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`According to still further features in the described preferred embodiments step
`
`(b) is effected at least twice.
`
`According to still further features in the described preferred embodiments
`
`removing comprises subjecting the tissue to a detergent solution.
`
`5
`
`According to still further features in the described preferred embodiments the
`
`detergent solution comprises TRITON-X-100.
`
`According to still further features in the described preferred embodiments the
`
`detergent solution further comprises ammonium hydroxide.
`
`According to still further features in the described preferred embodiments the
`
`IO
`
`Triton-X-100 is provided at a concentration selected from the range of 0.1-2 % (v/v).
`
`According to still further features in the described preferred embodiments the
`
`Triton-X-100 is provided at a concentration of 1 % (v/v).
`
`According to still further features in the described prefeJ;'fed embodiments the
`
`ammonium hydroxide is provided at a concentration selected from the range of 0.05-
`
`15
`
`1.0 % (v/v).
`
`According to still further features in the described preferred embodiments the
`
`ammonium hydroxide is provided at a concentration of 0.1 % (v/v).
`
`According to still further features in the described preferred embodiments
`
`subjecting the tissue to the detergent solution is effected for at least 24-48 hours.
`
`20
`
`According to still further features in the described preferred embodiments
`
`subjecting the tissue to the detergent solution is effected for 2-4 times.
`
`According to still further features in the described preferred embodiments the
`
`tissue comprises a myocardium tissue.
`
`According to still further features in the described preferred embodiments the
`
`25
`
`tissue comprises a vascular tissue.
`
`According to still further features in the described preferred embodiments the
`
`tissue comprises tissue segments.
`
`According to still further features in the described preferred embodiments each
`
`of the tissue segments is 2-4 mm thick.
`
`30
`
`According to still further features in the described preferred embodiments the
`
`cellular components comprise cell nuclei, nucleic acids, residual nucleic acids, cell
`
`membranes and/or residual cell membranes.
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`According to still further features in the described preferred embodiments the
`
`myocardium-derived decellularized ECM maintains mechanical and structural
`
`properties of a myocardium tissue ECM
`
`According to still further features in the described preferred embodiments the
`
`5 myocardium-derived decellularized ECM is capable of remodeling upon seeding with
`
`cells.
`
`According to still further features in the described preferred embodiments the
`
`myocardium-derived decellularized ECM maintains at least 90 % of a collagen
`
`content and at least 80 % of an elastin content of a myocardium tissue.
`
`IO
`
`According to still further features in the described preferred embodiments the
`
`myocardium-derived decellularized ECM is characterized by a stress value of at least
`
`0.4 MPa when strained to 40 %.
`
`According to still further features in the described preferred embodiments the
`
`myocardium tissue is a pig myocardium tissue.
`
`15
`
`According to still further features in the described preferred embodiments the
`
`at least one cell type is cardiomyocyte and the myocardium-derived decellularized
`
`ECM exhibits spontaneous beating.
`
`According to still further features in the described preferred embodiments the
`
`spontaneous beating is in concert.
`
`20
`
`According to still further features in the described preferred embodiments the
`
`at least one type of cells comprises cardiomyocytes.
`
`According to still further features in the described preferred embodiments the
`
`at least one type of cells comprises cardiac fibroblasts.
`
`The present invention successfully addresses the shortcomings of the presently
`
`25
`
`known configurations by providing a novel method of decellularizing natural tissues
`
`which results in matrices which are completely devoid of cellular components and
`
`thus non-immunogenic when implanted in a subject, maintain the structural and
`
`mechanical properties of the natural tissue ECMs and are remodeled when seeded
`
`with cells.
`
`30
`
`Unless otherwise defined, all technical and scientific terms used herein have
`
`the same meaning as commonly understood by one of ordinary skill in the art to
`
`which this invention belongs. Although methods and materials similar or equivalent
`
`to those described herein can be used in the practice or testing of the present
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`invention, suitable methods and materials are described below. In case of conflict, the
`
`patent specification, including definitions, will control.
