Three-dimensional tissue composition and method of use

Abstract

To provide tissue compositions for supporting cell growth, maintenance, and / or differentiation.SOLUTION: A tissue composition comprises: a. a scaffold having a generally flat orientation and comprising fibers of a first fiber type and fibers of a second fiber type in a knitted or woven configuration with pores therein, the first fiber type having a different composition and a different degradation profile from the second fiber type, the first fiber type comprising glycolide, lactide and trimethylene carbonate, the second fiber type comprising lactide and trimethylene carbonate; b. extracellular matrix (ECM) materials and ECM-generating cells disposed on or on and within the scaffold, the ECM and ECM-generating cells filling the pores in the scaffold; and c. induced pluripotent stem cell-derived cardiomyocytes, myocardial progenitor cells, or cardiomyocytes therein.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 521,137, filed on Jun. 16, 2017, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to tissue engineering, and more specifically, to three - dimensional engineered tissue compositions for enabling the growth, differentiation, and / or maintenance of one or more cell types. The tissue compositions herein can be constructed for in vitro or in vivo use, for example, for transplantation or surgical purposes. The tissue compositions herein can be adapted for short - term or long - term storage (e.g., cryopreservation).

Background Art

[0003] In recent years, three - dimensional cultures have increasingly been used to provide conditions similar to those predicted in vivo (e.g., appropriate structure and microenvironment) for cell growth, differentiation, and / or maintenance. Currently, significant efforts are being devoted to the development of three - dimensional cultures that mimic specific tissues. Such three - dimensional tissue cultures can be used for various purposes, such as for creating biological models for therapeutic purposes, for research and testing, etc.

Summary of the Invention

[0004] The present invention features engineered tissue compositions, e.g., three - dimensional tissue compositions that assist in cell growth, maintenance, and / or differentiation. The tissue compositions herein can be used for various purposes, including in vitro and in vivo applications, such as therapeutic purposes, e.g., surgical transplantation for treating a disease or condition.

[0005] For example, the present invention provides a tissue composition comprising a scaffold (scaffold as described herein) and an extracellular matrix (ECM) material disposed on or in and on the scaffold. The ECM can be of biological origin (e.g., produced by ECM-generating cells), synthetic origin, or can be characterized by a biological origin portion and a synthetic origin portion. Other features of the tissue composition are described below.

[0006] The present invention provides a tissue composition comprising a scaffold (scaffold as described herein) and an extracellular matrix (ECM) material disposed on or in and on the scaffold, wherein the ECM is cell-free. The ECM can be of biological origin (e.g., produced by ECM-generating cells), synthetic origin, or can be characterized by a biological origin portion and a synthetic origin portion. Other features of the tissue composition are described below.

[0007] The present invention also provides a tissue composition comprising a scaffold (scaffold as described herein) and an extracellular matrix (ECM) material and ECM-generating cells disposed on or in and on the scaffold. The ECM can be of biological origin (e.g., produced by ECM-generating cells), synthetic origin, or can be characterized by a biological origin portion and a synthetic origin portion. Other features of the tissue composition are described below.

[0008] The present invention also provides a tissue composition comprising a scaffold having pores therein, wherein at least a portion of the scaffold is composed of a slow-degrading material, the slow-degrading material being a material that is resorbed, absorbed, or degraded within a time frame of at least one month after the start of cell culture and / or transplantation (e.g., the slow-degrading material is not completely resorbed, absorbed, or degraded within one month after transplantation), and an extracellular matrix (ECM) material disposed on the scaffold, or on and within the scaffold. The present invention also provides a tissue composition comprising a scaffold having pores therein, wherein at least a portion of the scaffold is composed of a slow-degrading material, the slow-degrading material being a material that is resorbed, absorbed, or degraded within a time frame of at least two months after the start of cell culture and / or transplantation (e.g., the slow-degrading material is not completely resorbed, absorbed, or degraded within two months after transplantation), and an extracellular matrix (ECM) material disposed on the scaffold, or on and within the scaffold. The present invention also provides a tissue composition comprising a scaffold having pores therein, wherein at least a portion of the scaffold is composed of a slow-degrading material, the slow-degrading material being a material that is resorbed, absorbed, or degraded within a time frame of at least three months after the start of cell culture and / or transplantation (e.g., the slow-degrading material is not completely resorbed, absorbed, or degraded within three months after transplantation), and an extracellular matrix (ECM) material disposed on the scaffold, or on and within the scaffold. The present invention also provides a tissue composition comprising a scaffold having pores therein, wherein at least a portion of the scaffold is composed of a slow-degrading material, the slow-degrading material being a material that is resorbed, absorbed, or degraded within a time frame of at least six months after the start of cell culture and / or transplantation (e.g., the slow-degrading material is not completely resorbed, absorbed, or degraded within six months after transplantation), and an extracellular matrix (ECM) material disposed on the scaffold, or on and within the scaffold. In certain embodiments, the slow-degrading material comprises a protein, a polymer, or a plurality of fibers. In certain embodiments, the tissue composition does not fold over itself during transplantation (e.g., does not spontaneously fold over itself).In certain embodiments, when the user accidentally folds the tissue composition on itself (e.g., during transplantation), the tissue composition cannot remain folded on itself. In certain embodiments, at least a portion of the scaffold is non-resorbable. In some embodiments, the tissue composition has a cell layer, and the cell layer is 3 to 500 cell layers thick. In some embodiments, the tissue composition further comprises seeded cells seeded in and / or on the ECM, and the seeded cells are stem cells, embryonic stem cells, embryonic stem cell-derived cells, induced pluripotent stem cell-derived cells, progenitor cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, hepatocytes, pancreatic cells, lung cells, bone cells, umbilical cord cells, endothelial cells, central nervous system cells, gastrointestinal cells, endocrine cells, salivary cells, mesenchymal stem cells, fibroblasts or paracrine cells. Other features of the tissue composition are described below.

[0009] The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of recombinant peptides having pores therein, and an extracellular matrix (ECM) material disposed on, or on and within, the scaffold. In some embodiments, the tissue composition further comprises ECM-producing cells in the ECM, and the ECM-producing cells are alive, dead, or a portion of the ECM-producing cells are dead. In some embodiments, the tissue composition further comprises seeded cells seeded in or on the ECM, and the seeded cells are stem cells, embryonic stem cells, embryonic stem cell-derived cells, induced pluripotent stem cell-derived cells, progenitor cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, hepatocytes, pancreatic cells, lung cells, bone cells, umbilical cord cells, endothelial cells, central nervous system cells, gastrointestinal cells, endocrine cells, salivary cells, mesenchymal stem cells, fibroblasts or paracrine cells. Other features of the tissue composition are described below.

[0010] The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of fibers having pores therein, and an extracellular matrix (ECM) material disposed on the scaffold, or on and within the scaffold. The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of fibers having pores therein, an extracellular matrix (ECM) material disposed on the scaffold, or on and within the scaffold, and seeded cells seeded in and / or on the ECM. In certain embodiments, the seeded cells are stem cells, induced pluripotent stem cell-derived cells, progenitor cells, cardiac cells, skeletal muscle cells, smooth muscle cells, hepatocytes, pancreatic cells, lung cells, bone cells, umbilical cord cells, endothelial cells, central nervous system cells, gastrointestinal cells, endocrine cells, mesenchymal stem cells, fibroblasts, salivary cells or paracrine cells. Other features of the tissue composition are described below.

[0011] The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of recombinant peptides having pores therein, or a plurality of proteins having pores therein, or a plurality of polymers having pores therein, and an extracellular matrix (ECM) material disposed on the scaffold, or on and within the scaffold. The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of recombinant peptides having pores therein, or a plurality of proteins having pores therein, or a plurality of polymers having pores therein, an extracellular matrix (ECM) material disposed on the scaffold, or on and within the scaffold, and seeded cells seeded in and / or on the ECM. In certain embodiments, the seeded cells are stem cells, induced pluripotent stem cell-derived cells, progenitor cells, cardiac cells, skeletal muscle cells, smooth muscle cells, hepatocytes, pancreatic cells, lung cells, bone cells, umbilical cord cells, endothelial cells, central nervous system cells, gastrointestinal cells, endocrine cells, mesenchymal stem cells, fibroblasts, salivary cells or paracrine cells. Other features of the tissue composition are described below.

[0012] The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of fibers having pores inside, or a plurality of recombinant peptides having pores inside, or a plurality of proteins having pores inside, or a plurality of polymers having pores inside, and an extracellular matrix (ECM) material disposed on the scaffold, or on and inside the scaffold. The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of fibers having pores inside, or a plurality of recombinant peptides having pores inside, or a plurality of proteins having pores inside, or a plurality of polymers having pores inside, an extracellular matrix (ECM) material disposed on the scaffold, or on and inside the scaffold, and seeded cells seeded in and / or on the ECM. In certain embodiments, the seeded cells are stem cells, induced pluripotent stem cell-derived cells, progenitor cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, hepatocytes, pancreatic cells, lung cells, bone cells, umbilical cord cells, endothelial cells, central nervous system cells, gastrointestinal cells, endocrine cells, mesenchymal stem cells, fibroblasts, salivary cells or paracrine cells. Other features of the tissue composition are described below.

[0013] The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of fibers having pores inside, or a plurality of recombinant peptides having pores inside, or a plurality of proteins having pores inside, or a plurality of polymers having pores inside, an extracellular matrix (ECM) material disposed on the scaffold, or on and inside the scaffold, and seeded cells seeded in and / or on the ECM, wherein the seeded cells are cardiomyocytes. In certain embodiments, the tissue composition has a beat rate of 0 to 120 bpm. In certain embodiments, the tissue composition has a beat rate of 0 to 60 bpm. In certain embodiments, the tissue composition has a beat rate of 0 to 50. In certain embodiments, a drug or solution or other product is applied to the tissue composition to achieve a specific beat rate, such as a beat rate of 0 to 60, 0 to 50, etc. In certain embodiments, the cardiomyocytes constitute 3 to 60% of the area of the tissue composition, or 3 to 60% of the volume of the tissue composition, or 3 to 60% of the volume of the cell material in the tissue composition. Other features of the tissue composition are described below.

[0014] The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of fibers having pores inside, or a plurality of recombinant peptides having pores inside, or a plurality of proteins having pores inside, or a plurality of polymers having pores inside, an extracellular matrix (ECM) material disposed on the scaffold, or on and inside the scaffold, and seeded cells seeded in the ECM, wherein the seeded cells are skeletal muscle cells. The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of fibers having pores inside, or a plurality of recombinant peptides having pores inside, or a plurality of proteins having pores inside, or a plurality of polymers having pores inside, an extracellular matrix (ECM) material disposed on the scaffold, or on and inside the scaffold, and seeded cells seeded in the ECM, wherein the seeded cells are hepatocytes. The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of fibers having pores inside, or a plurality of recombinant peptides having pores inside, or a plurality of proteins having pores inside, or a plurality of polymers having pores inside, an extracellular matrix (ECM) material disposed on the scaffold, or on and inside the scaffold, and seeded cells seeded in the ECM, wherein the seeded cells are gastrointestinal cells. The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of fibers having pores inside, or a plurality of recombinant peptides having pores inside, or a plurality of proteins having pores inside, or a plurality of polymers having pores inside, an extracellular matrix (ECM) material disposed on the scaffold, or on and inside the scaffold, and seeded cells seeded in the ECM, wherein the seeded cells are pancreatic cells. The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of fibers having pores inside, or a plurality of recombinant peptides having pores inside, or a plurality of proteins having pores inside, or a plurality of polymers having pores inside, an extracellular matrix (ECM) material disposed on the scaffold, or on and inside the scaffold, and seeded cells seeded in the ECM, wherein the seeded cells are umbilical cord cells or umbilical cord blood cells.The present invention also provides tissue compositions comprising a scaffold constructed from a plurality of fibers having pores therein, or a plurality of recombinant peptides having pores therein, or a plurality of proteins having pores therein, or a plurality of polymers having pores therein, an extracellular matrix (ECM) material disposed on the scaffold or on and within the scaffold, and seeded cells seeded in the ECM, the seeded cells being smooth muscle cells. Other features of the tissue compositions are described below.

[0015] The present invention also provides a tissue composition comprising a scaffold constructed from a plurality of recombinant peptides that form a sponge-like configuration with pores, and an ECM material disposed on the scaffold, or on and throughout the scaffold. In certain embodiments, some of the pores overlap. In certain embodiments, the pores are clustered. In certain embodiments, the recombinant peptide is type I collagen. In certain embodiments, the recombinant peptide is one or a combination of peptides found in extracellular matrix materials.

[0016] The invention also provides tissue compositions comprising at least a scaffold and an ECM as described herein, wherein the tissue composition is in a closed culture system (e.g., grown in a closed culture system, maintained in a closed culture system, preserved in a closed culture system, e.g., cryopreserved in a closed culture system).

[0017] The invention also provides tissue compositions comprising at least a scaffold as described herein and an ECM, wherein the tissue composition is in a cryopreserved state, e.g., at −80° C. to −196° C., −90° C. to −196° C., etc. In certain embodiments, the tissue composition may be in a cryopreserved state for at least 3 days, at least 7 days, at least 14 days, at least 21 days, at least 60 days, at least 3 months, at least 6 months, at least 1 year, at least 2 years, etc.

[0018] The following features may apply to any of the aforementioned tissue compositions provided above, or to any of the tissue compositions described herein, or to any of the methods described herein.

[0019] In some embodiments, the ECM is generated prior to its deposition onto the scaffold. The ECM may be acellular, for example, in some embodiments, the ECM is all synthetically derived. In some embodiments, the ECM is bioderived (e.g., generated by ECM-producing cells) and subsequently decellularized. In some embodiments, the ECM is bioderived (e.g., generated by ECM-producing cells), and the ECM and the ECM-producing cells are seeded onto the scaffold. In some embodiments, the ECM-producing cells are seeded onto the scaffold, and the ECM-producing cells subsequently generate the ECM.

