other Applications," IEEE Proceedings of Micro Electro
Mechanical Systems Conference, pp. 88-93 (1995).
[0057] In this process, a metal layer is first evaporated onto a
planar substrate. A layer of photoresist is then deposited onto the
metal to form a patterned mold which leaves an exposed-metal region
in the shape of needles. By electroplating onto the exposed regions
of the metal seed layer, the mold bounded by photoresist can be
filled with electroplated material. Finally, the substrate and
photoresist mold are removed, leaving the finished mold array. The
molds produced by this process generally have diameters on the
order of 1 .mu.m or larger. See, e.g., Frazier, et al., "Two
dimensional metallic microelectrode arrays for extracellular
stimulation and recording of neurons", IEEE Proceedings of the
Micro Electro Mechanical Systems Conference, pp. 195-200
(1993).
[0058] Another method for forming molds made of silicon or other
materials is to use microfabrication techniques to make a mold
form, transferring that mold form to other materials using standard
mold transfer techniques, such as embossing or injection molding,
and reproducing the shape of the original mold form using the
newly-created mold to yield the final molds. Alternatively, the
creation of the mold form could be skipped and the mold could be
microfabricated directly, which could then be used to create the
final molds.
[0059] Another method of forming solid silicon molds is by using
epitaxial growth on silicon substrates, as is utilized by
Containerless Research, Inc. (Evanston, Ill., USA) for its
products.
[0060] The size distribution of the etched porous structure is
highly dependent on several variables, including doping kind and
illumination conditions, as detailed in Lehmann, "Porous Silicon--A
New Material for MEMS", IEEE Proceedings of the Micro Electro
Mechanical Systems Conference, pp. 1-6 (1996). Porous polymer molds
can be formed, for example, by micromolding a polymer containing a
volatilizable or leachable material, such as a volatile salt,
dispersed in the polymer, and then volatilizing or leaching the
dispersed material, leaving a porous polymer matrix in the shape of
the mold. Hollow molds can be fabricated, for example, using
combinations of dry etching processes (Laermer, et al., "Bosch Deep
Silicon Etching: Improving Uniformity and Etch Rate for Advanced
MEMS Applications," Micro Electro Mechanical Systems, Orlando,
Fla., USA, (Jan. 17-21, 1999); Despont et al., "High-Aspect-Ratio,
Ultrathick, Negative-Tone Near-UV Photoresist for MEMS", Proc. of
IEEE 10.sup.th Annual International Workshop on MEMS, Nagoya,
Japan, pp. 518-522 (Jan. 26-30, 1997)); micromold creation in
lithographically-defined polymers and selective sidewall
electroplating; or direct micromolding techniques using epoxy mold
transfers.
[0061] A chromium mask can be substituted for the solid molds using
a silicon nitride layer covered with chromium. Solid molds are then
etched, the chromium is stripped, and the silicon is oxidized. The
silicon nitride layer will prevent oxidation. The silicon nitride
is then stripped, leaving exposed silicon and oxide-covered silicon
everywhere else. The needle is then exposed to an ICP plasma which
selectively etches the silicon in a highly anisotropic manner to
form the interior hole of the needle. A second method uses solid
silicon as `forms` around which the actual needle structures are
deposited. After deposition, the forms are etched away, yielding
the hollow structures. Silica needles or metal needles can be
formed using different methods. The wafers are then oxidized to a
controlled thickness, the silicon nitride is then stripped and the
silicon core selectively etched away (e.g., in a wet alkaline
solution) to form a hollow silica mold.
[0062] In another embodiment, deep reactive ion etching is combined
with a modified black silicon process in a conventional reactive
ion etcher. First, designs are patterned through photoresist into
SiO.sub.2, such as on a silicon wafer. Then the silicon can be
etched using deep reactive ion etching (DRIE) in an inductively
coupled plasma (ICP) reactor to etch deep vertical holes or
channels. The photoresist is then removed. Next, a second
photolithography step patterns the remaining SiO.sub.2 layer. The
photoresist is then removed and the silicon wafer again deep
silicon etched completely through the wafer in the regions not
covered with SiO.sub.2). This process can be varied as follows.
After the wafer is patterned, the photoresist and SiO.sub.2 layers
are replaced with conformal DC sputtered chromium. The second ICP
etch is replaced with a SF.sub.6/O.sub.2 plasma etch in a reactive
ion etcher (RIE), which results in positively sloping outer
sidewalls. Henry, et al., "Micromachined Needles for the
Transdermal Delivery of Drugs," Micro Electro Mechanical Systems,
Heidelberg, Germany, pp. 494-498 (Jan. 26-29, 1998).