`
`In addition, the materials,
`
`methods, and examples are illustrative only and not intended to be limiting.
`
`5
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`The invention is herein described, by way of example only, with reference to
`
`the accompanying drawings. With specific reference now to the drawings in detail, it
`
`is stressed that the particulars shown are by way of example and for purposes of
`
`illustrative discussion of the preferred embodiments of the present invention only, and
`
`IO
`
`are presented in the cause of providing what is believed to be the most useful and
`
`readily understood description of the principles and conceptual aspects of the
`In this regard, no attempt is made to show structural details of the
`invention in more detail than is necessary for a fundamental understanding of the
`
`invention.
`
`invention, the description taken with the drawings making apparent to those skilled in
`
`15
`
`the art how the several forms of the invention may be embodied in practice.
`
`In the drawings:
`
`FIGs. 1 a-fare photographs depicting myocardium tissue segments from pig
`
`(Figures la-e) or rat (Figure If) hearts subjected to the decellularization process of the
`
`present invention. Figure la - The heart of an adult pig. The left ventricle wall is
`
`20 marked by a circle and the right atrium is marked by an arrow; Figure 1 b
`
`myocardium segments of 2-4 mm thick sliced from left ventricle; Figure 1 c
`
`myocardium segments after partial decellularization. Myocardium segments were
`
`subjected to 12 hours of proteolytic digestion in 0.05 % trypsin and two cycles of
`
`incubation in a detergent solution (1 % Triton-X-100 / 0.1 % ammonium hydroxide),
`
`25
`
`48 hours each. Cellular remnants are visible in the center of the segment (marked by
`
`an arrow); Figure 1 d - myocardium segments from the left ventricle after complete
`
`decellularization as described in Example I of the Examples section which follows.
`
`Preservation of vascular structures is demonstrated (marked by arrows); Figure le -
`
`30
`
`myocardium segments from right atrium after complete decellularization. Note that
`the three-dimensional (3D) structure of the inner wall is preserved; Figure lf - The
`heart of an adult rat after the complete decellularization process.
`
`FIG. 2 is a photomicrograph depicting Hematoxylin and Eosin (H&E) staining
`
`of a matrix after decellularization. Matrices after decellularization were frozen with
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`OCT medium and 5 µm frozen sections were stained with H&E. Note that no cell
`
`nuclei are present in the matrix. Magnification is x 40.
`
`FIGs. 3a-d are photomicrographs depicting the assessment of nuclear and
`
`nucleic acid removal using fluorescent DAPI staining. Matrices after a complete [2
`
`5
`
`cycles in 0.05 % trypsin (24 hours each) and 4 cycles in a detergent solution (1 %
`Triton-X-100 I 0.1 % ammonium hydroxide; 48 hours each); Figures 3a and b;] or a
`
`partial [12 hours digestion in 0.05 % trypsin and two cycles of 48 hours each in a
`
`detergent solution (1 % Triton-X-100 / 0.1 % ammonium hydroxide); Figure 3c and
`
`d)] decellularization process were washed in PBS and incubated for 20 minutes with 1
`
`10
`
`µg/ml DAPI.
`
`Samples were exposed to UV and examined by a fluorescent
`
`microscope. Note the absence of cell nuclei in the completely processed matrices
`
`(Figures 3a-b ), whereas some could be found in the partially processed ones (Figures
`
`3c-d). Also note that while in the partially processed matrices some residual non(cid:173)
`
`nuclear staining is seen (Figures 3c-d) indicating incomplete removal of cellular DNA
`
`15
`
`from broken nuclei, in the completely processed matrices no residual staining is seen
`
`(Figures 3a-b ). All samples were similarly exposed to UV light for photography.