[0020] In some embodiments, the ECM-producing cells are live cells. In some embodiments, the ECM-producing cells are dead cells. In some embodiments, some of the ECM-producing cells are dead. For example, in some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% of the ECM-producing cells are dead. In certain embodiments, the density of the ECM-producing cells within the scaffold is 5x10 5 cells / cm 2 ~5x10 7 cells / cm 2 . In some embodiments, the ECM-producing cells are genetically modified to express one or more genes.

[0021] In some embodiments, the scaffold (or a portion thereof) is constructed from a material that degrades within a time frame of 4 weeks or more after implantation, e.g., a portion of the scaffold that is absorbent / resorbable / degradable within a time frame of 4 weeks or more after implantation. In some embodiments, the scaffold (or a portion thereof) is constructed from a material that degrades within a time frame of 1 month or more after implantation, e.g., a portion of the scaffold that is absorbent / resorbable / degradable within a time frame of 1 month or more after implantation. In some embodiments, the scaffold (or a portion thereof) is constructed from a material that degrades within a time frame of 6 weeks or more after implantation, e.g., a portion of the scaffold that is absorbent / resorbable / degradable within a time frame of 6 weeks or more after implantation. In some embodiments, the scaffold (or a portion thereof) is constructed from a material that degrades within a time frame of 8 weeks or more after implantation, e.g., a portion of the scaffold that is absorbent / resorbable / degradable within a time frame of 8 weeks or more after implantation. In some embodiments, the scaffold (or a portion thereof) is constructed from a material that degrades within a time frame of 3 months or more after implantation, e.g., a portion of the scaffold that is absorbent / resorbable / degradable within a time frame of 3 months or more after implantation. In some embodiments, the scaffold (or a portion thereof) is constructed from a material that degrades within a time frame of 4 months or more after implantation, e.g., a portion of the scaffold that is absorbent / resorbable / degradable within a time frame of 4 months or more after implantation. In some embodiments, the scaffold (or a portion thereof) is constructed from a material that degrades within a time frame of 6 months or more after implantation, e.g., a portion of the scaffold that is absorbent / resorbable / degradable within a time frame of 6 months or more after implantation.

[0022] The ECM-producing cells can be fibroblasts, e.g., iPSC-derived fibroblasts, embryonic stem cell-derived fibroblasts, organ-derived fibroblasts (e.g., dermal fibroblasts), etc. In certain embodiments, the ECM-producing cells are non-fibroblasts.

[0023] Regarding the scaffold, the scaffold may be constructed from a plurality of fibers having a plurality of pores disposed between the fibers. In certain embodiments, the scaffold is composed of a plurality of proteins or a network of proteins. In some embodiments, the scaffold is composed of a plurality of polymers or a network of polymers. In certain embodiments, the scaffold is composed of a plurality of recombinant peptides or a network of recombinant peptides. In certain embodiments, the recombinant peptide is type I collagen. In certain embodiments, the recombinant peptide is one or a combination of ECM-related peptides. In certain embodiments, the recombinant peptide is for a sponge-like configuration or a film-like configuration. In some embodiments, the pores have a diameter of 50 μm to 500 μm. In some embodiments, the pores have a diameter of 50 μm to 1000 μm. The pores may be non-uniformly disposed throughout the scaffold. In certain embodiments, at least a portion of the scaffold is resorbable, absorbable or degradable within a time frame of up to 3 years after implantation into a subject. In certain embodiments, the ECM fills at least 80% of the pores. The tissue composition may be present in a flat orientation. In certain embodiments, the tissue composition can curl. In certain embodiments, the cell layer in the tissue composition is 3 to 500 cell layers thick. In certain embodiments, the scaffold is at least 50 μm thick. In some embodiments, the tissue composition is 200 to 1000 μm thick.

[0024] The tissue composition may be characterized by seeded cells seeded on or within the scaffold and / or ECM. In certain embodiments, the seeded cells are seeded as a solution. In certain embodiments, the seeded cells are seeded as spheroids. In certain embodiments, the seeded cells are seeded in the form of a cell sheet. In certain embodiments, the seeded cells are seeded in the form of a gel. In certain embodiments, the seeded cells are seeded in the form of a foam. In some embodiments, the seeded cells are stem cells (e.g., mesenchymal stem cells), induced pluripotent stem cell-derived cells, progenitor cells, terminally differentiated cells (e.g., hepatocytes, beta cells, endodermal cells, smooth muscle cells, skeletal muscle cells, salivary cells, epithelial cells, endothelial cells, cardiomyocytes or combinations thereof) or combinations thereof.

[0025] The seeded cells may form a layer, and the layer may be three or more cells thick, four or more cells thick, five or more cells thick, etc. The seeded cells may be derived from diseased or mutated tissue. The seeded cells may be genetically modified, for example, modified to express one or more genes.

[0026] The tissue composition may have a pulsation rate of 0 to 50 bpm. The tissue composition may have a pulsation rate of 0 to 120 bpm.

[0027] In certain embodiments, the tissue composition can be evaluated for one or more mechanical parameters, electrophysiological parameters, chemical parameters, biochemical parameters, or combinations thereof. Mechanical or electrophysiological parameters can include, but are not limited to, contraction rate, contraction / relaxation rate, constant contractile force, non-constant contractile force, displacement rate, displacement force, impulse directionality, impulse velocity, electric field potential, amplitude, capture threshold, chronotropic response, excitation sequence after stimulation, functional gap junction formation, response to electrical pacing, electric field potential amplitude, conduction velocity, propagation pattern, gap junction analysis, oxygen consumption, or combinations thereof.

[0028] As described above, the tissue composition may be cryopreserved. The tissue composition may retain its structural integrity when cryopreserved and subsequently thawed. The tissue composition may be centrifuged and retain its structural integrity. The composition may maintain its structural integrity when handled with forceps or by hand.

[0029] The present invention also provides a method of generating or manufacturing the tissue compositions of the present specification. The method may feature the use of a scaffold, an ECM, cells, and any of the other materials or features described herein. In some embodiments, the method may include applying an extracellular matrix (ECM) material to a scaffold and seeding a population of cells into the ECM. In some embodiments, the method includes applying an extracellular matrix (ECM) material to a scaffold, seeding a population of ECM-producing cells onto and / or into the ECM within the scaffold, and seeding a population of seeded cells onto and / or into the ECM. In some embodiments, the method includes applying extracellular matrix (ECM)-producing cells to a scaffold and culturing the ECM-producing cells to produce an ECM within the scaffold. Subsequently, a population of seeded cells may be seeded onto and / or into the ECM.

[0030] Regarding the foregoing method, in some embodiments, the ECM is generated prior to its deposition onto the scaffold. The ECM may be cell-free. In certain embodiments, the ECM is decellularized. In some embodiments, the ECM is produced by ECM-producing cells. In some embodiments, the ECM is synthetically derived. In some embodiments, a portion of the ECM is biologically produced and a portion is synthetically produced. In some embodiments, the ECM is seeded with ECM-producing cells, cultured for a period of time, and subsequently caused to produce an ECM. In some embodiments, the ECM is added to the scaffold, followed by the addition of ECM-producing cells. The tissue composition may feature living ECM-producing cells, dead ECM-producing cells, or a combination thereof. The ECM and / or ECM-producing cells fill a portion of the pores within the scaffold.

[0031] The tissue composition may be characterized by seeded cells. The seeded cells may be cultured for a certain period before use. In some embodiments, the seeded cells are not cultured for a certain period before use. The ECM-producing cells may be cultured for a certain period before seeding the seeded cells. In some embodiments, the seeded cells are cultured to differentiate the cells, for example, growth factors or differentiation factors may be added during the culture. In some embodiments, the seeded cells (or ECM-producing cells) are cultured to proliferate the cells, for example, specific growth factors may be added during the culture.

[0032] In some embodiments, the method is characterized by seeding ECM-producing cells and / or seeding seeded cells. This may include applying cells (e.g., ECM-producing cells, seeded cells) to the scaffold, centrifuging the cells and the scaffold, applying cells (e.g., ECM-producing cells, seeded cells) to the scaffold and rocking the cells and the scaffold, applying cells (e.g., ECM-producing cells, seeded cells) to the scaffold and moving the cells to the scaffold by gravity, applying the cells using a directional force such as in a spray, etc. The cells may be seeded in the suspension as spheroids, in the form of cell sheets, in the form of gels, in the form of foams, etc. The cell sheet may be generated by seeding the cells on a temperature-sensitive plate, a low-adhesion plate, or a plate containing a composition that allows detachment of the cells at a selected time. The composition that allows detachment may include coated liposomes, e.g., gold-coated liposomes, engineered liposomes, photoactivated liposomes, liposomes containing RGD ligands, etc.

[0033] In some embodiments, the scaffold or ECM contains an adhesion factor that helps attach the ECM-producing cells and / or seeded cells to the scaffold and / or ECM during seeding of the ECM-producing cells. The adhesion factors may include ligands, antibodies, magnetic beads, liposomes, liposomes coated with a material that attracts or attaches ECM-producing cells to the scaffold, or combinations thereof. In some embodiments, the adhesion factor is a foam.

[0034] The present invention also provides a method for reducing the heart rate of a contractile composition, such as a tissue composition characterized by cells such as cardiomyocytes that can contract. The method may be characterized by applying a drug or other composition to the tissue composition, and the drug or composition reduces the heart rate of the tissue composition. The present invention also provides a method for reducing the metabolic rate of a tissue composition. The method may be characterized by applying a drug or other composition to the tissue composition, and the drug or composition reduces the metabolic rate of the tissue composition. In some embodiments, the drug or composition reduces the heart rate of the tissue composition, thereby reducing the metabolic rate. The aforementioned method may improve the stability of the tissue composition during transportation. The method may improve the stability and / or lifespan or survival rate of the tissue composition during transplantation. The method may be characterized by changing the temperature of the tissue composition to reduce the heart rate or metabolic rate of the tissue composition.

[0035] The present invention also features a method of transplanting a tissue composition into a subject in need thereof. The tissue composition may be transplanted at a specific heart rate, such as a heart rate of 0 - 50 bpm, 10 - 50 bpm, 0 - 40 bpm, 50 - 120 bpm, 0 - 120 bpm, etc.

[0036] The present invention also provides a method of treating a disease or condition, characterized by transplanting the tissue composition of the present invention into a subject in need thereof.

[0037] The present invention also provides a tissue composition produced by any of the methods described herein.

[0038] Any feature or combination of features described herein is included within the scope of the present invention as long as the features included in such a combination do not conflict with each other as is apparent from the context, this specification, and the knowledge of those skilled in the art. Further advantages and aspects of the present invention will be apparent in the following detailed description and claims.

[0039] The features and advantages of the present invention will become apparent from consideration of the following detailed description presented in connection with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

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DETAILED DESCRIPTION OF THE INVENTION

[0058] <Terms> 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 disclosed invention belongs. The singular terms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the phrase "or" is intended to include "and" unless the context clearly dictates otherwise. The term "comprising" means that other elements may exist in addition to the recited defined elements. The use of "comprising" indicates inclusion rather than limitation. That is, the term "comprising" means "including primarily, but not necessarily solely". Further, variations of the phrase "comprising", such as "comprise" and "comprises", have corresponding same meanings. In one aspect, the techniques described herein related to the compositions, methods, and respective components thereof described herein that are essential to the present invention, whether essential or not, accept the inclusion of elements not specified ("comprising").

[0059] All embodiments disclosed herein can be combined with other embodiments unless the context clearly indicates otherwise.

[0060] Methods and materials suitable for the practice and / or testing of embodiments of the present disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the present disclosure pertains are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), “Guide to Protein Purification” in Methods in Enzymology (M.P. Deutscher, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2. nd It is described in various general and more specific references including Ed. (R.I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.), the disclosures of which are incorporated herein by reference in their entirety.

[0061] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, this specification, including explanations of terms, will control.

[0062] Although the disclosed techniques can be practiced or tested using methods and materials similar or equivalent to those described herein, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0063] To facilitate an overview of the various embodiments of the present disclosure, the following explanations are provided for certain terms.

[0064] The term "progenitor cell" refers to a cell having a cell phenotype that is more primitive than the cells that can arise by differentiation (e.g., at an earlier stage of the developmental pathway or progression than fully differentiated cells). Often, progenitor cells also have a significant or very high proliferative capacity. Progenitor cells can give rise to multiple different differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which the cells develop and differentiate.

[0065] As used herein, the term "stem cell" refers to an undifferentiated cell that has the ability to proliferate and give rise to a greater number of progenitor cells that can in turn give rise to a number of mother cells that can then differentiate or be capable of differentiating into daughter cells. The daughter cells themselves can be made to proliferate and produce progeny, and then, while retaining one or more cells with parental developmental potential, these progeny can be differentiated into one or more mature cell types. The term "stem cell" refers to a subset of progenitor cells that have the ability or potential to differentiate into a more specialized or differentiated phenotype under certain circumstances and that retain the ability to proliferate without substantially differentiating under certain circumstances. In one embodiment, the term "stem cell" generally refers to the naturally occurring mother cells from which the descendants (progeny) of the stem cell typically diversify progressively in the embryo and in tissues, and in many cases diversify in different directions by differentiation, for example by acquiring completely individual characteristics. Cell differentiation is a process that typically occurs through many cell divisions. Differentiated cells can be derived from pluripotent cells that themselves are derived from pluripotent cells such as. Each of these pluripotent cells can be considered a stem cell, but the range of cell types that each can give rise to can vary widely. Among the differentiated cells, some have the ability to give rise to cells with high developmental potential. Such ability may be natural or may be artificially induced by treatment with various factors. In many biological examples, stem cells are also "pluripotent" because they can generate progeny of multiple different cell types, but this is not necessary for "stem-ness". Self-renewal is another classical part of the definition of stem cells and is essential as used herein. Theoretically, self-renewal can occur by either of two main mechanisms. Stem cells may divide asymmetrically, with one daughter cell maintaining the stem cell state and the other daughter cell expressing some different other specific functions and phenotypes. Alternatively, some of the stem cells in a population can divide symmetrically into two stem cells, so that some of the stem cells in the population are maintained as a whole and the other cells in the population give rise only to differentiated progeny.