[0063] Metal shapes can be formed by physical vapor deposition of
appropriate metal layers on solid forms, which can be made of
silicon using the techniques described above, or which can be
formed using other standard mold techniques such as embossing or
injection molding. The metals are selectively removed using
electropolishing techniques, in which an applied anodic potential
in an electrolytic solution will cause dissolution of metals due to
concentration of electric field lines. Once the underlying silicon
forms have been exposed, the silicon is selectively etched away to
form structures. This process could also be used to make structures
made from other materials by depositing a material other than metal
on the needle forms and following the procedure described
above.
[0064] Molds formed of silicon dioxide can be made by oxidizing the
surface of the silicon mold forms, rather than depositing a metal
and then etching away the solid needle forms to leave the hollow
silicon dioxide structures. In one embodiment, hollow, porous, or
solid molds are provided with longitudinal grooves or other
modifications to the exterior surface of the molds.
[0065] Polymeric molds can be made using microfabricated molds. For
example, the epoxy molds can be made as described above and
injection molding techniques can be applied to form the structures.
These micromicromolding techniques are relatively less expensive to
replicate than the other methods described herein.
Solid Free Form Methods for Configuring the Molds
[0066] 3DP is described by Sachs, et al, "CAD-Casting: Direct
Fabrication of Ceramic Shells and Cores by Three Dimensional
Printing" Manufacturing Review 5(2), 117-126 (1992) and U.S. Pat.
No. 5,204,055 to Sachs, et al. 3DP is used to create a solid object
by ink-jet printing a binder into selected areas of sequentially
deposited layers of powder. Each layer is created by spreading a
thin layer of powder over the surface of a powder bed. The powder
bed is supported by a piston which descends upon powder spreading
and printing of each layer (or, conversely, the ink jets and
spreader are raised after printing of each layer and the bed
remains stationary). Instructions for each layer are derived
directly from a computer-aided design (CAD) representation of the
component. The area to be printed is obtained by computing the area
of intersection between the desired plane and the CAD
representation of the object. The individual sliced segments or
layers are joined to form the three dimensional structure. The
unbound powder supports temporarily unconnected portions of the
component as the structure is built but is removed after completion
of printing.
[0067] SFF methods other than 3DP that can be utilized to some
degree as described herein are stereo-lithography (SLA), selective
laser sintering (SLS), ballistic particle manufacturing (BPM), and
fusion deposition modeling (FDM). SLA is based on the use of a
focused ultra-violet (UV) laser which is vector scanned over the
top of a bath of a photopolymerizable liquid polymer material. The
UV laser causes the bath to polymerize where the laser beam strikes
the surface of the bath, resulting in the creation of a first solid
plastic layer at and just below the surface. The solid layer is
then lowered into the bath and the laser generated polymerization
process is repeated for the generation of the next layer, and so
on, until a plurality of superimposed layers forming the desired
device is obtained. The most recently created layer in each case is
always lowered to a position for the creation of the next layer
slightly below the surface of the liquid bath. A system for
stereolithography is made and sold by 3D Systems, Inc., of
Valencia, Calif., which is readily adaptable for use with
biocompatible polymeric materials. SLS also uses a focused laser
beam, but to sinter areas of a loosely compacted plastic powder,
the powder being applied layer by layer. In this method, a thin
layer of powder is spread evenly onto a flat surface with a roller
mechanism. The powder is then raster-scanned with a high-power
laser beam. The powder material that is struck by the laser beam is
fused, while the other areas of powder remain dissociated.
Successive layers of powder are deposited and raster-scanned, one
on top of another, until an entire part is complete. Each layer is
sintered deeply enough to bond it to the preceding layer. A
suitable system adaptable for use in making medical devices is
available from DTM Corporation of Austin, Tex.
[0068] BPM uses an ink-jet printing apparatus wherein an inkjet
stream of liquid polymer or polymer composite material is used to
create three-dimensional objects under computer control, similar to
the way an ink-jet printer produces two-dimensional graphic
printing. The device is formed by printing successive
cross-sections, one layer after another, to a target using a cold
welding or rapid solidification technique, which causes bonding
between the particles and the successive layers. This approach as
applied to metal or metal composites has been proposed by Automated
Dynamic Corporation of Troy, N.Y. FDM employs an x-y plotter with a
z motion to position an extrudable filament formed of a polymeric
material, rendered fluid by heat or the presence of a solvent. A
suitable system is available from Stratasys, Incorporated of
Minneapolis, Minn.