`
`FIGs. 4a-d are photomicrographs depicting assessment of cell membrane
`
`removal using fluorescent DiO staining. Matrices following partial [12 hours
`
`digestion in 0.05 % trypsin and two cycles of 48 hours each in a detergent solution (1
`20 % Triton-X-100 I 0.1 % ammonium hydroxide); Figures 4a and b] or complete [two
`
`cycles of 24 hours each in 0.05 % trypsin and four cycles of 48 hours each in a
`
`detergent solution (1 % Triton-X-100 I 0.1 % ammonium hydroxide); Figures 4c and
`
`d] decellularization process were washed in PBS and incubated in the dark at room
`
`temperature for two hours with 5 µg/ml DiO stain. Samples were inspected by a
`
`25
`
`fluorescent microscope with a blue filter. Figures 4c and 4d represent the same field
`
`with (Figure 4c) or without (Figure 4d) the additional exposure to a white light. All
`
`size bars represent 100 µm. Note the presence of membrane residues in the partially
`
`processed matrices (Figures 4a-b) and the complete absence of membrane residues in
`
`the completely processed decellularized matrices (Figures 4c-d). All samples were
`
`30
`
`similarly exposed to fluorescence for photography.
`
`FIGs. 5a-b are bar graphs depicting preservation of collagen (Figure 5a) and
`
`elastin (Figure 5b) after complete decellularization of myocardial tissue segments.
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`Complete decellularization was performed according to the decellularization protocol
`
`described in Example 1 of the Examples section which follows and included two
`
`cycles of 24 hours each in 0.05 % trypsin and four cycles of 48 hours each in I %
`
`Triton-X-100/ 0.1 % ammonium hydroxide. Fresh myocardial tissue segments (fresh)
`
`5
`
`and myocardium-derived decellularized ECM matrices
`
`( decellularized) were
`
`lyophilized and the total collagen and elastin contents were measured. Results are
`
`presented as the average (± SD) amount of collagen or elastin [in milligrams (mg)]
`per 100 mg of original fresh tissue ( dry weight, n = 5 in each case). Note that about
`90 % of the collagen and about 80 % of the elastin were preserved in the matrices
`
`10
`
`following complete decellularization.
`
`FIGs. 6a-c are photomicrographs depicting SEM analysis of myocardium(cid:173)
`
`derived decellularized matrices. Matrices were fixed in 2.5 % glutaraldehyde,
`
`dehydrated in ascending concentrations of ethanol and subjected to SEM analysis.
`
`Note the highly fibrous and porous matrix with various thicknesses of collagen fibers
`
`15
`
`and high crosslinking levels. Size bars represent 25 µm (Figure 6a), 8 µm (Figure 6b)
`
`and 2.5 µm (Figure 6c ).
`
`FIG. 7 is a bar graph depicting the glycosaminoglycan (GAG) content in the
`
`myocardium-derived decellularized matrix of the present invention. GAG content
`
`was quantified from lyophilized samples of the decellularized matrix of the present
`
`20
`
`invention and a commercial bovine tendon type I collagen (Sigma) using the safranin
`
`0 assay by extrapolation from a chondroitin sulfate standard curve. Bovine serum
`albun1in (BSA) served as a negative control. Results are presented as average ± SD of
`microgram GAG per mg sample as determined in six samples in each case. Note the
`
`significantly high GAG content in the myocardium-derived decellularized matrix of
`
`25
`
`the present invention as compared to the commercial collagen type I matrix.
`
`FIGs. 8a-c are graphs depicting mechanical properties of the myocardium(cid:173)
`
`derived decellularized matrices of the present invention. Matrices were decellularized
`
`according to the protocol described in Example 1 of the Examples section that
`
`included two cycles of 24 hours each in 0.05 % trypsin and four cycles of 48 hours
`
`30
`
`each in l % Triton-X-100 / 0.1 % ammonium hydroxide. Figure 8a- Cyclic strain.
`
`Matrices were pulled from "rest point" (0 stress, 0 strain) at a constant strain rate of
`
`0.05 mm per second to 15 % strain and released to the rest point at the same rate.