[0066] The term "embryonic stem cell" is used to refer to pluripotent stem cells of the inner cell mass of an embryonic blastocyst (see U.S. Patent No. 5,843,780 and U.S. Patent No. 6,200,806, which are incorporated herein by reference). Such cells can also be obtained from the inner cell mass of a blastocyst derived from somatic cell nuclear transfer (see, for example, U.S. Patent No. 5,945,577, U.S. Patent No. 5,994,619, and U.S. Patent No. 6,235,970, which are incorporated herein by reference). Distinctive features of embryonic stem cells define the phenotype of embryonic stem cells. Thus, if a cell has one or more of the distinctive features of embryonic stem cells that enable it to be distinguished from other cells, that cell has the phenotype of an embryonic stem cell. Exemplary characteristic features of embryonic stem cells include, but are not limited to, gene expression profiles, proliferative ability, differentiation ability, karyotype, responsiveness to specific culture conditions, and the like.

[0067] The term "adult stem cell" or "ASC" is used to refer to any pluripotent stem cell derived from non-embryonic tissues such as fetal, juvenile, and adult tissues. Stem cells have been isolated from a variety of adult tissues such as blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells can be characterized based on gene expression, factor responsiveness, and morphology in culture. As noted above, it has been found that stem cells are present in virtually every tissue. Thus, it is understood that the techniques described herein can isolate stem cell populations from virtually any animal tissue.

[0068] As used herein, the term "iPS cell" or "induced pluripotent stem cell" refers to pluripotent cells artificially derived (e.g., induced by complete or partial reprogramming) from differentiated somatic cells (non-pluripotent cells). Pluripotent cells can differentiate into cells of any of the three developmental germ layers.

[0069] The term "derived from" when applied to a cell "derived from" another cell or tissue means that the cell has been isolated from the tissue mentioned or has differentiated from a reference tissue or cell type. Thus, a cell "derived from" the tissue of a particular individual has been isolated or differentiated from the tissue of that individual. The individual can include an individual having a predetermined state. Induced pluripotent stem cells are derived from an individual, for example, a postnatal human individual, in many cases somatic tissues of an adult. Similarly, embryonic stem cells are derived from an embryo. Cells derived from iPS cells refer to cells differentiated from iPS cells. Alternatively, a cell can be converted from one cell type to a different cell type by a process called differentiation conversion or direct reprogramming. Alternatively, with respect to iPS cells, cells (e.g., iPS cells) can be induced from differentiated cells by a process called dedifferentiation or reprogramming in the art.

[0070] As used herein, the term "pluripotent" refers to cells that can give rise to all types of cells in the body except germ cells. As used herein, the term "pluripotency" or "pluripotent state" refers to cells that have the ability to differentiate into all three germ layers, namely, the endoderm (gut tissue), mesoderm (including blood, muscle, and blood vessels), and ectoderm (such as skin and nerves), and typically have the potential to divide in vitro for extended periods, for example, for more than one year or more than 30 passages. Pluripotency is also demonstrated by the expression of embryonic stem (ES) cell markers, but a preferred test for pluripotency is the demonstration of the ability to differentiate into cells of any of the three germ layers, as detected, for example, using the nude mouse teratoma formation assay. iPS cells are pluripotent cells. Pluripotent cells further differentiate into pluripotent cells that give rise to cells with specific functions. For example, multipotent cardiovascular stem cells give rise to cells of the heart, such as cardiomyocytes, and other cells involved in the vascular structure of the heart. Cells useful for in vitro differentiation into the muscle cells or cardiomyocytes disclosed herein include, for example, iPS cells and multipotent cardiovascular stem cells. The main advantage of the use of iPSCs or other stem cells for generating muscle cells or cardiomyocytes for the compositions and methods disclosed herein is the ability to prepare large numbers of such cells, for example, by expanding such cells from a particular human patient or subject. This is in contrast to methods and compositions that rely on the isolation and use of adult heart cells.

[0071] As used herein, the term "differentiation" refers to the process by which a cell moves further down a developmental pathway, becomes more specialized, and begins to express markers and phenotypic characteristics associated with cells closer to terminally differentiated cells. The pathway by which cells progress from less committed cells to cells increasingly committed to a particular cell type and ultimately to terminally differentiated cells is called progressive differentiation or progressive commitment. Cells that have become more specialized (e.g., have begun to progress along a pathway of progressive differentiation) but have not yet become terminally differentiated are called partially differentiated. Differentiation is a developmental process by which cells assume a more specialized phenotype, for example, acquire one or more characteristics or functions different from other cell types. In some cases, the differentiated phenotype refers to the cell phenotype at the mature endpoint of a developmental pathway (the so-called terminally differentiated cell). In many, but not all, tissues, the process of differentiation is associated with exit from the cell cycle. In these cases, terminally differentiated cells lose their ability to proliferate or have it greatly restricted. However, in the context of this specification, the terms "differentiation" or "differentiated" refer to cells that are more specialized in their fate or function than at a particular point in their development. For example, in the context of this application, differentiated cells include ventricular cardiomyocytes differentiated from cardiovascular progenitor cells, which in some examples can be derived from the differentiation of ES cells or alternatively from the differentiation of induced pluripotent stem (iPS) cells or in some embodiments from a human ES cell line. Thus, such ventricular cardiomyocytes are more specialized than when they had the phenotype of cardiovascular progenitor cells but may not be more specialized than when the cells existed as mature cells from which the iPS cells were derived (e.g., prior to reprogramming of the cells to form iPS cells).

[0072] "Differentiated" cells, compared to progenitor cells, have one or more phenotypic differences and characteristics of a more mature or specialized cell type compared to their progenitor cells. Phenotypic differences include, but are not limited to, morphological differences as well as differences in gene expression and biological activity, including not only the presence or absence of expressed markers but also differences in the amount of markers and differences in the co-expression patterns of a set of markers.

[0073] As used herein, "growing" and "growth" refer to an increase (proliferation) in the number of cells within a population due to cell division. Cell growth is generally understood to result from the coordinated activation of multiple signaling pathways in response to the environment, including growth factors and other mitogens. Cell growth can also be promoted by the release from the action of intracellular or extracellular signals, and by mechanisms that block cell growth or have an adverse effect on cell growth.

[0074] The term "tissue" refers to a group or layer of similarly specialized cells that together perform a particular specialized function.

[0075] As used herein, the phrase "cardiovascular condition, disease or disorder" is intended to include any disorder characterized by inadequate, undesirable or abnormal cardiac function, such as arrhythmias, ischemic heart disease, hypertensive heart disease and pulmonary hypertensive heart disease, valvular heart disease, congenital heart disease, and any condition that causes congestive heart failure in a subject, particularly a human subject. Inadequate or abnormal cardiac function can be the result of disease, injury and / or aging. By way of background, the response to myocardial injury follows a well-defined pathway in which some cells die and others, while not yet dead, enter a hibernating state of dysfunction. This is followed by infiltration of inflammatory cells and deposition of collagen as part of the scar, all of which occur in parallel with new blood vessel growth and a certain degree of continuous cell death. As used herein, the term "ischemia" refers to any local tissue ischemia due to a decrease in blood inflow. The term "myocardial ischemia" refers to a circulatory disorder caused by coronary artery atherosclerosis and / or inadequate oxygen supply to the myocardium. For example, acute myocardial infarction represents an irreversible ischemic attack on myocardial tissue. This attack results in an occlusive (e.g., thrombotic or embolic) event in the coronary circulation, creating an environment in which myocardial metabolic demands exceed the oxygen supply to the myocardial tissue.

[0076] The terms "disease" or "disorder" refer to a change in the state of the body or a part of an organ that blocks or interferes with the performance of their functions and / or causes symptoms such as discomfort, dysfunction, pain, etc. or even death to the affected person or a person in contact with the affected person. A disease or disorder may also be related to pain, malaise, illness, malady, disorder, sickness, complaint, discomfort or affliction.

[0077] As used herein, the terms "treat", "treatment" or "treating" refer to both therapeutic and prophylactic means, the purpose of which is to prevent or delay the onset of a disease, such as delaying the manifestation of cardiac dysfunction, or to reduce at least one adverse effect or symptom of a cardiovascular symptom, disease or disorder, for example, any disorder characterized by inadequate or undesirable cardiac function. The adverse effects or symptoms of cardiac dysfunction are well known in the art and include, but are not limited to, dyspnea, chest pain, palpitations, dizziness, syncope, edema, cyanosis, pallor, fatigue and death. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced as defined by the term herein. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. That is, "treatment" includes not only improvement of symptoms or reduction of disease markers, but also cessation or delay or worsening of progression of symptoms predicted in the absence of treatment. Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the degree of the disease, stabilization of the disease state (e.g., not worsening), delay in the progression of the disease, improvement or alleviation of the disease state, and remission (partial or complete). "Treatment" may also mean extending the survival period compared to the survival period predicted in the absence of treatment. Persons in need of treatment include those already diagnosed with cardiac symptoms and those who are likely to develop cardiac symptoms due to genetic susceptibility or other factors such as weight, diet and health.

[0078] The term "scaffold" refers to a support structure for cells and / or cell materials. The support structure may be characterized by fibers and pores, but the scaffold is not limited to such compositions. For example, the scaffold may be in the form of a film, sponge or solution. The scaffold may be constructed from various materials such as, for example, fibers, peptides (e.g., recombinant peptides), lipids, carbohydrates, etc.

[0079] The present invention features engineered tissue compositions, e.g., three-dimensional tissue compositions that assist in cell growth and / or maintenance and / or differentiation, etc. The tissue compositions herein can be used for various purposes including in vitro applications and in vivo applications, e.g., surgical implants for treating diseases or conditions, research and testing, etc. The tissue compositions herein can be designed to withstand short-term or long-term storage (e.g., cryopreservation), transportation. In certain embodiments, the tissue composition can withstand a specific length of time at room temperature.

[0080] In summary, the engineered tissue compositions of the present invention comprise a scaffold and an extracellular matrix (ECM) material. The ECM material can be produced by an ECM-producing cell type such as, but not limited to, fibroblasts, any other suitable ECM-producing cell type, or a combination of ECM-producing cell types. The ECM material can be produced prior to application to the scaffold or the ECM material can be produced directly on the scaffold (or part is pre-made and part is produced directly on the scaffold). In certain embodiments, the ECM material is cell-free or contains cells (e.g., live cells, dead cells, combinations thereof). In certain embodiments, the ECM material is characterized by a bio-derived material, a synthetic-derived material or a combination thereof. In certain embodiments, the tissue composition further comprises ECM-producing cells, non-ECM-producing cells, and / or additional seeding cells, e.g., cells derived from a specific tissue of interest, stem cells, etc. In certain embodiments, the tissue composition further comprises additional factors such as growth factors, drugs or components that enhance cell attachment to the ECM, etc.

[0081] Figure 1 shows a non-limiting example of the components that make up the tissue composition of the present invention. For example, in certain embodiments, the tissue composition includes a scaffold and an ECM material (the ECM is acellular, e.g., no cells are present). In some embodiments, the ECM material includes dead cells, e.g., one live cell. In some embodiments, the ECM material is a by-product of live cells (such as currently living cells, dead cells, combinations thereof, etc.). In certain embodiments, the tissue composition includes a scaffold, an ECM material, and a cell population that is an ECM-producing cell, a non-ECM-producing cell, or a combination thereof. In certain embodiments, the tissue composition includes a scaffold, an ECM material, and a population of seeded cells (e.g., a cell type of interest). In certain embodiments, the tissue composition includes a scaffold, an ECM material, a cell population that is an ECM-producing cell, a non-ECM-producing cell, or a combination thereof, and a population of seeded cells (e.g., a cell type of interest). The foregoing examples of tissue compositions may include additional factors such as, for example, growth factors, drugs, compositions for enhancing cell attachment, etc.

[0082] <scaffold> The scaffolds used in the engineered tissue compositions herein can be constructed from various types of materials, materials of various sizes, various configurations, etc. In certain embodiments, the scaffold includes a plurality of fibers, and the fibers are arranged together (e.g., woven) to provide a plurality of pores disposed therebetween. The scaffold is not limited to a fibrous configuration. In certain embodiments, the scaffold includes a film, a sponge, a gel, a solution, etc. Non-limiting examples of alternative scaffolds are polymers, proteins, recombinant peptides, e.g., sponges or films constructed from human type I collagen. Sponges and films are further described below.

[0083] In an embodiment with a scaffold of fibrous structure, the fibers of the scaffold may have a substantially uniform diameter or the fibers may have various diameters. For example, in some embodiments, the scaffold includes fibers of a first fiber type and fibers of a second fiber type, and the first fiber type has a diameter different from that of the second fiber type. The scaffold may include more than the first fiber type and the second fiber type. For example, it should be noted that the scaffold may include the first fiber type, the second fiber type, and a third fiber type, or further a fourth fiber type, or further a fifth fiber type, and so on. A scaffold having multiple fiber types may be arranged in various configurations including, but not limited to, printed or spun fibers or knits or weaves having a fixed pattern of fiber type arrangement, printed or spun fibers or knits or weaves having a random fiber type arrangement, and the like. In some embodiments, small fibers extend from one or more large fibers. In some embodiments, the fibers are printed, spun, knitted, or woven loosely so that there is space for cells to be located therebetween. The fibers may be arranged in a configuration and / or orientation that allows a specific cell arrangement.

[0084] As described above, the fibers of the scaffold may be arranged in a woven or knitted configuration. The woven or knitted configuration may include, but is not limited to, plain weave, twill weave, alternating twill weave, knit weave, flat Dutch weave, Dutch twill weave, reverse Dutch weave, hexagonal watermark weave, warp knitting, or a combination thereof. For construction purposes, the scaffold may be woven, knitted, spun, extruded, or printed. In some embodiments, the scaffold has a ring-shaped configuration.