[0069] Cells for Forming Vascularized Tissue Layers
[0070] Although described herein with particular reference to
formation of vascularized tissue, it should be understood that the
channels can be used for form lumens for passage of a variety of
different fluids, not just blood, but also bile, lymph, urine, and
other body fluids, and for the guided regeneration or growth of
other types of cells, especially nerve cells. The tissue layer may
include some lumens for forming vasculature and some for other
purposes, or be for one purpose, typically providing a blood supply
to carry oxygen and nutrients to and from the cells in the
tissue.
[0071] The tissue will typically include one or more types of
"functional" or parenchymal cells, such as cells having specific
metabolic functions, like hepatocytes, pancreatic cells, kidney,
brain, reproductive tissue cells, cells forming intestine, nerve
cells, bone, muscle, heart, skin cells, etc. The vasculature will
typically be formed from endothelial cells.
[0072] Cells can be obtained by biopsy or harvest from a living
donor, cell culture, or autopsy. Cells can be dissociated using
standard techniques such as digestion with collagenase, then seeded
immediately into the mold or after being maintained in cell
culture. Cells can be normal cells or genetically engineered, from
the patient into which the tissue is to be implanted, or from a
suitable donor.
[0073] Methods for Seeding Cells into Molds
[0074] A method and materials to create complex vascularized living
tissue in three dimensions from a two-dimension microfabricated
mold has been developed. The method involved creating a two
dimensional surface having a branching structure etched into the
surface. As shown in FIG. 2a, in a preferred embodiment, the
pattern in the mold 10 formed of a silicon wafer 11 begins with one
or more large channels 12 which serially branch into a large array
of channels as small as individual capillaries 14a, 14b, 14c, etc.,
then converge to one or more large channels 16. The cross section
of the single "arterial" channel 12 and "venous" channel 16 are
shown in FIG. 2b. The cross-sections of the portion of mold 10
containing the "capillary" channels 14a, 14b, 14c, etc., is shown
in FIG. 2c. The mold is shown in cross-section in FIG. 2d, with a
depth of approximately five microns.
[0075] The etched surface serves as a template within a mold formed
with the etched surface for the circulation of an individual tissue
or organ. As shown in FIG. 4A, the mold pieces 30 and 32 are fitted
together to make an enclosure 34, and the cells cultured. The
vascular cells form vascular channels 36 based on the pattern
etched in the mold, as shown in FIG. 4B. Once formed and sustained
by their own matrix, the top 32 of the mold is removed, and the
organ or tissue specific cells are then added to the etched
surface, where they attach and proliferate to form a thin,
vascularized sheet of tissue 36. As shown in FIG. 4C, the tissue
can then be gently lifted from the mold using techniques such as
fluid flow and other supporting material, as necessary.
[0076] Construction of Tissue or Organ Equivalents
[0077] After formation of the tissue layers, the tissue can be
systematically folded and compacted into a three-dimensional
vascularized structure, as shown in FIG. 5. The two-dimensional
surface of the mold can be varied to aid in the folding and
compacting process. For example, the surface can be changed from
planar to folded accordian like. It can be stacked into multiple
coverging plates. It could be curvilinear or have multiple
projections.
[0078] FIGS. 3A-G are perspective views of ways in which a single
tissue layer 36 (FIG. 3A) can be folded (FIGS. 3B and 3C) or
stacked (FIG. 3D), or expanded to form a balloon shape (FIG. 3E),
funnel (FIG. 3F), or large lumen (FIG. 3G).
[0079] This structure can then be implanted into animals or
patients by directly connecting the blood vessels to flow into and
out of the device, as depicted in FIG. 6. Immediate perfusion of
oxygenated blood occurs, which allows survival and function of the
entire living mass.
[0080] Different types of tissue, or multiple layers of the same
type of tissue, can be placed adjacent to each other prior to
folding and compacting, to create more complex or larger
structures. For example, a tubular system can be layered onto a
vascular system to fabricate glomerular tissue and collecting
tubules for kidneys. Bile duct tubes can be onlaid over
vascularized liver or hepatocyte tissue, to generate a bile duct
drainage system. Alveolar or airway tissue can be placed on lung
capillaries to make new lung tissue. Nerves or lymphatics can be
added using variations of these same general techniques. The
two-dimensional surface of the mold can also be varied to aid in
the folding and compacting process. For example, the surface can be
changed from planar to folded accordian like. It can be stacked
into multiple coverging plates. It could be curvilinear or have
multiple projections.