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`Results are presented as the stress [in mega Pasqual (MPa) units] as a function of the
`
`percentage of strain as measured for six decellularized matrix samples. Each colored
`
`curve represents an average (of six samples) of a separate strain-release cycle
`
`[(straining to 15 % strain (arrow pointing up) and releasing back to rest point (arrow
`
`5
`
`pointing down)] and the bold black line represents an average of all samples in all 6
`
`cycles. No significant decrease in elasticity is observed as indicated by retaining
`
`maximal stress during the 6 cycles of straining to 15 %. Figure 8b - Strain -
`
`relaxation. Matrices were quickly pulled (0.5 mm per second) to 20 % strain and kept
`there for 10 minutes. Results presented as the load (in Newton [N] units) as a
`
`10
`
`function of time [in seconds (s)] as measured for 6 decellularized matrices (each
`
`represented by a colored curve, bold black line indicating average of the six samples).
`
`No significant decrease in elasticity is observed as indicated by minimal decrease in
`
`load over time. Figure Sc - Strain to break. Matrices were slowly pulled (strain rate
`
`of 0.05 mm per second) until tom. The experiment was performed on 6 decellularized
`
`15 matrices. Shown is a representative graph of the stress (in MPa units) as a function of
`
`percentage of strain for one decellularized matrix. Note the high strength and
`
`flexibility as indicated by withstanding a stress of up to .0.42 MPa when pulled to 40
`
`% strain.
`
`FIGs. 9a-g are SEM (Figures 9a-d) and QuantomiX™ WET-SEM™ (Figures
`
`20
`
`9e-g) analyses of cardiac
`
`fibroblasts
`
`seeded on
`
`the myocardium-derived
`
`decellularized matrices of the present invention. Adult sheep cardiac fibroblasts were
`seeded at a concentration of approximately 104 cells per 1 cm2 matrix and following
`28 days of static culturing the matrices were subjected to SEM or WET-SEM
`
`analyses. Size bars represent the following: Figure 9a - 8 µm; Figure 9b - 25 µm;
`
`25
`
`Figure 9c - 80 µm; Figure 9d - 250 µm; Figure 9e - 10 µm; Figure 9f - 20 µm;
`
`Figure 9g - 500 µm. Note the significant cell density following 28 days in culture
`(Figures 9a-d) and the remodeling of the matrix by the fibroblasts into about .1 mm3
`
`spheroids (Figures 9d and f). Also note the new collagen fibers surrounding the cells
`
`populating the scaffold (indicated by arrows in Figure 9e).
`
`30
`
`FI Gs. I 0a-e are fluorescent photomicrographs depicting cardiac fibroblast
`
`cells cultured on the decellularized matrices of the present invention. Cardiac
`
`fibroblasts were stained with the DiO stain, following which the fibroblasts were
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`seeded on the decellularized matrices.
`
`Shown are the stained cells on the
`
`decellularized matrices at various time points after seeding: Figure I 0a -
`
`IO hours
`
`(Magnification x 20); Figure 1 Ob - 4 days (Magnification x 1 0); Figure 1 0c - 12 days
`
`(Magnification x 4); Figure 10d - 18 days (Magnification x 4; Figure IOe - 24 days
`
`5
`
`(Magnification x 4). Note that three weeks after seeding the matrices began to shrink
`
`and formed dense cell populated spheres (Figures l 0d and e ).
`
`FIGs. 1 la-d are photomicrographs depicting histochemical H&E staining of
`
`seeded matrices. Decellularized myocardium-derived matrices were seeded with
`
`cardiac fibroblasts and 14 (Figures l la-b) or 21 (Figures 1 lc-d) days post seeding the
`
`IO matrices were either fixed in paraformaldehyde and embedded in paraffin blocks
`
`(Figures 11 a and c) or frozen in OCT block (Figures 11 b and d) and sections of 5 µm
`
`were prepared and stained with H&E. Note that 14 days post seeding the cells were
`
`distributed throughout the scaffold (Figures lla-b) and that 21 days post seeding the
`
`scaffolds shrunk and the cells were populated more densely (Figures 11 c-d).