[0085] The fiber diameter may be of various sizes. For example, in some embodiments, at least a portion of the fibers have a diameter such as 5 μm to 100 μm, 10 μm to 500 μm, 100 μm to 1 mm (e.g., fiber bundle), etc. The present invention is not limited to the aforementioned fiber diameters.

[0086] In some embodiments, all of the fibers are constructed from a single material. In some embodiments, a portion of a fiber is constructed from a first material and a portion of the fiber is constructed from a second material different from the first material. In some embodiments, a portion of a fiber is constructed from a first material, a portion of the fiber is constructed from a second material different from the first material, and a portion of the fiber is constructed from a third material different from the first and second materials. The present invention is not limited to three different material types. The scaffold may be constructed from four, five, six, etc. different materials. In some embodiments, one or more fibers of the scaffold are constructed from two or more materials, for example, individual fibers are made from a combination of materials.

[0087] Materials used to construct scaffolds (e.g., scaffolds having other components such as fibers and / or peptides) include, but are not limited to, polyglycolide, polylactide, polyhydroxybutyrate, poly(anhydrides), poly(dioxanone), poly(trimethylene carbonate), polyglactin, poly(lactic acid), polyvinylidene fluoride, polyester, silicone, polyurethane, polymethylmethacrylate, polypropylene, polyethylene, polyglycaprone-25 monofilament, polycarbonate, polyamide, polyester, polystyrene, polyacrylate, polyvinyl, polytetrafluoroethylene, thermonox, nitrocellulose, collagen, fibrin, elastin, silk, metal, TMC, polyester, gelatin, dextran, protein, peptide, or combinations thereof. For example, in some embodiments, the first material comprises at least glycolide, lactide, and trimethylene carbonate, and the second material comprises at least lactide and trimethylene carbonate.

[0088] As described above, in certain embodiments, the scaffold is in the form of a film. In certain embodiments, the scaffold is in solution. In certain embodiments, the scaffold is a sponge. With respect to the sponge configuration, in some embodiments, the sponge has a substantially uniform diameter. In certain embodiments, the sponge has a non-uniform diameter. In certain embodiments, the sponge is layered having a plurality of levels or layers (e.g., a lower level, an upper level, etc.), where one layer may have a first uniformity and another layer may have a second uniformity. The sponge may be characterized by grooves or ridges. For example, one layer, such as an upper level or a lower layer, may be characterized by ridges or grooves.

[0089] The scaffold may be constructed from a variety of materials including, but not limited to, peptides (e.g., recombinant peptides), carbohydrates, lipids, etc. In certain embodiments, the scaffold includes both recombinant peptides and fibers (e.g., absorbent / degradable fibers, non-absorbent / degradable fibers, or combinations thereof). Non-limiting examples of recombinant peptides used to create a film scaffold or a sponge scaffold or a solution scaffold include type I collagen.

[0090] The scaffold may be constructed from bio-derived materials, synthetic-derived materials, or a combination of synthetic-derived and bio-derived materials.

[0091] The scaffold may be characterized by grooves and ridges, e.g., parallel grooves and ridges, organized or patterned grooves and ridges, randomly organized grooves and ridges. The configuration of the scaffold can serve to enhance cell growth or differentiation / maturation. The configuration of the scaffold can serve to organize cells in a particular direction or orientation, e.g., to align cells for a particular purpose such as muscle contraction.

[0092] The scaffold may be constructed with various thicknesses. For example, in some embodiments, the scaffold has a thickness of at least 25 μm. In some embodiments, the scaffold has a thickness of at least 40 μm. In some embodiments, the scaffold has a thickness of at least 50 μm. In some embodiments, the scaffold has a thickness of at least 100 μm. In some embodiments, the scaffold has a thickness of at least 250 μm. In some embodiments, the scaffold has a thickness of at least 500 μm. In some embodiments, the scaffold has a thickness of at least 1 mm. In some embodiments, the scaffold has a thickness of at least 2 mm. In some embodiments, the scaffold is 30 or 40 μm to 820 or 850 μm. In some embodiments, the scaffold has a thickness of 50 μm to 500 μm. In some embodiments, the scaffold has a thickness of 500 μm to 1 mm. In some embodiments, the scaffold has a thickness of 1 mm to 2 mm. The present invention is not limited to the aforementioned thicknesses, for example, 2 - 3 mm, 3 - 4 mm, 4 - 5 mm, 5 - 6 mm, etc. For example, the thickness may be 45 μm to 1070 μm, 45 - 499 μm, 246 - 1070 μm, etc. The aforementioned thicknesses can be applied to the engineered tissue composition (for example, a scaffold having an ECM with cells in some cases). For example, in certain embodiments, the engineered tissue composition has a thickness of 200 - 1000 microns.

[0093] Some or all of the components of the scaffold, such as fibers, peptides, etc., may be resorbable, absorbable, or degradable. For example, when proliferative ECM-producing cells replicate on and within the scaffold, one or more of the components may be resorbed / absorbed / degraded / dissolved. In an example where the scaffold includes two or more different fiber types, in certain embodiments, the fibers of the first fiber type may be resorbable / absorbable / degradable, and the fibers of the second fiber type may not be resorbable / absorbable / degradable. In certain embodiments, all of the scaffold components may be resorbable / absorbable / degradable to some extent, for example, the first fiber type may be resorbable / absorbable / degradable at a different rate than the second fiber type. The scaffold may be characterized by a degradation profile (time-limited, hierarchical) in which portions of the scaffold degrade at a particular time. Any suitable degradable material or combination thereof may achieve the desired degradation profile.

[0094] In the following examples, starting from the surgical implantation of the tissue composition, the degradation profile (when the scaffold is resorbed / absorbed / degraded) is measured. In some embodiments, some or all of the scaffold is resorbed / absorbed / degraded within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, etc. In certain embodiments, the scaffold is not completely resorbed / absorbed / degraded (the scaffold is non-absorbable).

[0095] The mechanical properties of the scaffold (such as stiffness, etc.) may change as the components of the scaffold (such as other materials like fibers and / or peptides) are resorbed / absorbed / degraded.

[0096] While not wishing to limit the present invention to any theory or mechanism, a scaffold that does not curl or fold significantly upon itself during surgical implantation can provide advantages such as, for example, making it easier for a surgeon to implant a tissue composition if it does not fold upon itself.

[0097] FIG. 2 shows a non-limiting example of the characteristics of a scaffold used in the tissue composition of the present invention. For example, as described above, in certain embodiments, the scaffold comprises absorbent / degradable fibers, non-absorbent / degradable fibers, or combinations thereof. In certain embodiments, the scaffold comprises a sponge (e.g., constructed from recombinant peptides, e.g., human type I collagen). In some embodiments, the scaffold comprises a film (e.g., constructed from recombinant peptides, e.g., human type I collagen). In some embodiments, the scaffold is in solution. In certain embodiments, the scaffold comprises both recombinant peptides (forming a film or sponge) and fibers (e.g., absorbent / degradable fibers, non-absorbent / degradable fibers, or combinations thereof). In certain embodiments, the scaffold comprises bio-derived components (e.g., naturally produced by cells), synthetic-derived components, or combinations thereof. In certain embodiments, the scaffold has additional features or properties that enhance cell and / or ECM attachment. For example, in certain embodiments, the scaffold has a hydrophilicity adapted to allow cell and / or ECM attachment. In certain embodiments, the scaffold has a surface roughness adapted to allow cell and / or ECM attachment. Non-limiting examples of compositions or features that can enhance cell attachment can include specific textures or roughnesses, specific hydrophilic compounds or features, specific ligands, RGD-coated materials, and the like.

[0098] <Mechanical properties of the scaffold> The scaffold can be anisotropic, e.g., the mechanical properties of the scaffold may be different in one direction than in other directions. Or, the scaffold can be isotropic. The scaffold has various mechanical properties related to strength and flexibility.

[0099] Generally, there is a difference between the properties of the substrate (e.g., scaffold only) and the properties of the tissue composition (having fibroblasts and ECM). For example, if the scaffold is characterized by degradable components (e.g., fibers and / or peptides) (e.g., fibers or peptides that degrade during culture), the scaffold can be stiffer than what is ultimately used as the tissue composition (e.g., for in vivo use). In some embodiments, the scaffold can be selected from materials in the GPa range, but when the tissue composition is implanted, it can be in the MPa range. Similarly, the scaffold can be selected in the MPa range, but when the tissue composition is implanted, it can be in the kPa range. In some embodiments, the implanted material (properties of the tissue composition) can be less than 100 MPa.

[0100] The elastic modulus (rigidity) can be from 20 kPa to 100 GPa. The scaffold may have a bursting strength such as 20 N / cm to 200 N / cm, 50 N / cm to 100 N / cm, 75 N / cm to 90 N / cm, etc. The scaffold may have a parallel / vertical tear resistance of 10 N / 5 N to 50 N / 40 N. The scaffold may have a parallel / vertical tear resistance of 30 / 31 N to 350 N / 36 N. In some embodiments, the scaffold has a longitudinal rigidity of 1 N / mm to 50 N / mm. In some embodiments, the scaffold has a longitudinal rigidity of 4 N / mm to 30 N / mm. In some embodiments, the scaffold has a transverse rigidity of 0.5 N / mm to 5 N / mm. In some embodiments, the scaffold has a longitudinal rigidity different from the transverse rigidity. In some embodiments, the scaffold has the same longitudinal rigidity as the transverse rigidity. In some embodiments, the scaffold has a maximum longitudinal force of 10 kPa to 100 MPa. In some embodiments, the scaffold has a maximum transverse force of 10 kPa to 100 MPa. In some embodiments, the scaffold has a rigidity of 5 to 3000 kPa. In some embodiments, the scaffold has a rigidity of 3000 to 4600 kPa. In some embodiments, the scaffold has a rigidity greater than 4600 kPa. The scaffold may be characterized by fibers having different rigidities, for example, fibers having a rigidity of 1 to 20 MPa and fibers having a rigidity exceeding 10 MPa. As described above, the rigidity of the scaffold before culturing cells may be different from that of the final product, for example, the tissue composition. In some embodiments, the tissue composition (e.g., the final product) may be from 20 kPa to 50 MPa, but the present invention is not limited to these values.

[0101] Mechanical properties (e.g., rigidity, etc.) can change during processes such as manufacturing, cell growth, cell differentiation, transplantation, etc. The present invention is not limited to the mechanical property parameters described herein.

[0102] In certain embodiments, the engineered tissue composition enables electrical signal transmission.

[0103] <scaffold pores> The density of the pores, for example, the number of pores per unit area (e.g., 1 mm of the scaffold) 2The number of pores per hit) can help the cells grow efficiently across the entire scaffold. In some embodiments, the pores are dense and / or are arranged on top of slightly overlapping pores. However, the present invention is not limited to dense pores or pores arranged on top of slightly overlapping pores. Furthermore, the pore density can depend on the type of material used for the scaffold, the thickness of the scaffold, the type of weave of the scaffold, etc. In some embodiments, the scaffold is 1 cm 2 has 1 to 1,000 pores per hit. In some embodiments, the scaffold is 1 cm 2 has 10 to 1,000 pores per hit. In some embodiments, the scaffold is 1 cm 2 has 100 to 1,000 pores per hit. In some embodiments, the scaffold is 1 mm 2 has 100 to 1,000 pores per hit. In some embodiments, the scaffold is 1 mm 2 has 100 to 500 pores per hit. In some embodiments, the scaffold is 1 mm 2 has 200 to 1,000 pores per hit. The pore density can also change over time. For example, in some embodiments, the components of the scaffold (e.g., fibers and / or peptides, etc.) decompose (e.g., biodegrade, are resorbed, are absorbed, etc.), which can result in a pore density different from what was originally present in the scaffold.

[0104] In some embodiments, the pores are uniformly arranged throughout the scaffold. In some embodiments, the pores are randomly arranged throughout the scaffold. In some embodiments, the pores are arranged in a pattern throughout the scaffold. The pores can be of various shapes (e.g., cross-sectional shape), for example, rectangular, rounded rectangular, but not limited to, other geometric shapes including hexagonal, circular, elliptical, figure-eight, etc., or irregular shapes or combinations of shapes. Thus, the pores can be described as having a height, width, length, diameter, area, etc. In some embodiments, the pores of the scaffold (e.g., one or more of the pores) are 0.1 μm 2 ~100 μm 2 、1 μm 2 ~1000 μm 2 、100 μm2 ~5000 μm 2 、0.1 μm 2 ~0.01 mm 2 、0.1 μm 2 ~0.1 mm 2 、0.1 μm 2 ~1 mm 2 、0.1 μm 2 ~2 mm 2 、0.1 μm 2 ~10 mm 2 and have areas such as these. In some embodiments, the average pore diameter is about 104,540 μm 2 or the like. In some embodiments, the average pore diameter is 5,000 μm 2 ~1,000,000 μm 2 or more. In some embodiments, the pores have a diameter of 50 μm to 90 μm. In some embodiments, the pores have a diameter of 50 μm to 200 μm. In some embodiments, the pores have a diameter such as 200 μm to 400 μm, 200 μm to 500 μm, etc. In some embodiments, the pores have a diameter of 500 μm to 1000 μm. In some embodiments, the pores have a diameter of 500 μm to 1500 μm. In some embodiments, the pores have a diameter of 800 μm to 1200 μm. In some embodiments, the pores have a diameter of 800 μm to 1000 μm.

[0105] In some embodiments, the scaffold retains at least 50% of its mechanical strength over at least 4 weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, etc.

[0106] In some embodiments, the scaffold degrades in 3 weeks or more. In some embodiments, the scaffold degrades in 4 weeks or more after transplantation. In some embodiments, the scaffold degrades in 6 weeks or more after transplantation. In some embodiments, the scaffold degrades in 8 weeks or more after transplantation. In some embodiments, the scaffold degrades in 10 weeks or more after transplantation.