EXAMPLE 1
Micromaching of Template to Tissue Engineer Branched Vascularized
Channels for Liver Fabrication
[0081] Micromachining technologies were used on silicon and pyrex
surfaces to generate complete vascular systems that may be
integrated with engineered tissue before implantation. Trench
patterns reminiscent of branched architecture of vascular and
capillary networks were etched using standard photolithographic
techniques onto silicon and pyrex surfaces to serve as templates.
Hepatocytes and endothelial cells were cultured and subsequently
lifted as single-cell monolayers from these two dimensional molds.
Both cell types were viable and proliferative on these surfaces. In
addition, hepatocytes maintained albumin production. The lifted
monolayers were then folded into compact three-dimensional tissues.
The goal is to lift these branched vascular networks from two
dimensional templates so that they can be combined with layers of
parenchymal tissue, such as hepatocytes, to form three dimensional
conformations of living vascularized tissue for implantation.
Materials and Methods
[0082] Micromachining Techniques
[0083] Templates for the formation of sheets of living vascularized
tissue were fabricated utilizing micromachining technology. For the
present work, a single level etch was utilized to transfer a
vascular network pattern into an array of connected trenches in the
surface of both silicon and pyrex wafers.
[0084] In this prototype, a simple geometry was selected for
patterning the vascular network. Near the edge of each wafer, a
single inlet or outlet was positioned, with a width of 500 .mu.m.
After a short length, the inlet and outlet branched into three
smaller channels of width 250 .mu.m; each of these branched again
into three 125 .mu.m channels, and finally down to three 50 .mu.m
channels. Channels extend from the 50 .mu.m channels to form a
capillary network, which comprises the bulk of the layout. In
between these inlet and outlet networks lies a tiled pattern of
diamonds and hexagons forming a capillary bed and filling the
entire space between the inlet and outlet. In one configuration,
the capillary width was set at 25 .mu.m, while in the other,
capillaries were fixed at 10 .mu.m. This geometry was selected
because of its simplicity as well as its rough approximation to the
size scales of the branching architecture of the liver. Layout of
this network was accomplished using CADENCE software (Cadence,
Chelmsford, Mass.) on a Silicon Graphics workstation. A file with
the layout was generated and sent electronically to Align-Rite
(Burbank, Calif.), where glass plates with electron-beam-generated
patterns replicating the layout geometry were produced and returned
for lithographic processing.
[0085] Starting materials for tissue engineering template
fabrication were standard semiconductor grade silicon wafers
(Virginia Semiconductor, Powhatan, Va.), and standard pyrex wafers
(Bullen Ultrasonics, Eaton, Ohio) suitable for MEMS processing.
Silicon wafers were 100 mm diameter and 525 microns thick, with
primary and secondary flats cut into the wafers to signal crystal
orientation. Crystal orientation was <100>, and wafers were
doped with boron to a resistivity of approximately 5 W-cm. The
front surface was polished to an optical finish and the back
surface ground to a matte finish. Pyrex wafers were of composition
identical to Corning 7740 (Corning Glass Works, Corning N.Y.), and
were also 100 mm in diameter, but had a thickness of 775 microns.
Both front and back surfaces were polished to an optical finish.
Prior to micromachining, both wafer types were cleaned in a mixture
of 1 part H.sub.2SO.sub.4 to 1 part H.sub.2O.sub.2 for 20 minutes
at 140.degree. C., rinsed 8 times in deionized water with a
resistivity of 18 MW, and dried in a stream of hot N.sub.2 gas.
[0086] For silicon and pyrex wafers, standard photolithography was
employed as the etch mask for trench formation. Etching of pyrex
wafers requires deposition of an intermediate layer for pattern
transfer which is impervious to the etch chemistry. A layer of
polysilicon of thickness 0.65 .mu.m over the pyrex was utilized for
this purpose. This layer was deposited using Low Pressure Chemical
Vapor Deposition (LPCVD) at 570.degree. C. and 500 mTorr via the
standard silane decomposition method. In the case of silicon,
photoresist alone could withstand limited exposure to two of the
three etch chemistries employed. For the third chemistry, a 1.0
.mu.m layer of silicon dioxide was thermally deposited at
1100.degree. C. in hydrogen and oxygen.