`
`15
`
`FIGs. 12a-b are bar graphs depicting the viability (in percentages) of
`
`fibroblasts (Figure 12a) or cardiomyocytes (Figure 12b) after seeding on the
`
`decellularized matrices of the present invention. Cells were statically seeded at a
`concentration of 104 cells per l-cm2 scaffolds (decellularized matrices). Every second
`change of medium (e.g. every 4-6 days) the cells were transferred to new wells and
`
`20
`
`alamarBlue was added to the medium (l/15 v/v). After 3 hours of incubation with
`
`alamarBlue, samples of 100 µl from each well were taken for fluorescent reading at
`
`535 nm / 590 nm. Values were normalized according to a standard curve of
`
`fluorescence per cell (not shown). Results are presented as the viability (in
`
`percentages, relative to the initial viability measured for each sample) as a function of
`
`25
`
`days post-seeding.
`
`FIGs. 13a-b are photographs of a native (Figure 13a) and a lyophilized,
`
`decellularized - porcine blood vessel (Figure 13b). Note the clean, vasculature-free
`
`vessel obtained following the decellularization process described in Example 4 of the
`
`Examples section which follows.
`
`30
`
`FIGs. 14a-b are photomicrographs of H&E staining depicting a natural (Figure
`
`14a) and a decellularized (Figure 14b) artery. Arrows mark the elastin fibers. Note
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`
`that the decellularized artery preserves the collagen and elastin structure of the natural
`
`artery tissue. Magnification is x 4.
`
`FIG. 15 is a bar graph depicting the collagen and elastin contents in the distal,
`
`center and proximal areas of decellularized arteries as percentages of dry artery
`
`5
`
`weight.
`
`FIGs. 16a-d are SEM images of native (Figures 16a-c) and decellularized
`
`(Figure 16d) arteries. Figure 16a - Im.age of an artery at low magnification ( size bar =
`
`1 mm); Figure 16b - Higher magnification of the outer surface of the artery shown in
`
`Figure 16a demonstrating layers of cells (size bar = 20 µm); Figure 16c - Higher
`
`10 magnification of the inner surface of the artery shown in Figure 16a demonstrating a
`
`monolayer of cells (size bar= 50 µm); Figure 16d - Image of a decellularized artery,
`
`demonstrating the complete absence of cells following the decellularization process
`
`(size bar= 8 µm).
`
`FIG. 17 is an image of an agarose gel electrophoresis of DNA samples
`
`15
`
`extracted from native (lane b) or decellularized (lane c) arteries. Lane a - molecular
`
`weight size marker in kilo base pair (kb). Note that while the native artery exhibits an
`
`intense DNA band (lane b ), no DNA is seen in the decellularized matrix [including
`
`absence oflow molecular weight DNA in the decellularized matrix (not shown)].
`
`FI Gs. l 8a-c are photomicrographs of H&E staining (Figures 18a-b) or a -
`
`20
`
`actin immunohistochemistry (Figure 18c; actin in dark purple) of a collagen
`
`decellularized artery scaffold seeded with smooth muscle cells. Magnification is x 1 0
`
`in Figures 18a and c and x 40 in Figure 18b.
`
`FIGs. 19a-f are photomicrographs depicting recellularized porcine carotid
`
`artery (PCA) with cells expressing red fluorescent protein (RFP) or green fluorescent
`
`25
`
`protein (GFP). Figure 19a - Expression of RFP by endothelial cells four weeks after
`
`seeding (Magnification x 40); Figure 19b - Smooth muscle cells (SMC) expressing
`
`GFP four weeks post seeding (Magnification x 40); Figure 19c - Wet SEM image of
`
`Figure 19a (Size bar= 20 µm); Figure 19d- Wet SEM image of Figure 19b (Size bar
`= 20 µm); Figure l 9e-f - Masson stained SMC seeded scaffold following 3 months in
`culture (Size bar = 100 µm).