[0107] The scaffold may be characterized by two different fibers that (relative to each other) one is resorbed quickly and the other is resorbed slowly to enable two-stage resorption.

[0108] <Extracellular matrix material> As described above, the engineered tissue composition of the present invention comprises an extracellular matrix (ECM) material. The ECM can be produced by ECM-producing cells, but the present invention is not limited to ECM produced by ECM-producing cells. In certain embodiments, the ECM comprises only synthetic-derived materials. In certain embodiments, the ECM comprises a biologically-derived material (e.g., ECM produced by ECM-producing cells). In certain embodiments, the ECM comprises a combination of a biologically-derived material and a synthetic-derived material. As an example, materials such as synthetically produced collagen and fibronectin (and the like, e.g., materials described herein) may be combined to form an ECM without the need for ECM-producing cells.

[0109] In some embodiments, 0-10% (according to area or volume) of the ECM or tissue composition is synthetic-derived. In some embodiments, 10-25% (according to area or volume) of the ECM or tissue composition is synthetic-derived. In some embodiments, 25-40% (according to area or volume) of the ECM or tissue composition is synthetic-derived. In some embodiments, 40-60% (according to area or volume) of the ECM or tissue composition is synthetic-derived. In some embodiments, 60-75% (according to area or volume) of the ECM or tissue composition is synthetic-derived. In some embodiments, 75-90% (according to area or volume) of the ECM or tissue composition is synthetic-derived. In some embodiments, 50-95% (according to area or volume) of the ECM or tissue composition is synthetic-derived.

[0110] Components of the ECM include, but are not limited to, collagen (e.g., type I collagen, type III collagen), elastin, fibronectin, laminin, tenascin, proteoglycan, glycosaminoglycan (e.g., Veriscan, Decorin, Betaglycan, Syndecan), etc. (see Naughton, 2002, Ann N Y Acad Sci, 961:372-85). In some embodiments, exogenous gelatin is deposited on the scaffold. In some embodiments, exogenous collagen, fibronectin, fibrin are added (or other suitable ECM components).

[0111] Although not wishing to limit the present invention to any theory or mechanism, a specific amount of ECM is thought to be beneficial for the engineered tissue composition to be effective (e.g., effective for seeding cells, differentiating seeding cells, surgical implantation, etc.).

[0112] In certain embodiments, ECM is produced by seeding ECM-producing cells within and / or on the scaffold (e.g., on and / or within the pores and components of the scaffold), and the ECM-producing cells subsequently proliferate, expand within and / or on and through the scaffold, and produce ECM. The ECM-producing cells may move along the components of the scaffold (e.g., fibers, peptides, etc.) and further within the pores (e.g., together with the produced ECM). In the seeding process of ECM-producing cells (and / or other cells herein), various steps may be utilized to enhance cell attachment to the scaffold, including, but not limited to, centrifugation or other suitable forces (e.g., electrical forces) or combinations thereof.

[0113] In certain embodiments, the ECM is generated by ECM-producing cells before being applied to the scaffold. As an example, following the generation of the ECM by the ECM-producing cells, an ECM material may be applied to the scaffold together with the ECM-producing cells. Alternatively, in certain embodiments, the ECM material generated by the ECM-producing cells may be decellularized and subsequently applied to the scaffold. In certain embodiments, a cell population is seeded into and / or onto the scaffold prior to the application of the ECM.

[0114] It should be noted that the ECM-producing cells may be alive or dead (or a combination of live and dead cells). For example, the tissue composition may include a scaffold, an ECM, and live ECM-producing cells (e.g., fibroblasts or other ECM-producing cell types or combinations thereof). In certain embodiments, the tissue composition includes a scaffold, an ECM, and dead ECM-producing cells. In certain embodiments, the tissue composition includes a scaffold, an ECM, a population of live ECM-producing cells, and a population of dead ECM-producing cells.

[0115] The ECM-producing cells can be fibroblasts, such as human dermal fibroblasts. However, the present invention is not limited to fibroblasts. In some embodiments, the ECM-producing cells include fibroblasts, osteoblasts, chondrocytes, glial cells, neural stem cells, cardiomyocytes, myofibroblasts, etc., or combinations thereof. As described herein, in certain embodiments, the ECM-producing cells can be genetically engineered to produce, for example, a specific ECM and / or growth factor, and / or can be manipulated to proliferate.

[0116] The ECM-producing cells may be derived from a suitable source or host. For example, in some embodiments, the ECM-producing cells are human cells. In some embodiments, the ECM-producing cells are primate cells. In some embodiments, the ECM-producing cells are cells derived from mice, rats, goats, rabbits, horses, dogs, cats, or any other host. In certain embodiments, the ECM-producing cells are genetically modified to be universal (non-immunogenic) cells.

[0117] As described above, the ECM-producing cells can be fibroblasts. In certain embodiments, the fibroblasts are iPSC-derived fibroblasts. In some embodiments, the fibroblasts are skin-derived fibroblasts, such as dermal neonatal fibroblasts. In some embodiments, the fibroblasts are blood-derived fibroblasts. In some embodiments, the fibroblasts are heart-derived, muscle-derived, liver-derived, pancreas-derived, adipose tissue-derived, central nervous system (CNS)-derived, or lung-derived fibroblasts.

[0118] In certain embodiments, the ECM-producing cells are wild-type cells. In certain embodiments, the ECM-producing cells are genetically modified, for example, engineered to express one or more genes of interest. In certain embodiments, the ECM-producing cells are a combination of wild-type cells and genetically modified cells.

[0119] The ECM-producing cells can form a layer on the scaffold. (In certain embodiments, the ECM cells seeded on / within the scaffold are already within the ECM. In certain embodiments, the cells within the ECM are alive and / or dead.) In some embodiments, the ECM-producing cells are disposed within and / or on the scaffold. The ECM-producing cells can exist as an aggregate within or on the scaffold, form one or more layers on the scaffold, introduce an alternative arrangement within or on the scaffold, or combinations thereof. The tissue composition of the present invention may have a layer of cells, for example, 3 to 500 cell layers. The cell layer may be composed of ECM-producing cells, non-ECM-producing cells, or combinations thereof.

[0120] When the ECM-producing cells proliferate and produce ECM within and on the scaffold, the ECM-producing cells and / or the ECM fill at least a portion of the pores of the scaffold. The ECM-producing cells and / or the ECM can then fill all of the pores of the scaffold. It should be noted that in some embodiments, a cell-free ECM material that fills a part or all of the pore area of the scaffold is used.

[0121] Examples of the tissue compositions of the present invention are shown in FIGS. 3A, 3B, 3C, 3D, 3E and 3F. FIG. 3A shows fibroblasts and extracellular matrix (ECM) between and surrounding fiber bundles of a particular scaffold. The fibroblasts proliferate and produce ECM to fill the pores, providing an additional support structure for the attachment and proliferation of additional cell types throughout the pores. FIGS. 3B and 3C show scaffolds made of recombinant peptides (collagen I) filled with human dermal fibroblasts and locally seeded with iPSC-derived cardiomyocytes. The fibroblasts penetrate the pores of the scaffold. The cardiomyocytes are present on the surface of the monolayer culture. FIG. 3D shows a scaffold made of a recombinant peptide (collagen I) in a "hollow" configuration. The scaffold is locally seeded with human dermal fibroblasts. The fibroblasts do not embed in the scaffold, but rather form cell layers on the top and bottom. One side also contains iPSC-derived cardiomyocytes within the monolayer culture. FIG. 3E shows a tissue composition (magnification 20x) containing fibroblasts and cardiomyocytes seeded on a scaffold made of polyglycolic acid (PGA) and trimethylene carbonate (TMC). FIG. 3F shows a tissue composition (magnification 20x) containing fibroblasts and cardiomyocytes seeded on a scaffold made of polyglycolic acid (PGA).

[0122] Regarding tissue compositions containing scaffolds made of recombinant peptides, in certain embodiments, the recombinant peptide scaffold is 300 to 500 μm thick. In certain embodiments, the cardiomyocytes are later 20 to 50 μm thick. In certain embodiments, the pores are 50 to 90 μm in diameter. The present invention is not limited to the aforementioned dimensions.

[0123] In some embodiments, 100% of the pore area is filled by ECM-producing cells and / or ECM. In some embodiments, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 50%, etc. of the pore area is filled by ECM-producing cells and / or ECM.

[0124] In some embodiments, at least 50% of the pore area is filled (filled by ECM-producing cells and / or ECM) within 2 to 10 days, within 3 to 10 days, within 4 to 10 days, within 5 to 10 days, within 8 to 10 days, within 5 to 15 days, within 8 to 15 days, within 10 to 15 days, within 12 to 15 days, within 5 to 25 days, within 10 to 25 days, within 15 to 25 days, within 20 to 25 days, etc. after seeding the ECM-producing cells.

[0125] The time taken for the pores to be filled may depend on certain factors, such as the method of seeding the cells, for example, whether the tissue composition is agitated during the seeding process, the agitation speed, whether centrifugation is used during the seeding process, etc. As an example, in certain embodiments, using agitation, at least 50% of the pore area is filled (filled by ECM-producing cells and / or ECM) within 5 to 10 days after seeding the ECM-producing cells, whereas in certain embodiments without using agitation, at least 50% of the pore area is filled (filled by ECM-producing cells and / or ECM) within 14 to 17 days after seeding the ECM-producing cells. The foregoing examples are not meant to limit the present invention in any way and serve only as an example to illustrate that agitation may have the potential to accelerate the time required to fill at least 50% of the pore area.

[0126] When ECM-producing cells proliferate and produce ECM within the scaffold, the cells undergo morphological changes (see, e.g., FIGS. 3A and 4). FIG. 4 shows images of tissue compositions over several days (1, 14, 17, and 28 days) after seeding human neonatal dermal fibroblasts (HDFs), where the pores are filled over time. At the 17-day time point, the spindle shape of the HDFs is elongated. By 28 days, the cell population becomes homogeneous, and the cell population does not show a spindle shape but instead shows a pebble-like shape. The present invention is not limited to the time frame of the morphological changes described in FIG. 4. For example, the elongated spindle shape can be formed within less than 17 days, such as within 16 days, within 15 days, within 14 days, within 13 days, within 12 days, within 11 days, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, less than 4 days, within 16 days or less, within 15 days or less, within 14 days or less, within 13 days or less, within 12 days or less, within 11 days or less, within 10 days or less, within 9 days or less, within 8 days or less, within 7 days or less, within 6 days or less, within 5 days or less, etc. In certain embodiments, the pebble-like shape can be formed within less than 28 days, such as within 27 days, within 26 days, within 25 days, within 24 days, within 23 days, within 22 days, within 21 days, within 20 days, within 19 days, within 18 days, within 17 days, within 16 days, within 15 days, within 14 days, within 13 days, within 12 days, within 11 days, within 10 days, within 9 days, within 8 days, less than 8 days, within 26 days or less, within 25 days or less, within 24 days or less, within 23 days or less, within 22 days or less, within 21 days or less, within 20 days or less, within 19 days or less, within 18 days or less, within 17 days or less, within 16 days or less, within 15 days or less, within 14 days or less, within 13 days or less, within 12 days or less, within 11 days or less, within 10 days or less, within 9 days or less, within 8 days or less, etc. The time required for the morphological changes to occur can depend on the manufacturing method.

[0127] The engineered tissue composition has a final density of 5x10 5 cells / cm 2 ~5x10 6 cells / cm 2 of ECM-producing cells. In some embodiments, the engineered tissue composition has 1×10 5 cells / cm 2 ~1×10 7cells / cm 2 has a final density of ECM-producing cells. In some embodiments, the engineered tissue composition has a final density of 1×10 4 cells / cm 2 ~1×10 8 cells / cm 2 of ECM-producing cells. The present invention is not limited to the aforementioned final density of ECM-producing cells.

[0128] In some embodiments, additional factors are added to the ECM, scaffold, and / or ECM-producing cells. Additional factors may be added to enhance the production of ECM, or for other purposes, such as enhancing cell growth, maintenance, and / or differentiation, enhancing cell and / or ECM attachment, etc. By way of non-limiting example, ascorbic acid, which helps to promote ECM deposition, may be added. In some embodiments, the additional factor is for enhancing the attachment of ECM-producing cells and / or for enhancing the attachment of seeded cells.

[0129] In some embodiments, exogenous growth factors are added together with or in combination with the ECM and / or ECM-producing cells and / or scaffolds. Growth factors (e.g., those secreted by fibroblasts, those added exogenously) can improve the growth (of fibroblasts themselves, seeded cells), seeding efficiency (of fibroblasts themselves, seeded cells), incorporation of fibroblasts into scaffolds, ECM production, etc. Growth factors include, but are not limited to, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), angiopoietin-1 matrix deposition factors (e.g., transforming growth factor (TGF-b1), transforming growth factor (TBG-b3)), cell division promoting factors (e.g., platelet-derived growth factor A (PDGF-A), insulin-like growth factor 1 (IGF-1), erythropoietin (EPO), heparin-binding epidermal growth factor (HBEGF), platelet-derived growth factor a (TGFa)), angiogenesis factors (e.g., angiogenin, angiopoietin-2), endothelial growth factor, leptin, platelet-derived growth factor BB (PDGF-BB), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), Secreted protein acid and rich in cysteine (SPARC), interleukin 6 (IL-6), interleukin 8 (IL-8), inflammatory cytokines (e.g., interferon γ, interleukin 1a, interleukin 1b, interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein 1, granulocyte colony-stimulating factor (GCSF), tumor necrosis factor a (TNFa)), etc. (See Naughton, 2002, Ann N Y Acad Sci, 961:372-85; Lancaster et al., 2010, Tissue Eng Part A, 16(10):3065-73). Growth factors derived from other cells, such as therapeutic cells, may also be added. For example, cardiomyocytes secrete specific factors for stimulating in vitro signaling, maturation, myokine activity, and in vivo myogenesis, etc., and the above growth factors can also be added exogenously or via seeding of another cell type.