[0087] Once the wafers were cleaned and prepared for processing,
images of the prototype branching architecture were translated onto
the wafer surfaces using standard MEMS lithographic techniques. A
single layer of photoresist (Shipley 1822, MicroChem Corp., Newton,
Mass.) was spun onto the wafer surfaces at 4000 rpm, providing a
film thickness of approximately 2.4 .mu.m. After baking at
90.degree. C. for 30 minutes, the layer of photoresist was exposed
to uv light using a Karl Suss MA6 (Suss America, Waterbury, Vt.)
mask aligner. Light was passed through the lithographic plate
described earlier, which was in physical contact with the coated
wafer. This method replicates the pattern on the plate to an
accuracy of 0.1 .mu.m. Following exposure, wafers were developed in
Shipley 319 Developer (MicroChem Corp., Newton, Mass.), and rinsed
and dried in deionized water. Finally, wafers were baked at
110.degree. C. for 30 minutes to harden the resist, and exposed to
an oxygen plasma with 80 Watts of power for 42 seconds to remove
traces of resist from open areas.
[0088] Silicon wafers were etched using three different
chemistries, while pyrex wafers were processed using only one
technique. For pyrex, the lithographic pattern applied to the
polysilicon intermediate layer was transferred using a brief
(approximately 1 minute) exposure to SF.sub.6 in a
reactive-ion-etching plasma system (Surface Technology Systems,
Newport, United Kingdom). Photoresist was removed, and the pattern
imprinted into the polysilicon layer was transferred into trenches
in the silicon using a mixture of 2 parts HNO.sub.3 to 1 part HF at
room temperature. With an etch rate of 1.7 microns per minute, 20
micron deep trenches were etched into the pyrex wafers in
approximately 12 minutes. Since the chemistry is isotropic, as the
trenches are etched they become wider. Processing with the layout
pattern with 25 .mu.m wide capillary trenches tended to result in
merging of the channels, while the use of 10 .mu.m wide trenches
avoided this phenomenon. Interferometric analysis of the channels
after etching showed that surface roughness was less than 0.25
.mu.m. Once channel etching of pyrex wafers was completed,
polysilicon was removed with a mixture of 10 parts HNO.sub.3 to 1
part HF at room temperature, and wafers were re-cleaned in 1 part
H.sub.2SO.sub.4 to 1 part HF.
[0089] Three different chemistries were employed to etch silicon in
order to investigate the interaction between channel geometry and
cell behavior. First, a standard anisotropic plasma etch chemistry,
using a mixture of SF.sub.6 and C4F.sub.8 in a switched process
plasma system from STS.sup.24, was used to produce rectangular
trenches in silicon. Narrower trenches are shallower than deep
trenches due to a phenomenon known as RIE lag. A second process
utilized a different plasma system from STS, which produces
isotropic trenches with a U-shaped profile. While the process is
isotropic, widening of the trenches is not as severe as is
experienced in the isotropic pyrex etching process described
earlier. In both of these plasma etching cases, trenches were
etched to a nominal depth of 20 .mu.m. For the third process,
anisotropic etching in KOH (45% w/w in H.sub.2O at 88.degree. C.),
the intermediate silicon dioxide layer mentioned above was
employed. First, the silicon dioxide layer was patterned using HF
etching at room temperature. The KOH process produces angled
sidewalls rather than the rectangular profile or U-shaped profile
produced by the first two recipes, respectively. Crystal planes in
the <111>orientation are revealed along the angled sidewalls,
due to anisotropic properties of the KOH etch process as a function
of crystal orientation. Due to the self-limiting nature of the
channels produced by this process, trench depth was limited to 10
.mu.m. After completion of the silicon wafer etching, all layers of
photoresist and silicon dioxide were removed, and wafers were
cleaned in 1 part H.sub.2SO.sub.4:1 part H.sub.2O.sub.2 at
140.degree. C., followed by rinsing in deionized water and drying
in nitrogen gas.
[0090] For this set of experiments, no attempt was made to alter
the surface chemistry of the silicon and pyrex wafers. Prior to
processing, silicon wafers were uniformly hydrophobic, while pyrex
wafers were equally hydrophilic, as determined by observations of
liquid sheeting and sessile drop formation. After processing,
unetched surfaces appeared to retain these characteristics, but the
surface chemistry within the channels was not determined.
[0091] Animals
[0092] Adult male Lewis rats (Charles River Laboratories,
Wilmington, Mass.), weighing 150-200 g, were used as cell donors.
Animals were housed in the Animal Facility of Massachusetts General
Hospital in accordance with NIH guide lines for the care of
laboratory animals. They were allowed rat chow and water ad libitum
and maintained in 12-hour light and dark cycle.