`
`30
`
`FIGs. 20a-f are photomicrographs of H&E staining (Figures 20a-c) or SMC
`
`actin immunostaining (Figures 20d-f) of decellularized artery scaffolds following 4
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`weeks of seeding and culturing with SMCs. Figures 20a and d - Static seeding and
`
`culture; Figures 20b and e - Centrifugal seeding and static culture; Figures 20c and f -
`
`Centrifugal seeding and dynamic culture. H&E stains the cell nuclei in purple and the
`
`extracellular space in pink. Actin immunostaining stains the actin protein in green
`
`5
`
`and the cell nuclei in blue. Note that in the scaffold seeded by centrifugal seeding
`
`(Figures 20b and e) the cell penetration through the scaffold is more efficient than in
`
`the scaffold seeded by static seeding (Figures 20a and d). Also note that in scaffold
`
`seeded by the centrifugal seeding and cultured using dynamic culturing (Figures 20 c
`
`and t) cell penetration is significantly more efficient than in scaffolds seeded by
`
`10
`
`centrifugal seeding and cultured by static culturing (Figures 20b and e ). Size bars
`
`represent 100 µm in Figures 20a-c and 50 µm in Figures 20d-£
`
`FIGs. 21a-c are photomicrographs depicting procollagen I immunostaining of
`
`decellularized artery scaffolds following 4 weeks of seeding and culturing with
`
`SMCs. Figure 21a - Static seeding and culture; Figure 21b - Centrifugal seeding and
`
`15
`
`static culture; Figure 21 c - Centrifugal seeding and dynamic culture. Cell nuclei are
`stained in purple and pro-collagen I is stained in brown. Note that vast amount of
`
`collagen secreted by cells that were seeded using a centrifugal method and cultured
`
`using a dynamic method (Figure 21c, marked by an arrow). Size bars represent 100
`
`µm.
`
`20
`
`FIGs. 22a-c are images depicting RT-PCR analysis of elastin (Figure 22a),
`
`collagen III (Figure 22b) and GAPDH (Figure 22c) performed on mRNA samples
`
`derived from SMCs seeded on the decellularized artery scaffolds. Lane 1 - static
`
`seeding and culture; lane 2 - centrifugal seeding and static culture; lane 3 - centrifugal
`
`seeding and dynamic culture. Note that the mRNA level of elastin is significantly
`
`25
`
`higher in scaffolds seeded using the centrifugal seeding and cultured by the dynamic
`
`culture (Figure 22a, lane 3) as compared to scaffolds seeded using the centrifugal
`
`seeding and cultured by static culture (Figure 22b, lane 2) or scaffolds seeded and
`
`cultured using the static method (Figure 22a, lane 1 ). The level of the GAPDH
`
`mRNA indicates that equal amounts of RNA were used in all assays.
`
`30
`
`FI Gs. 23a-d are photomicrographs depicting H&E staining (Figures 23a and c)
`
`and CD3 l
`
`immunostaining
`
`(Figures 23b and d) of coated artery-derived
`
`decellularized scaffolds seeded with HUVEC following 9 days in culture. Figures
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`23a-b - scaffolds coated with PBS; Figures 23c-d - scaffolds coated with corneal
`
`matrix (CM). CD3 l immunostaining stains CDl in green and cell nuclei in blue.
`
`Note that in the CM - coated scaffolds (Figure 23d) the cells penetrate the scaffold
`
`more efficiently that in the PBS - coated scaffolds (Figure 23b) as indicated by the
`
`5
`
`deeper layers of nuclei stained in blue. Also note that in the CM - coated scaffolds
`
`(Figure 23d) the endothelial cells form a more continuous layer than in the PBS -
`
`coated scaffolds (Figure 23b) as indicated by the green labeling. Size bars represent
`
`50µm.
`
`FIG. 24 is a graph depicting the proliferation of SMCs on artery-derived
`
`10
`
`decellularized scaffolds at different time points. Cells were seeded and cultured using
`
`the indicated methods: blue -
`
`static seeding, static culturing; pink -
`
`centrifugal
`
`seeding, static culturing; green - centrifugal seeding, dynamic culturing. Proliferation
`
`was measured using Alamar-Blue reagent and results are presented as the number of
`cells x 106 as a function of time (in days) post seeding. N = 4, * p < 0.05.