[0130] In some embodiments, the scaffold constitutes 1 to 70% of the tissue composition by volume. In some embodiments, the scaffold constitutes 1 to 80% of the tissue composition by volume. In some embodiments, the scaffold constitutes 5 to 50% of the tissue composition by volume. In some embodiments, the ECM and ECM-producing cells constitute at least 20% of the tissue composition by volume. In some embodiments, the ECM and ECM-producing cells constitute 20 to 50% of the tissue composition by volume. In some embodiments, the ECM and ECM-producing cells constitute 50 to 75% of the tissue composition by volume. In some embodiments, the ECM and ECM-producing cells constitute 50 to 99% of the tissue composition by volume. In some embodiments, the ECM and ECM-producing cells constitute 30 to 99% of the tissue composition by volume. The amount of the tissue composition (versus the scaffold) composed of the ECM and ECM-producing cells can depend on various factors, such as the amount of ECM-producing cells seeded at the start of the culture, the predicted degradation rate of the scaffold material, the amount of the seed cell population, etc. For example, the amount of the tissue composition composed of the seed cells can range from 3 to 60% of the tissue composition.

[0131] As an example, to evaluate the mass of cells and ECM relative to the scaffold mass, a specific tissue composition (including ECM and ECM-producing cells) constructed with a specific scaffold material (e.g., double fiber / slowly degrading scaffold, lactide scaffold, polyglactin scaffold) was analyzed. First, the scaffold was weighed, and then the tissue composition (fully cultured HDF scaffold composition) was weighed both in the wet state and the dry state. For the double fiber scaffold cultured with cells and ECM, the wet weight was 0.058 g / cm2 and the dry weight was 0.028 g / cm2, whereas for the double fiber scaffold alone, the wet weight was 0.021 g / cm2 and the dry weight was 0.016 g / cm2. Thus, the mass of the ECM and ECM-depositing cells was 0.037 g / cm2. The dry weight was 0.012 g / cm2.

[0132] The manipulated tissue composition can generally be flat. The manipulated tissue composition may itself have a curl, or the microscopic features of the tissue composition may have convex or concave components. While not wishing to limit the present invention to any theory or mechanism, the concave structure may potentially be beneficial for cell seeding, attachment, and incorporation.

[0133] Figure 5 shows non-limiting examples of the characteristics of the ECM used in the tissue compositions of the present invention. For example, as described above, in certain embodiments, the ECM is of biological origin (e.g., from ECM-producing cells), synthetic origin, or a combination thereof. In certain embodiments, the ECM-producing cells are wild-type, genetically modified, or the ECM-producing cells are characterized by a population of wild-type cells and a population of genetically modified cells. In certain embodiments, the ECM is cell-free. In certain embodiments, the ECM includes living or dead ECM-producing cells characterized by a population of living cells and a population of dead cells. In certain embodiments, the ECM includes growth factors, drugs, and / or other compositions useful for ECM production, cell attachment, ECM attachment to a scaffold, etc.

[0134] <Seeded cells> The tissue compositions of the present invention may include seeded cells. The seeded cells can be of any suitable cell type (and from any suitable host, or genetically modified to be a universal cell). For example, in some embodiments, the seeded cells are human cells. In some embodiments, the seeded cells are mouse cells, rat cells, goat cells, rabbit cells, horse cells, dog, cat, or cells from any other host. The seeded cells can be related to blood, heart tissue, skeletal muscle tissue, liver tissue, pancreatic tissue, lung tissue, bone tissue, umbilical cord tissue, endothelial tissue, central nervous system tissue, gastrointestinal tissue, endocrine cells, paracrine cells, enzyme-secreting cells, their stem cells, their progenitor cells, prokaryotes, eukaryotes, or other oxygen-releasing particles, or combinations thereof. Note that the seeded cells can be derived from the same donor as the ECM-producing cells. In certain embodiments, the seeded cells are derived from a donor different from the donor of the ECM-producing cells.

[0135] The seeded cells can be proliferative, non-proliferative, or a combination thereof. The seeded cells can be stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem cells), primary cells, progenitor cells, etc. For example, the seeded cells can be human induced pluripotent stem cell-derived cells (hiPSC), e.g., human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). The seeded cells can be terminally differentiated cells, e.g., terminally differentiated cardiomyocytes, hepatocytes, beta cells, endoderm, smooth muscle cells, salivary cells, etc.

[0136] The seeded cells can be mature or immature. For example, cardiac progenitor cells can express one or more markers such as, but not limited to, MESP1, GATA4, ISL1, NKX2.5, etc., or a combination thereof. Cardiomyocytes during the developmental stage can express one or more markers such as, but not limited to, CTNT, MHC, MLC, sarcomeric actin, etc., or a combination thereof. iPSCs can express one or more markers such as, but not limited to, Oct-4, LIN-23, etc., or a combination thereof.

[0137] The seeded cells can be wild-type cells. In certain embodiments, the seeded cells are genetically modified to express one or more genes of interest. In certain embodiments, the seeded cells include a population of wild-type cells and a population of genetically modified cells. For example, the genes of interest can include, but are not limited to, thymosin β-4 (TB4), akt murine thyoma viral oncogene homolog (AKT1), stromal cell-derived factor 1α (SDF-1), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), erythropoietin (EPO), etc. The present invention is not limited to the aforementioned genes, nor is it limited to genetically modified cells that express genes for specific therapeutic purposes. In some embodiments, the seeded cells can include additional cells or particles such as prokaryotes, eukaryotes, or particles engineered to generate oxygen spontaneously or in response to an external stimulus.

[0138] Cells obtained from a specific disease state or genetic state may be seeded. For example, the seed cells can be cells having an abnormality associated with a specific disease state or symptom. In some embodiments, the seed cells are cells derived from tissue in an abnormal state (e.g., after stress or trauma or an event such as myocardial infarction). Non-limiting examples of cells having a specific gene mutation or cells associated with a specific disease state or symptom include congenital cardiomyopathy, acquired cardiomyopathy, arrhythmogenic cardiomyopathy (e.g., long QT syndrome, short QT syndrome (SQTS), Brugada syndrome, catecholamine-induced polymorphic ventricular tachycardia (CPVT), arrhythmogenic right ventricular cardiomyopathy (ARVC)), dilated cardiomyopathy (e.g., hypertrophic cardiomyopathy, left ventricular non-compaction, transthyretin amyloidosis, hereditary hemochromatosis, RASopathy (also known as Noonan spectrum disorder), heart failure, etc.). In some embodiments, the disease state or symptom is an acquired symptom such as dilated ischemic cardiomyopathy and non-ischemic cardiomyopathy, hypertensive heart disease. In some embodiments, the disease state or symptom is an acquired congenital disease or congenital + arrhythmia. In some embodiments, the disease state or symptom is diabetes, cancer, muscular dystrophy, congenital, hereditary or acquired symptoms affecting the digestive tract, symptoms affecting skeletal muscle, smooth muscle, etc. The present invention is not limited to the foregoing symptoms.

[0139] The ECM-producing cells and / or ECM and / or other factors of the tissue composition can (where appropriate) cause differentiation and / or maturation of the seeded cells. Growth factors (e.g., fibroblast-derived, exogenous) can help improve one or more of seeding, incorporation into the scaffold, proliferation, and differentiation of the seeded cells in vitro or in vivo. In certain cells having the ability to differentiate into two or more cell types, the microenvironment of the tissue composition (e.g., ECM-producing cells, ECM, growth factors, etc.) can help promote the differentiation pathway. In some embodiments, exogenous factors are added to enhance differentiation in a specific direction.

[0140] The tissue composition of the present invention may further include enhancing cells. Non-limiting examples of enhancing cells may include secretory cells, paracrine cells, enzyme cells, β cells, gastrointestinal cells, or combinations thereof. The enhancing cells may be in a specific ratio to the seeded cells and / or ECM-producing cells. The ratio of the cells may depend on the cell type and the desired result. This ratio may also depend on the clustering (e.g., cell bundles) of the enhancing cells. Alternatively, this ratio may depend on the proliferation of the cells (the proliferation of the cells ultimately affects this ratio). The spheroids / embryoid bodies may be prepared in advance or generated spontaneously during the preparation. In some embodiments, the ratio of the enhancing cells to the seeded cells or ECM-producing cells is from 1:10 to 10:1. In some embodiments, the ratio of the enhancing cells to the seeded cells or ECM-producing cells is from 1:5 to 5:1. In some embodiments, the ratio of the enhancing cells to the seeded cells or ECM-producing cells is from 1:20 to 20:1. In some embodiments, the ratio of the enhancing cells to the seeded cells or ECM-producing cells is from 1:25 to 25:1. In some embodiments, the ratio of the enhancing cells to the seeded cells or ECM-producing cells is from 1:50 to 50:1. In some embodiments, the ratio of the enhancing cells to the seeded cells or ECM-producing cells is from 1:100 to 100:1. The present invention is not limited to the aforementioned ratios.

[0141] The tissue composition of the present invention may further include proliferative cells different from the ECM-producing cells, such as adherent proliferative cells, such as mesenchymal stem cells, pre-vascular cells, endothelial cells, progenitor cells, and the like. It should be noted that there is a specific microenvironment for each cell type. Therefore, it is highly likely that each cell type used provides a specific niche to facilitate the escape or incorporation of additional cell types.

[0142] As described above, the seed cells can be cardiomyocytes or cardiomyocytes derived from human induced pluripotent stem cells. In such tissue compositions, the cardiomyocytes can develop, for example, in a synchronized manner and contract spontaneously. The cardiomyocytes enable the propagation of electrical signals. In some embodiments, the scaffold directs contraction. As described above, in some embodiments, the fibers of the scaffold form grooves, which can help the scaffold direct seeding or contraction of the cells.

[0143] In the example of cardiomyocytes, in certain embodiments, the cardiomyocytes contract at a rate of 0 beats per minute to 30 beats per minute, 20 beats per minute to 60 beats per minute, 30 beats per minute to 50 beats per minute, 30 beats per minute to 200 beats per minute, 40 beats per minute to 270 beats per minute, 20 beats per minute to 300 beats per minute, 40 beats per minute to 80 beats per minute, etc. For example, it should be noted that factors such as, but not limited to, temperature, time after cryopreservation, time after seeding, etc. can affect the heart rate. The heart rate can be zero or can vary during culture.

[0144] The seed cells can be present in a specific ratio relative to the ECM-producing cells. The exact ratio can depend on the cell type of the seed cells. For example, in some embodiments, the ratio of seed cells to ECM-producing cells is 1:10 to 10:1. In some embodiments, the ratio of seeded cells to ECM-producing cells is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc. In some embodiments, the ratio of seed cells to ECM-producing cells is 1:5 to 5:1. In some embodiments, the ratio of seed cells to ECM-producing cells is 1:20 to 20:1. In some embodiments, the ratio of seed cells to ECM-producing cells is 1:25 to 25:1. In some embodiments, the ratio of seed cells to ECM-producing cells is 1:50 to 50:1. In some embodiments, the ratio of seed cells to ECM-producing cells is 1:100 to 100:1. In some embodiments, in scaffolds such as the hollow one in Figure 3D, the ratio of seeded cells to ECM-producing cells is 1:1. Similarly, the amount of seed cells deposited on the tissue composition can depend on the cell type of the seed cells. In some embodiments, 0.5×10 6 cells / cm 2 ~5×106 cells / cm 2 The seeding cells are seeded so as to have a final density of. In some embodiments, 1×10 4 cells / cm 2 ~1×10 7 cells / cm 2 The seeding cells are seeded so as to have a final density of. In some embodiments, 1×10 2 cells / cm 2 ~1×10 7 cells / cm 2 The seeding cells are seeded so as to have a final density of.

[0145] The arrangement of the seeding cells may be a layer. Alternatively, the arrangement of the seeding cells on the scaffold may be, for example, a bundle, aggregate or group of densely packed cells such as embryoid bodies, cardiospheres (or a combination of a layer and a bundle). The cell bundles may be of various sizes and may be mixed with single or layered cells. In certain embodiments, the tissue composition is characterized by 3 to 500 cell layers.

[0146] In some embodiments, cell bundles (e.g., embryoid bodies) are seeded, for example, with the cells pre-clustered prior to seeding. In some embodiments, the cells may form the above bundles or aggregates after the seeding process. Such bundles of cells (embryoid bodies) may exhibit their own microenvironment.

[0147] FIG. 6 shows a non-limiting example of the characteristics of seeding cells (which may include a single population of cells or a combination of cell populations) that can be used in the three-dimensional tissue compositions of the present specification.

[0148] <Characteristics and Variations of Tissue Compositions> In certain embodiments, a tissue composition includes a biomaterial (e.g., a scaffold only, a scaffold with seeded cells and no ECM-producing cells, a scaffold with seeded cells and ECM-producing cells, a scaffold with ECM-producing cells, etc.) and a proliferative cell population. For example, a tissue composition may include a scaffold and ECM (regardless of the presence or absence of ECM-producing cells) and a proliferative cell population. In certain embodiments, a tissue composition includes a biomaterial and a non-proliferative cell population. For example, a tissue composition may include a scaffold and ECM (regardless of the presence or absence of ECM-producing cells) and a non-proliferative cell population. A tissue composition can have a cell layer that is 3 to 500 cell layers thick, but the invention is not limited to this configuration or range of cell layers. The tissue compositions herein can be cultured with factors (e.g., FGF), other proliferative cytokines, growth factors, or combinations thereof to achieve a desired thickness.

[0149] The biomaterial can be absorbable, non-absorbable, or a combination thereof. The biomaterial can be characterized as a synthetically derived material, a biologically derived material, or a combination thereof. The biomaterial may include pre-seeded ECM-producing cells (e.g., fibroblasts). In certain embodiments, the biomaterial does not include ECM-producing cells (e.g., fibroblasts). In certain embodiments, the biomaterial includes pores. In certain embodiments, the biomaterial does not include pores. In certain embodiments, the biomaterial can be characterized by ligands, antibodies, magnetic-based particles, etc. for attracting cells to the biomaterial, or combinations thereof. In certain embodiments, the culture plates, bioreactors, or other materials used to generate the tissue composition are characterized by an anti-adhesion material on at least a portion of their surface to deter cells from adhering to the surface and instead cause them to adhere to the biomaterial.