[0093] Cell Isolations
[0094] Male Lewis rats were used as hepatic cell donors. HCs were
isolated using a modification of the two-step collagenase perfusion
procedure as previously by Aiken et al., J Pediatr Surg 25, 140
(1990); Seglen PO Methods Cell Biol 13, 29 (1976). Briefly, the
animals were anesthetized with Nembutal Sodium Solution (Abbott
Laboratories, North Chicago, Ill.), 50 mg/kg, and the abdomen was
prepared in sterile fashion. A midline abdominal incision was made
and the infrahepatic inferior vena cava was cannulated with a
16-gauge angiocatheter (Becton Dickinson). The portal vein was
incised to allow retrograde efflux and the suprahepatic inferior
vena cava was ligated. The perfusion was performed at a flow rate
of 29 ml/min initially with a calcium-free buffer solution for 5 to
6 minutes, then with a buffer containing collagenase type 2
(Worthington Biomedical Corp., Freehold, N.J.) at 37.degree. C. The
liver was excised after adequate digestion of the extracellular
matrix and mechanically agitated in William's E medium (Sigma, St.
Louis, Mo.) with supplements to produce a single cell suspension.
The suspension was filtered through a 300 micron mesh and separated
into two fractions by centrifugation at 50 g for 2 minutes at
4.degree. C. The pellet containing the viable HC fraction was
resuspended in William's E medium and further purified by an
isodensity Percoll centrifugation. The resulting pellet was then
resuspended in Hepatocyte Growth Medium, and cell counts and
viabilities of HCs were determined using the trypan blue exclusion
test.
[0095] The endothelial cells were derived from rat lung
microvessels and they were purchased directly from the vendor,
Vascular Endothelial Cell Technologies (Rensellaer, N.Y.).
[0096] Hepatocyte Culture Medium
[0097] William's E medium supplemented with 1 g sodium pyruvate
(Sigma, St. Louis, Mo.) and 1% glutamine-penicillin-streptomycin
(Gibco BRL, Gaithersburg, Md.) were used during the cell isolation
process. The plating medium was Dulbecco's modified eagle medium
(Gibco BRL) supplemented with 10% fetal bovine serum, 1%
penicillin-streptomycin, 44 mM sodium-bicarbonate, 20 mM HEPES, 10
mM niacinamide, 30 microgram/ml L-proline, 1 mM ascorbic acid 2
phospate, 0.1 microM dexamethasone (Sigma),
insulin-transferrin-sodium selenite (5 mg/L-5 mg/L-5 microgram/L,
Roche Molecular Biomedicals, Indianapolis, Ind.), and 20 ng/ml
epidermal growth factor (Collaborative Biomedical Products,
Bedford, Mass.).
[0098] Endothelial Cell Culture Medium
[0099] Dulbecco's modified eagle medium (Gibco BRL) was
supplemented with 10% fetal bovine serum, 1%
penicillin-streptomycin, 25 mg of ascorbic acid (Sigma), 10 mg
L-alanine (Sigma), 25 mg L-proline (Sigma), 1.5 microgram cupric
sulfate (Sigma), glycine (Sigma) and 1 M Hepes buffer solution
(Gibco BRL). The media was supplemented with 8 mg of ascorbic acid
every day. Cell attachment and lifting from non-etched silicon and
pyrex wafers Silicon and pyrex were both tested as possible
substrates for the culture and lifting of endothelial cells and
hepatocytes. Prior to cell seeding, the pyrex wafers were
sterilized with 70% ethanol (Fisher, Pittsburg, Pa.) overnight and
washed three times with sterile phosphate buffered saline (Gibco
BRL). Silicon wafers were first soaked in acetone for 1 hr,
followed a methanol rinse for 15 minutes, and overnight
sterilization in 100% isopropyl alcohol. Rat lung microvascular
endothelial cells was cultured on non-coated pyrex and silicon
surfaces, as well as wafers coated with vitrogen (30 microgram/ml),
Matrigel.RTM. (1%), or Gelatin (10 mg/ml). Once isolated, the cells
were resuspended in endothelial cell culture medium, seeded
uniformly onto the wafer at a density of 26.7.times.10.sup.3
cells/cm.sup.2, and cultured at 5% CO.sub.2 and 37.degree. C. After
reaching confluence, the ability of the monolayer of endothelial
cells to lift from the wafers was tested using a cell scrapper to
promote detachment.
[0100] The rat hepatocytes were also cultured on non-coated pyrex
and silicon, as well as wafers coated with a thin and thick layers
of vitrogen (30 microgram/ml and 3 microgram/ml) and Matrigel (1%)
in order to determine the optimal methods for lifting hepatocyte
sheets. Once isolated, the hepatocytes were resuspended in
hepatocyte growth media, seeded onto the wafer at a density of
111.3.times.10.sup.3 cells/cm.sup.2, and cultured at 5% CO.sub.2
and 37.degree. C. Cell attachment and growth was observed daily
using microscopy and cell lifting occurred spontaneously.