`FI Gs. 25a-d are photomicrographs depicting H&E staining (Figures 25a-c) or
`
`15
`
`Masson's trichrome staining (Figure 25d) of sections of artery-derived decellularized
`
`scaffolds which were subject to centrifugal seeding and dynamic culturing with
`
`SMCs. Figure 25a - 1 day post-seeding; Figure 25b - 3 weeks post-seeding; Figures
`
`25c and d - 7 weeks post-seeding. Masson's trichrome staining stains the cell nuclei
`
`20
`
`in brown, the elastin and SMCs in red-purple and the collagen in blue. Size bars
`
`represent 50 µm.
`
`FIGs._ 26a-d are photomicrographs depicting the assessment of the immune
`
`response to implanted artery-derived decellularized scaffolds.
`
`Implanted scaffolds
`
`were harvested one (Figures 26a-b) or two (Figures 26c-d) weeks post implantation
`
`25
`
`and tissue sections were stained with H&E. Figures 26a and c - low magnification of
`
`x 100; Figures 26b and d - high magnification of x 400. Note the depth of cell
`
`penetration and thickness of capsule at two weeks post implantation (Figures 26c and
`
`d). In Figure 26d, arrow head pointing at a neutrophil cell; thick arrow pointing at a
`
`fibroblast; and the thin arrow pointing at a lymphocyte cell.
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`30
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`15
`
`DESCRIPTION OF THE PREFERRED EMBODIMENTS
`
`The present invention is of a method of generating completely decellularized
`
`ECMs from natural tissues such as myocardium or vascular tissues which are non(cid:173)
`
`immunogenic when implanted in a subject, preserve the structural and mechanical
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`5
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`properties of the natural tissue ECM and are remodeled upon seeding with cells.
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`Specifically, the present invention can be used for tissue regeneration and/or repair
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`applications such as of myocardial or vascular tissues.
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`The principles and operation of the method of generating the decellularized
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`ECM according to the present invention may be better understood with reference to
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`10
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`the drawings and accompanying descriptions.
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`Before explaining at least one embodiment of the invention in detail, it is to be
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`understood that the invention is not limited in its application to the details set forth in
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`the following description or exemplified by the Examples. The invention is capable
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`of other embodiments or of being practiced or carried out in various ways. Also, it is
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`15
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`to be understood that the phraseology and terminology employed herein is for the
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`purpose of description and should not be regarded as limiting.
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`Heart failure is a main contributor to morbidity and mortality in the Western
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`world. The main reason for the morbidity and mortality associated with heart failure
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`is the inability of cardiomyocytes to proliferate and regenerate following injuries such
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`20
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`as caused by myocardial infarction (MI). Thus, the current treatment regimens for
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`malfunctioning heart tissues rely on heart transplantation. However, due to the
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`limited availability of donated hearts, there is a need to develop engineered cardiac
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`tissues which can replace injured or diseased hearts.
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`One preferred approach of tissue engineering is the use of decellularized
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`25
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`natural tissues. Prior art studies describe various methods of decellularization of
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`natural tissues (See for example, U.S. Pat. Appl. Nos. 20040076657, 20030014126,
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`20020114845, 20050191281, 20050256588 and U.S. Pat. Nos. 6,933,103, 6,743,574,
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`6,734,018 and 5,855,620; which are fully incorporated herein by reference).
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`However, none of the prior art methods resulted in complete decellularized matrices
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`30 which are non-immunogenic when implanted in a subject, maintain the mechanical
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`and structural properties of the tissue ECM and are remodeled upon seeding with
`In addition, to date, there is no report of a decellularized matrix which is
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`cells.
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`derived from a myocardium tissue.
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`While reducing the present invention to practice, the present inventors have
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`uncovered a novel method of decellularizing a natural tissue so as to obtain a matrix
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`which is completely devoid of cellular components and exhibits mechanical and
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`structural properties that are suitable for tissue regeneration.