[0150] Non-limiting examples of proliferative cells include cardiomyocyte progenitor cells, fibroblasts, mesenchymal stem cells (MSCs), and other progenitor cells (e.g., skeletal muscle progenitor cells, smooth muscle progenitor cells, neural progenitor cells, hepatic progenitor cells, etc.). Non-limiting examples of non-proliferative cells include cardiomyocytes, neurons, pancreatic cells, etc.

[0151] The tissue compositions of the present invention, for example, those produced by proliferative cells, can be induced to continue growing when seeded on a scaffold. Specific compositions (e.g., growth factors, peptides, etc.) may be used in this process. The tissue compositions of the present invention, for example, those produced by proliferative cells, may be capable of growing on a construct and can be induced to differentiate into a specific cell type at a specific time. For example, in a tissue composition containing myocardial progenitor cells, the myocardial progenitor cells can be promoted to differentiate when appropriate factors are introduced.

[0152] In certain embodiments, another cell population, such as an endothelial cell population, a cardiomyocyte population, a mesenchymal stem cell (MSC) population, etc., may be seeded onto the tissue composition (e.g., that produced by the above-mentioned proliferative cells).

[0153] The tissue compositions herein can be constructed in various ways. For example, in certain embodiments, a cell sheet or spheroid is generated and then transferred to a biomaterial. The cell sheet can be generated in various ways. For example, the cell sheet may be generated by seeding cells onto a temperature-responsive plate, a low-adhesion plate, or a plate having a composition (e.g., a ligand) that allows detachment of cells or tissues at a selected time. The temperature-responsive plate is designed to release adherent cells when placed at a specific temperature (e.g., 20 - 25°C). When the seeded cells reach their appropriate confluence and / or morphology, the cells can be separated from the plate (e.g., a temperature-responsive plate, a low-adhesion plate, a plate having a ligand, etc.) as a cell sheet and then transferred to a biomaterial. Spheroids may be generated, for example, using centrifugation techniques, low-adhesion plates, orbital shaking, etc. The spheroids can be pelleted and then seeded onto a biomaterial.

[0154] Although not wishing to limit the present invention to any theory or mechanism, it is believed that the use of cell sheets or spheroids can enhance the seeding efficiency of tissue compositions. This can be advantageous when dealing with expensive cell types and / or non-proliferative cell types.

[0155] The present invention also features a tissue composition constructed by seeding cells on a specific surface and subsequently attaching a biomaterial (e.g., a scaffold only, a scaffold having seeded cells and no ECM-producing cells, a scaffold having seeded cells and ECM-producing cells, a scaffold having ECM-producing cells, etc.) to the cells. A similar tissue composition may be constructed by first attaching the biomaterial to a plate and then seeding the cells. In certain embodiments, the tissue composition features a layer of biomaterial and cells. Methods of attaching a biomaterial to seeded cells may include, but are not limited to, centrifugation. In certain embodiments, the biomaterial includes one or more components (e.g., ligands, etc.) for attracting seeded cells. In certain embodiments, the seeded cells include one or more components (e.g., ligands) for attracting the biomaterial.

[0156] The surface can be a culture dish or a culture surface having features that allow for the removal of the tissue composition. For example, in some embodiments, the surface is a temperature-sensitive culture plate. The temperature-sensitive plate is designed to release attached cells (e.g., the seeded cells of the tissue composition) when placed at a specific temperature (e.g., 20-25 °C).

[0157] In certain embodiments, the plate includes an attachment component for temporarily attaching cells and / or biological materials to the plate. For example, in certain embodiments, the attachment component is an engineered liposome (e.g., a gold-coated liposome) having a ligand such as RGD. The ligand attaches the gold liposome to the plate, and the cells and / or biological materials attach to the liposome. Once the tissue composition is ready to be harvested, the gold-coated liposome can be activated and opened by resonant light, thereby detaching the tissue composition from the plate. The present invention is not limited to gold-coated liposomes. While not wishing to limit the present invention to any theory or mechanism, one advantage of using an attachment component (such as a gold-coated liposome) is that the tissue composition can be constructed in that shape by patterning the attachment component (e.g., a gold-coated liposome) on the plate into a specific shape. The attachment component can also enable the stability and controlled release of cells and specific substances that can be contained within the liposome, such as factors necessary for growth and / or differentiation.

[0158] The present invention also features a method of reducing the metabolic rate of a tissue composition. For example, the present invention features a method of reducing the beating rate of a tissue composition such as a tissue composition featuring cardiomyocytes. The method can feature introducing a drug that reduces the beating rate to a specific desired beating rate into the tissue composition. In certain embodiments, the method features temperature control. In certain embodiments, the beating rate is reduced for storage and / or stability, e.g., stability during transportation. The method of reducing the beating rate of a tissue composition can be applied to any suitable tissue composition herein, such as a tissue composition featuring a scaffold, ECM, ECM-producing cells, and cardiomyocytes; a tissue composition featuring a cell sheet with or without a scaffold, etc.

[0159] Furthermore, the present invention features a tissue composition (e.g., any suitable tissue composition herein, other contractile grafts, etc.) having a pulsation rate of 10 to 20 bpm. The present invention also features a tissue composition (e.g., any suitable tissue composition herein, other contractile grafts, etc.) having a pulsation rate of 20 to 30 bpm. The present invention also features a tissue composition (e.g., any suitable tissue composition herein, other contractile grafts, etc.) having a pulsation rate of 10 to 30 bpm. The present invention also features a tissue composition (e.g., any suitable tissue composition herein, other contractile grafts, etc.) having a pulsation rate of 30 to 40 bpm. The present invention also features a tissue composition (e.g., any suitable tissue composition herein, other contractile grafts, etc.) having a pulsation rate of 20 to 40 bpm. The present invention also features a tissue composition (e.g., any suitable tissue composition herein, other contractile grafts, etc.) having a pulsation rate of 40 to 50 bpm. The present invention also features a tissue composition (e.g., any suitable tissue composition herein, other contractile grafts, etc.) having a pulsation rate of 30 to 50 bpm. The present invention also features a tissue composition (e.g., any suitable tissue composition herein, other contractile grafts, etc.) having a pulsation rate of 0 to 50 bpm. The present invention also features a tissue composition (e.g., any suitable tissue composition herein, other contractile grafts, etc.) having a pulsation rate of 0 to 100 bpm. While not wishing to limit the present invention to any theory or mechanism, tissue compositions having a low pulsation rate have a lower metabolic load compared to tissue compositions having a high pulsation rate, and the low metabolic load can help extend the shelf life of the tissue composition (e.g., the time the graft can be set at room temperature before being transplanted), so a low pulsation rate (e.g., a pulsation rate of 10 to 50 bpm) is considered to be potentially advantageous. The low metabolic load can also be beneficial in an ischemic environment because it requires fewer nutrients for the tissue composition to be healthy and functional.

[0160] Methods for adjusting the pulsation rate of a tissue composition include, but are not limited to, the use of beta blockers or other exogenous agents.

[0161] The tissue composition of the present invention is constructed to withstand short-term and / or long-term storage, for example, cryopreservation. Cryopreservation may refer to a temperature of -80°C to -196°C or -90°C to -196°C. The ability to cryopreserve the tissue composition helps to enable storing the tissue until use as required and transporting the tissue composition from one location to another.

[0162] The tissue composition may also be constructed to withstand a specific length of time at room temperature (or a temperature below 37°C). The ability to survive at a temperature below 37°C for a specific length of time can be beneficial when the tissue composition is out of the incubator before use. As an example, the tissue composition may be taken out of the incubator and transported to the operating room for use in the transplantation process but not immediately transplanted, and thus may be exposed to room temperature for a long time.

[0163] <Properties of the tissue composition> The engineered tissue composition of the present invention (e.g., characterized by cardiomyocytes) can be evaluated for one or more mechanical parameters, electrophysiological parameters, chemical parameters, biochemical parameters (such as growth factors, metabolites, ion channels, etc.) or combinations thereof. Mechanical parameters or electrophysiological parameters may include, but are not limited to, contraction rate, contraction / relaxation rate, constant contractile force, non-constant contractile force, displacement rate, displacement force, impulse directionality, impulse velocity, electric field potential, amplitude, capture threshold, chronotropic response, excitation sequence after stimulation, functional gap junction formation, response to electrical pacing, electric field potential amplitude, conduction velocity, propagation pattern, gap junction analysis, or combinations thereof.

[0164] Similarly, the engineered tissue composition (e.g., characterized by cardiomyocytes) can be subjected to multi-electrode array mapping for real-time electrophysiological measurements. The contraction rate, systolic / diastolic displacement, systolic contraction rate, and / or diastolic relaxation rate may be detected using a microscope. As described above, the cardiomyocytes can be paced. Pacing can be achieved by an applied external field stimulus.

[0165] Tissue compositions, such as tissue compositions containing cardiomyocytes, may be constructed to have a specific pulsation rate. In certain embodiments, the pulsation rate is 0 to 100 bpm. In certain embodiments, the pulsation rate is 10 to 30 bpm. In certain embodiments, the pulsation rate is 20 to 40 bpm. In certain embodiments, the pulsation rate is 30 to 60 bpm. In certain embodiments, the pulsation rate is 40 to 70 bpm. In certain embodiments, the pulsation rate is 50 to 80 bpm. In certain embodiments, the pulsation rate is 60 to 90 bpm. In certain embodiments, the pulsation rate is 70 to 100 bpm. The present invention is not limited to the foregoing examples of pulsation rates.

[0166] The mechanical properties of the engineered tissue composition (e.g., characterized by cardiomyocytes) can depend on the scaffold material used. For example, displacement, strain rate, displacement velocity, etc. can all depend on the scaffold material. In some embodiments, the engineered tissue composition (e.g., characterized by cardiomyocytes) has a voltage amplitude of 0.1 mV to 1 mV across the entire engineered tissue composition at an electrode spacing of 1 mm to 1.5 cm.

[0167] The tensile strength of the tissue composition can be determined by its composition, e.g., the ratio of scaffold, ECM, cells, etc.

[0168] In certain embodiments, the relative expression of markers can be evaluated to determine the amount of a particular cell type of interest (e.g., cardiomyocytes, skeletal muscle cells, smooth muscle cells, etc.) relative to ECM-producing cells (e.g., fibroblasts). The ratio of the cell type of interest to the ECM-producing cells changes over time based on changes in the tissue composition, e.g., when the cells of interest proliferate, when a particular cell population dies, when the cells differentiate over time, etc. Non-limiting examples of markers that can be evaluated include CD90, vimentin, FSP-1, collagen I, α-SMA, HSP47, etc.

[0169] <Platform> The present invention also features a platform having the tissue composition of the present invention. For example, the present invention features a single-well plate (having a single well), and the engineered tissue composition of the present invention is deposited in the well therein. The present invention also features a multi-well plate having two or more wells, and the engineered tissue composition of the present invention is deposited in at least one of the wells therein. The multi-well plate may have 2 wells, 4 wells, 6 wells, 8 wells, 12 wells, 24 wells, 48 wells, 96 wells, more than 96 wells, 2 - 12 wells, 12 - 24 wells, 24 - 48 wells, 48 - 96 wells, etc. The present invention also features a platform for an engineered tissue composition having wells within any other suitable culture device such as a well, trough, or tube, tray, etc. The present invention is not limited to a culture dish as a platform for culturing, maintaining, and / or storing tissue compositions.

[0170] The present invention also features a closed-system platform for generating, maintaining, and / or storing the tissue compositions herein. For example, the closed system may feature encapsulation of the tissue composition, e.g., a scaffold and ECM contained therein and optionally other components described herein. In a closed system, the culture medium can be aseptically exchanged. The tissue composition can also be frozen using encapsulation and thawed when ready. In certain embodiments, multiple tissue compositions can be contained within an encapsulation or multiple encapsulations can be connected together to create a new encapsulation. For example, in some embodiments, up to 6 tissue compositions are contained within an encapsulation. In some embodiments, up to 10 tissue compositions are contained within an encapsulation. In some embodiments, up to 20 tissue compositions are contained within an encapsulation. In some embodiments, more than 20 compositions are contained within an encapsulation.

[0171] <Method of Use> The tissue compositions of the present invention can be used for various purposes, such as in vivo use, in vitro use, for example, transplantation into patients, in vitro assays, cell differentiation platforms, and the like. For example, the present invention features a method of repairing tissue (e.g., tissue affected by a disease or condition, trauma, etc.), and the tissue compositions of the present invention are transplanted into the affected tissue to enhance the function of the affected tissue. As a non-limiting example, a heart tissue composition (characterized by cardiomyocytes) may be transplanted into the target heart tissue, and the engineered tissue composition enhances the function of the heart tissue. Diseases or conditions can include (but are not limited to) arrhythmias, heart failure, myocardial infarction, arrhythmogenic cardiomyopathy (e.g., long QT syndrome (LQTS), short QT syndrome (SQTS), Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), arrhythmogenic right ventricular cardiomyopathy (ARVC), dilated cardiomyopathy (e.g., hypertrophic cardiomyopathy, left ventricular non-compaction, transthyretin amyloidosis, hereditary hemochromatosis, RASopathy (also known as Noonan spectrum disorder)), any other acquired heart disease or injury, or any other congenital heart disease or injury.

[0172] The present invention also features a method of differentiating cells by seeding the above cells onto the engineered tissue compositions of the present invention. Compounds derived from ECM-producing cells of the tissue composition, or other factors therein, cause cell differentiation. The present invention also features a method of enhancing cell maturation by seeding the above cells onto the engineered tissue compositions of the present invention. Compounds derived from ECM-producing cells of the tissue composition, or other factors therein, can cause cell maturation. The present invention also features a method of driving a specific phenotype or genotype of cells by seeding the above cells onto the engineered tissue compositions of the present invention. Compounds derived from ECM-producing cells of the tissue composition, or other factors therein, can cause cell development and express a specific phenotype or genotype.