[0101] After determining which method for culturing was best for
lifting the hepatocytes and endothelial cells in an intact layer,
both membranes were fixed in 10% buffered formalin for 1 hr and
harvested for histological study, and the hepatocytes were stained
immunohistochemically.
[0102] Immunohistochemical Staining
[0103] The hepatocyte cell monolayer membrane was fixed in 10%
buffered formalin and processed for hematoxylin-eosin and
immunohistochemical staining using a labeled streptavidin biotin
method (LSAB2 kit for rat specimen, DAKO, Carpinteria, Calif.). The
primary antibody was rabbit anti-albumin (ICN, Costa Mesa, Calif.).
Three-micron sections were prepared and deparafinized. The
specimens were treated with peroxidase blocking buffer (DAKO) to
prevent the nonspecific staining. Sections were stained with
albumin diluted with phosphate buffered saline, followed by
biotinylated anti-rabbit antibody and HRP conjugated streptavidin.
Sections were treated with DAB as substrate and were counterstained
with hematoxylin.
[0104] Albumin Production
[0105] To assess hepatocyte function, albumin concentration in the
culture medium was measured every 24 hours for 5 days pre-cell
detachment using an enzyme linked immunosorbent assay (n=5), as
described by Schwereet al., Clinica Chemica Acta 163, 237 (1987).
In brief, a 96 well microplate was coated with anti-rat albumin
antibody (ICN). After blocking non-specific responses with a 1%
gelatin solution, each sample was seeded onto the plate and
incubated for 1 hour. This was followed by another 1 hour
incubation with peroxidase conjugated anti-rat albumin antibody
(ICN). Finally, the substrate was added and extinction was measured
with a microplate reader at 410 nm. R.sup.2 of the standard curve
was >0.99.
[0106] Statistical Analysis
[0107] All data was expressed as mean .+-.SD. Statistical analysis
was performed with a paired t-test. Statistical significance was
determined as when the p value of each test was less than 0.05.
[0108] Cell Attachment to Etched Silicon and Pyrex Wafers
[0109] Endothelial cells and hepatocytes were also seeded onto
etched silicon and pyrex wafers. Prior to cell seeding, the pyrex
wafers were sterilized with 70% ethanol (Fisher) overnight and
washed three times with sterile phosphate buffered saline (Gibco
BRL). Silicon wafers were first soaked in acetone for 1 hr,
followed a methanol rinse for 15 minutes, and overnight
sterilization in 100% isopropyl alcohol. Onto these wafers were
seeded rat lung microvascular endothelial cells at a density of
26.7.times.10.sup.3 cells/cm.sup.2, or rat hepatocytes at a density
of 111.3.times.10.sup.3 cells/cm.sup.2. These cells were cultured
at 5% CO.sub.2 and 37.degree. C., and their attachment and growth
observed daily using microscopy.
[0110] Implantation of Hepatocyte Sheets into the Rat Omentum
[0111] Hepatocytes were cultured on silicon wafers coated with a
thin layer of vitrogen (30 microgram/ml), and lifted in sheets.
Retrorsine is a drug known to inhibit the regeneration of the
normal liver by producing a block in the hepatocyte cell cycle with
an accumulation of cells in late S and/or G.sub.2 phase (Peterson J
E J Pathol Bacteriol 89, 153 (1965)). This drug was administered
into the peritoneal cavity of two rats at a dose of 3 mg/ml/100 g
on day 0, and after two weeks. Three weeks later, a portacaval
shunt was created, and the following week a hepatocyte sheet,
lifted after four days culture on vitrogen coated silicon (30
microgram/ml), was implanted onto the microvasculature of the rat
omentum and rolled into a three-dimensional cylinder, and a 60%
hepatectomy was performed. The rolled omentum with hepatocytes was
harvested at four weeks and at three months after implantation and
analyzed using histology.
Results
[0112] Micromachining
[0113] A schematic of the vascular branching network design used as
a template for micromachining is shown in FIG. 9a. This pattern was
transferred to silicon and pyrex wafers using the processes
described in the Materials and Methods section. Typical trench
depths of 20 microns on silicon and 10 microns on glass were
achieved utilizing these processes. An optical micrograph of a
portion of the capillary network etched into a silicon wafer is
shown in FIG. 9b. In FIG. 9c, a Scanning Electron Micrograph
cross-section of an angled trench etched using the anisotropic
etching process described earlier is shown. This process resulted
in excellent adhesion and enhanced lifting of living tissue.