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`5
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`As described in the Examples section which follows, decellularization
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`according to the teachings of the present invention of myocardium or artery tissues
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`resulted in matrices which are completely devoid of all cellular components (Figure 2
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`and Example 1; Figures 16a-d and Example 4), are non-immunogenic when implanted
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`in a subject (Figures 26a-d, Example 4), maintain the ECM composition of the natural
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`10
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`tissue (e.g., at least 90 % of the collagen and 80 % of the elastin; Figures 5a-b, 7 and
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`Example 2; Figure 15 and Example 4), exhibit mechanical [e.g., elasticity and rigidity
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`(Figures 8a-c, Example 2 and Table 1, Example 4)] and structural (Figures 6a-c and
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`Example 2; Figures 14a-b and Example 4) properties of the tissue ECM and are
`In
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`remodeled upon seeding with cells (Figures 9a-f, l0a-e, l la-d; Example 3).
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`15
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`addition, when seeded with cardiomyocytes, the myocardium-derived decellularized
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`matrices of the present invention exhibited spontaneous pulsatile beating in concert,
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`similar to that of natural myocardium tissues (Example 3).
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`Thus, according to one aspect of the present invention there is provided a
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`method of generating a decellularized extracellular matrix (ECM) of a tissue. The
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`20 method is effected by (a) subjecting the tissue to a hypertonic buffer to thereby obtain
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`increased intercellular space within the tissue; (b) subjecting the tissue resultant of
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`step (a) to an enzymatic proteolytic digestion to thereby obtain digested cellular
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`components within the tissue; and subsequently ( c) removing the digested cellular
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`components from the tissue; thereby generating the decellularized ECM of the tissue.
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`25
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`As used herein the phrase "decellularized ECM of a tissue" refers to the
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`extracellular matrix which supports tissue organization ( e.g., a natural tissue) and
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`underwent a decellularization process (i.e., a removal of all cells from the tissue) and
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`is thus completely devoid of any cellular components.
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`The phrase "completely devoid of any cellular components" as used herein
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`30
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`refers to being more than 99 % (e.g., 100 %) devoid of the cellular components
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`present in the natural (e.g., native) tissue. As used herein, the phrase "cellular
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`components" refers to cell membrane components or intracellular components which
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`make up the cell. Examples of cell components include cell structures ( e.g.,
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`organelles) or molecules comprised in same. Examples of such include, but are not
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`limited to, cell nuclei, nucleic acids, residual nucleic acids ( e.g., fragmented nucleic
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`acid sequences), cell membranes and/or residual cell membranes (e.g., fragmented
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`membranes) which are present in cells of the tissue. It will be appreciated that due to
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`5
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`the removal of all cellular components from the tissue, such a decellularized matrix
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`cannot induce an immunological response when implanted in a subject.
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`The phrase "extracellular matrix (ECM)" as used herein, refers to a complex
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`network of materials produced and secreted by the cells of the tissue into the
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`surrounding extracellular space and/or medium and which typically together with the
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`10
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`cells of the tissue impart the tissue its mechanical and structural properties.
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`Generally, the ECM includes fibrous elements (particularly collagen, elastin, or
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`reticulin), cell adhesion polypeptides ( e.g., fibronectin,
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`laminin and adhesive
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`glycoproteins), and space-filling molecules [usually glycosaminoglycans (GAG),
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`proteoglycans].
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`15
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`A tissue-of-interest ( e.g., myocardium) may be an autologous or preferably a
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`non-autologous tissue ( e.g., allogeneic or even xenogeneic tissue, due to non(cid:173)
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`immunogenicity of the resultant decellularized matrix). The tissue is removed from
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`the subject [ e.g., an animal, preferably a mammal, such as a pig, monkey or
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`chimpanzee, or alternatively, a deceased human being (shortly after death)] and
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`20
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`preferably washed in a sterile saline solution (0.9 % NaCl, pH= 7.4), which can be
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`supplemented with antibiotics such as Penicillin/Streptomycin 250 units/ml.
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`Although whole tissues can