[0173] It should be noted that the tissue compositions of the present invention (e.g., microenvironments, e.g., compounds derived from ECM-producing cells) can cause seeded cells to develop / differentiate into multiple different cell populations at various ratios. For example, a group of progenitor cells can differentiate into two or more cell populations. As another example, the seeded cells can differentiate into cardiomyocytes (e.g., 80-90%), endothelial cells (e.g., 2-10%), and smooth muscle cells (e.g., 2-10%).

[0174] The tissue compositions of the present invention can also be used for transplantation. The methods can be characterized by open surgical procedures, minimally invasive procedures, percutaneous procedures, robotic procedures, etc.

[0175] The methods and compositions herein can be used for various subjects, such as humans / primates, pigs, rats, dogs, horses, cats, etc.

[0176] Since the engineered tissue compositions of the present invention can mimic natural tissues, the engineered tissue compositions can be used for testing compounds or other components for harmful or toxic effects, or for detecting beneficial or therapeutic effects. For example, the present invention features a method for detecting the effect (e.g., harmful, toxic, beneficial, therapeutic) of a test component (e.g., drug, small molecule, cell, cell product, etc.) on a specific tissue, e.g., the tissue of an engineered tissue composition. The tissue can be, for example, heart tissue. In some embodiments, the method includes introducing the test component into the engineered tissue composition. In some embodiments, the engineered tissue composition is stimulated in some way. The method may further include measuring one or more physical parameters, mechanical parameters, electrophysiological parameters, biochemical parameters, etc. Depending on the results of the test, it can be determined whether the test component has a harmful or beneficial effect. In some embodiments, the method further includes measuring displacement, strain, force, conduction velocity, millivoltage amplitude, or a combination thereof.

[0177] Using at least two different electrodes (along the x-axis or along the y-axis), the directionality of the impulse is measured. Since the excitation sequence can be determined, the directionality of the impulse can be determined. Using at least two different electrodes (along the x-axis or along the y-axis), the velocity of the impulse is measured. The electric field potential is measured using monopolar electrogram traces from individual electrodes or by combining electrodes to form bipolar traces. The voltage / electric field potential can be recorded and subsequently the amplitude can be analyzed. The capture threshold is measured using unique software developed by the inventors. A construct can be stimulated at a specific location via a multi-electrode array and the minimum voltage required to cause depolarization of the cell network of the construct can be determined. The chronotropic response is measured using the same unique software developed by the inventors. The heart can be stimulated at a determined rate and the response can be monitored. A tissue composition (e.g., a graft) can be stimulated at a known location and the subsequent excitation of the construct along multiple axes can be recorded. By pacing and introducing programmed additional stimuli, the inducibility of sustained ventricular tachycardia can be measured.

Example

[0178] [Example 1] Example 1 describes an example of a method for generating the tissue composition of the present invention. The present invention is not limited to the features of this example in this specification. (1) Human dermal fibroblasts (HDF) are taken out from liquid nitrogen, warmed, and cultured in a culture dish. (2) HDF is passaged about 8 times (it can be passaged up to about 20 times), and each passage is performed when the cells reach about 80% confluence. (3) At an appropriate passage, the cells are harvested (the cells may be removed from the culture dish using trypsin or the like, or the cells may be scraped off). (4) A scaffold is placed in a tissue culture dish (for example, a 60 mm dish). The scaffold may be of any appropriate shape and size, and it should be noted that the tissue culture dish is not limited to a circular 60 mm dish. For example, the scaffold and the dish may be rectangular, square, etc. (5) HDF is applied to the scaffold. The number of cells (and volume, thus the concentration of the cells) is known. It should be noted that the cryopreserved vial of HDF can be warmed and seeded directly onto the scaffold without passaging. (6) Gravity, rocking, and / or centrifugation may be used for seeding HDF. (7) The scaffold-HDF culture may be cultured for a specific period, for example, 30 days. Once the fibroblasts are prepared, another population of cells (for example, seeded cells such as cardiomyocytes) can be seeded using, for example, centrifugation. (8) The seeded cells can be cultured on the tissue composition for a specific period, for example, 2 days. (9) As described herein, the tissue composition can optionally be cryopreserved (for example, using a controlled-rate freezing protocol), and the tissue composition can be used immediately for various purposes, such as surgical transplantation, in vitro drug testing, etc.

[0179] [Example 2] Example 2 describes the tissue composition of the present invention. The present invention is not limited to the methods and features described in Example 2. Tests were conducted using cardiac progenitor cells. The inventors surprisingly found that the cardiomyocyte layer in the construct (tissue composition) was much thicker than previously described, for example, the cell layer was 1 - 50 cells thick (see Figure 7A). Cardiac progenitor cells are proliferative and continue to replicate during the culture period. When this graft (tissue composition) was transplanted into a rodent model of chronic heart failure (CHF), cardiac function was improved (see Figures 7B, 7C and Tables 1, 2 and 3). In some embodiments, the tissue composition of the present invention is an allogeneic cryopreserved heart graft that can be transplanted onto the epicardium of the heart. Some data indicate that these grafts can reduce the susceptibility to ventricular tachycardia. Furthermore, these progenitor cells have the ability to differentiate into cardiac-specific mesodermal lineage cells (endothelial cells, smooth muscle cells, and cardiomyocytes). While not wishing to limit the present invention to any theory or mechanism, the growth factor environment of the fibroblast graft may serve to promote and induce cardiomyocyte differentiation in vitro.

[0180] As described above, it was surprising that the cells continued to divide even after transplantation. While not wishing to limit the present invention to any theory or mechanism, it is hypothesized that cardiac progenitor cells may have excellent resistance to hypoxia and may survive better in a hypoxic or post-infarct environment than terminally differentiated cardiomyocytes. The final cell ratio of the graft is 5:1 to 100:1.

[0181] Referring to Table 1, hemodynamic evaluations were performed at the end of the test for sham, CHF, and progenitor cell transplantation treated with CPC (hiPSC-derived myocardial progenitor cells) using a solid Millar catheter. Transplantation of myocardial progenitor cell heart grafts reduces EDP and Tau, while increasing PDP. These values lead to an improvement in the filling capacity of the heart and ultimately suggest that in patients treated with these grafts, the condition may improve, resulting in a decrease in the New York Heart Association cardiac function classification. The data are mean ± standard error. CHF, n = 6; sham, n = 10; CPC, n = 13. Abbreviations: HR = heart rate, EDP = end-diastolic pressure, SysP = systolic pressure, dP / dt = change in blood pressure over time, PDP = Peak Developed Pressure, CHF = chronic heart failure.

Table 1

[0182] Referring to Table 2, non-invasive echocardiographic evaluations were performed at 3 weeks after treatment. Transplantation of myocardial progenitor cell grafts reduces maladaptive remodeling (LVid-sys / dia and LVv-sys / dia), while increasing EF and FS. The data are mean ± standard error. CHF, n = 6; CPC. Abbreviations: EF = ejection fraction; FS = fractional shortening of the left ventricular internal diameter; sys = systolic; dia = diastolic; LVid = left ventricular internal dimension; LVv = left ventricular volume; AW = anterior wall.

Table 2

[0183] Referring to Table 3, non-invasive echocardiographic evaluations were performed at 3 weeks after treatment. Transplantation of myocardial progenitor cell grafts reduces maladaptive remodeling (LVid-sys / dia and LVv-sys / dia), while increasing EF and FS. The data are mean ± standard error. CHF, n = 6; CPC. Abbreviations: EF = ejection fraction; FS = fractional shortening of the left ventricular internal diameter; sys = systolic; dia = diastolic; LVid = left ventricular internal dimension; LVv = left ventricular volume; AW = anterior wall.

Table 3

[0184] [Example 3] Example 3 describes the tissue composition of the present invention. The present invention is not limited to the methods and features described in Example 3. Example 3 describes a slow-degrading mesh scaffold seeded with fibroblasts and cardiomyocytes and evaluated for functional benefits in heart failure rats. The tissue composition was cryopreserved and reconstituted prior to transplantation. The data show functional improvement after transplantation and at 3-7 weeks post-transplantation.

[0185] A tissue composition (hiPSC-CM graft) (1.7 cm in diameter) was produced by culturing human neonatal dermal fibroblasts (hNDF) and human iPSC-derived cardiomyocytes (hiPSC-CM) within a bioabsorbable mesh (see Figure 8A). The grafts were cryopreserved at -196°C for 2-4 weeks, thawed, reconstituted, and transplanted into CHF rats. Myocardial infarction (MI) was induced in immunocompetent rats by permanent ligation of the left coronary artery (LCA) (time = 0). The rats were allowed to recover for 3 weeks to develop chronic heart failure. Subsequently, the tissue composition (hiPSC-CM graft) was transplanted via median sternotomy (time = 3 weeks). Echocardiography was performed at 3, 6, and 10 weeks post-infarction (0, 3, and 7 weeks post-graft transplantation). Hemodynamics and ex vivo pressure-volume cures were obtained at the end point of the study (end point of the study time = 10 weeks).

[0186] The tissue composition of the present invention (hiPSC-CM graft) used in this specification maintained spontaneous and synchronous contractions before freezing (65 ± 10 bpm) and after thawing (60 ± 10 bpm). The grafts were maintained up to 40 days after thawing without deterioration of the composition or heart rate (see Figure 8B). The tissue composition (hiPSC-CM graft) used reduced maladaptive left ventricular (LV) remodeling by decreasing LV volume (systole / diastole) and LV diameter (systole / diastole) (p < 0.05), and showed a tendency to improve ejection fraction and fractional shortening of the left ventricular internal diameter from 3 weeks to 7 weeks after transplantation compared to CHF controls (see Figure 8C). The grafts decreased LV end-diastolic pressure (24 ± 6 mmHg (CHF control) vs. 18 ± 3 mmHg (tissue composition used)) and showed a tendency to improve LV dP / dt(-) and dP / dt(+) compared to the control (see Table 4. Hemodynamic evaluation was performed at the end point of the study (7 weeks after hiPSC-CM graft transplantation, 10 weeks after MI). The hiPSC-CM grafts improved LV EDP and showed a tendency to improve dP / dt(+). Sham, n = 6; CHF, n = 6; hiPSC-CM graft, n = 10). Furthermore, the tissue composition (hiPSC-CM graft) used decreased LV EDP operative volume by 36% as shown by the leftward shift of the diastolic pressure-volume relationship (see Figure 8D).

Table 4

[0187] Example 3 shows that the tissue composition of the present invention can be cryopreserved, reconstituted, and transplanted into CHF rats. The treated rats showed recovery of maladaptive LV remodeling and continued functional improvement from 3 weeks to 7 weeks after treatment. While not wishing to limit the present invention to any theory or mechanism, it is believed that the ability to cryopreserve allows for long-term storage after large batch production, thereby providing cost and practicality advantages.

[0188] The disclosures of the following U.S. patents are hereby incorporated by reference in their entirety: U.S. Patent No. 4,963,489; U.S. Patent Application Publication No. 2009 / 0269316; International Publication No. 2013 / 151755 Pamphlet; International Publication No. 2011 / 102991 Pamphlet; U.S. Patent Application Publication No. 2014 / 0178450; U.S. Patent No. 8,802,144; International Publication No. 2009 / 102967 Pamphlet; U.S. Patent No. 9,119,831; International Publication No. 2010 / 042856 Pamphlet; U.S. Patent Application Publication No. 2008 / 0075750; U.S. Patent No. 9,587,222.

[0189] Various modifications of the present invention will be apparent to those skilled in the art from the foregoing description in addition to those described herein. Such modifications are also intended to be included within the scope of the appended claims. Each reference cited in this application is hereby incorporated by reference in its entirety.

[0190] Although the preferred embodiments of the present invention have been shown and described, it will be readily apparent to those skilled in the art that modifications may be made thereto without exceeding the scope of the appended claims. Accordingly, the scope of the present invention is limited only by the claims. The reference numbers listed in the claims are exemplary only and are for the convenience of the examiner at the Patent Office and are not limiting in any way. In some embodiments, the figures presented in this patent application are drawn to scale, including angles, ratios of dimensions, etc. In some embodiments, the figures are only representative and the claims are not limited by the dimensions of the figures. In some embodiments, the description of the present invention described herein using the phrase "comprising" includes embodiments that may be described as "consisting of", and thus meets the written description requirement for claiming one or more embodiments of the present invention using the phrase "consisting of".

[0191] The reference numbers listed in the claims are for the sole purpose of facilitating the examination of the present patent application and are illustrative only, and are not intended to limit the claims to specific features having corresponding reference numbers in the drawings in any way.

Claims

1. A tissue composition comprising: a. A scaffold having a generally flat orientation comprising a first fiber type and a second fiber type that are knitted or woven and have pores therein, wherein the first fiber type has a composition and a degradation profile different from that of the second fiber type, the first fiber type comprises glycolide, lactide, and trimethylene carbonate, and the second fiber type comprises lactide and trimethylene carbonate, and the scaffold; b. An extracellular matrix (ECM) material and ECM-producing cells disposed on or on and within the scaffold, wherein the ECM and ECM-producing cells fill the pores of the scaffold, the extracellular matrix (ECM) material and ECM-producing cells; c. Seeded cells seeded in and / or on the ECM, which are induced pluripotent stem cell-derived cardiomyocytes, cardiac progenitor cells, or cardiomyocytes, the seeded cells; A tissue composition comprising the above.

2. The tissue composition according to claim 1, wherein the scaffold has a pore size of 500 μm to 1200 μm.

3. The tissue composition according to claim 1, wherein the tissue composition has a pulsation rate of 0 to 50 bpm or 0 to 120 bpm.

4. The tissue composition according to claim 1, wherein the tissue composition pulsates synchronously.

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