[0114] Growth and Lifting of Cells from the Silicon and Pyrex
Wafers
[0115] The adhesion and growth of endothelial cells and hepatocytes
on several different substrate surfaces was compared. On all pyrex
wafers, coated or non-coated, the endothelial cells proliferated
and grew to confluence within four days. These cells did not lift
spontaneously, and when scraped, did not lift as a single sheet. In
addition, when the non-coated silicon wafers were seeded with
endothelial cells, the cell sheet fragmented upon lifting. On the
other hand, endothelial cells seeded onto silicon surfaces coated
with vitrogen (30 microgram/ml), Matrigel (1%), and gelatin (10
mg/ml) did lift with the use of mechanical means (i.e. a cell
scraper), and provided an intact monolayer sheet of endothelial
cells. Upon observation, there were no significant differences in
the effects of the three coatings on the detached cell sheets.
[0116] Hepatocytes also attached and spread well on all coated and
non-coated pyrex wafers, and did not lift spontaneously or in
sheets when scraped after several days of growth. However, when
seeded onto silicon wafers, they lifted spontaneously on all the
non-coated and coated wafers. The hepatocyte sheets lifted from the
non-coated wafers after 3 days, but were very fragile and
fragmented easily. The monolayers that lifted from the thin and
thickly coated vitrogen substrates (30 microgram/ml and 3
microgram/ml) lifted after 4 days in culture to form an intact
hepatocyte layer. Cells lifted from the Matrigel (1%) coated
silicon wafers after 5 days in culture. There were no significant
differences in appearance between the cell sheets lifted from the
vitrogen and Matrigel coated wafers.
[0117] Histological assessment of the detached cell monolayers of
both hepatocytes and endothelial cells manifested promising
results. Hemotoxylin and Eosin (H&E) staining of both showed
that all cells were viable and that most were undergoing mitoses.
The endothelial cells were observed to be primarily attenuated and
to form a single-celled alignment. The monolayer of hepatocytes
showed each cell to be of a spheriod configuration with
eosinophilic floculent cytoplasm and a large nucleus with a bright
red nucleolus, similar to that seen in the native liver. Moreover,
cellular attachments were less attenuated than the endothelial
cells. Thus, these results are reminiscent of each of the cell
types' specific functions. In biological systems, the endothelium
functions to provide a thin, smooth outer surface of a barrier and
a transport channel and so it is understandable that these cells
are observed here to be primarily attenuated and in a single-celled
array. The hepatocytes have more of a tendency to form tissue and
so less of a single-celled array and more of a rounded
multi-layered array is seen.
[0118] Albumin secretion into the hepatocyte culture medium at day
2, 3, 4, and 5 was 165.96.+-.29.87, 164.44.+-.17.22,
154.33.+-.18.46, 115.47.+-.18.09 (microgram/day, Graph 1),
respectively. Though there was a statistically significant
difference between day 4 and day 5, no significant differences were
observed between day 2, day 3, and day 4 (p<0.05 by the paired
t-test). Hence, this data shows that cells cultured on silicon
wafers were able to maintain a fairly constant albumin production
rate until day 4.
[0119] Moreover, through immunohistochemical staining of the
detached hepatocyte monolayers, many cells were stained positive
for albumin indicating further that hepatocyte function was
maintained on silicon wafers.
[0120] Implantation of Hepatocyte Sheet into the Rat Omentum
[0121] H&E staining of hepatocyte sheets implanted into rat
omentum demonstrated that all cells were viable and showed
proliferation at four weeks and three months. The implanted
hepatocyte monolayer sheets, when harvested, were over 5 cell
layers thick in most areas.
[0122] This study demonstrates that silicon microfabrication
technology can be utilized to form large sheets of living tissue.
It also demonstrates the feasibility of etching ordered branching
arrays of channels that allow living endothelial cells to line the
luminal surface of the channels. In addition, it has been shown
that organized sheets of engineered hepatocyte tissue and
endothelial tissue can be lifted from the surface of silicon or
pyrex wafers and can be folded into a compact three dimensional
configuration. The hepatocyte sheets have then been placed into
rats on the highly vascular surface of the omentum. That structure
has then been rolled into a three dimensional cylinder as a model
for an engineered vasculature. Vascularized hepatic tissue was
formed as a permanent graft.
[0123] Modifications and variations of the method and devices
described herein will be obvious to those skilled in the art and
are intended to be encompassed by the following claims.
* * * * *