WO2012119074A1

WO2012119074A1 - Apparatus and method for organizing three-dimensional cell structures using stiffness gradients and sacrificial gels

Info

Publication number: WO2012119074A1
Application number: PCT/US2012/027483
Authority: WO
Inventors: Devin Michael NEAL; Mahmut Sakar; Haruhiko Harry Asada
Original assignee: Massachusetts Institute Of Technology
Priority date: 2011-03-03
Filing date: 2012-03-02
Publication date: 2012-09-07

Abstract

The invention generally relates to systems, devices, apparatuses and methods for organizing cells in three-dimensions using matrix stiffness gradients. In particular, the invention relates to systems, devices, apparatuses and methods for organizing myogenic cells using matrix stiffness gradients. In some aspects, the invention relates to systems, devices, apparatuses and methods for generating muscle tissue.

Description

APPARATUS AND METHOD FOR ORGANIZING THREE-DIMENSIONAL CELL STRUCTURES USING STIFFNESS GRADIENTS AND SACRIFICIAL GELS

RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S.C. § 119(e) to U.S. provisional application, U.S.S.N. 61/448,944, filed March 3, 2011, the entire contents of which are incorporated herein by reference.

FIELD OF INVENTION

[0002] The invention relates to systems, devices, apparatuses and methods for organizing cell structure.

BACKGROUND

[0003] Cellular differentiation is the process by which a progenitor cell is transformed into a cell type having one or more specialized functions. The process of cell differentiation is influenced by a variety of extracellular inputs including growth factors, cytokines, and other molecules that modulate different signaling pathways in cells. Cell-cell interactions and cell-matrix interactions also influence cell signaling and modulate the differentiation process. As an example, myogenesis is the process by which progenitor cells differentiate to produce muscle cells and fibers. Myogenesis involves a proliferative expansion of myogenic precursor cells (e.g. , satellite cells, myoblasts), synthesis of a fibronectin-rich matrix, migration and alignment of cells, and fusion of aligned cells into multi-nucleated fibers, called myotubes. Myogenesis involves an interplay of various signaling molecules, cell-cell interactions, and cell matrix interactions. New devices are needed for examining and influencing cell function, including differentiation, e.g. , myogenesis, and for identifying factors (e.g. , test agents) that influence cell function, e.g., differentiation.

SUMMARY OF THE INVENTION

[0004] The present invention provides cell culture systems for organizing cells in three- dimensions. In some embodiments, the invention provides cell culture devices with matrix stiffness gradients for organizing cells in three-dimensions. In certain embodiments, the cell culture devices include a primary matrix and one or more regions within the primary matrix that comprise a secondary matrix, in which the stiffness of the primary matrix is different

2602367.1 than the stiffness of the secondary matrix. In particular embodiments, the secondary matrix comprises cells. The cells may organize and arrange themselves within the secondary matrix as a result of the difference in stiffness between the secondary matrix and the primary matrix. Accordingly, various characteristics of the primary matrix and secondary matrix (including, e.g. , structural properties (e.g., stiffness, permeability, etc.), size, and shape) may be tuned to control the organization and arrangement of the cells within the device. In addition, various types of cells may be cultured in the device. In some embodiments, muscle cells (e.g. , myogenic cells) are cultured in the device. In some embodiments, combinations of different cells are cultured in the device, either within the same region or within different regions. For example, non-muscle cells (e.g. , endothelial cells) may be cultured within the same region or within different regions as muscle cells.

[0005] In other aspects, the invention provides methods for culturing cells based on stiffness gradients. The methods typically involve the use of a cell culture device that includes a primary matrix and one or more regions within the primary matrix that comprise cells within a secondary matrix. The cell culture device is typically maintained under conditions that support viability of the cells (e.g. , temperature, 0₂ concentration, pH, etc.). For example, the device may be maintained in vitro or may be implanted in a subject and maintained in vivo to support the viability of the cells in the device. Any of the cell culture devices disclosed herein may be used in the inventive methods.

[0006] The methods may involve influencing cells to modulate one or more phenotypic characteristics within the device. Cells may be influenced to modulate their shape, size, or three-dimensional organization within the matrix. Cells may be induced to enter into the cell cycle or exit from the cell cycle (e.g., arrest in GO). In some embodiments, the methods involve inducing the cells to differentiate within the secondary matrix. In some

embodiments, the methods involve inducing the cells to form a tissue or organ-like structure within the secondary matrix. The cells may be induced to differentiate by, for example, contacting the cells with a growth factor, cytokine, or other agent that induces differentiation of the cells. In some cases, various characteristics of the secondary matrix (including, e.g. , its chemical properties (e.g. , substituent molecules (e.g. , synthetic polymers, natural polymers, biopolymers, etc.), degree of cross-linking, pH, ionic strength, hydrophilicity, polarity, etc.), its structural properties (e.g. , stiffness), its size, and its shape) can be tuned to induce differentiation. [0007] The invention, in some aspect, relates to methods for producing myotubes. For example, methods are provided that involve (a) culturing myogenic cells in a device comprising a primary matrix and one or more regions within the primary matrix that comprise a secondary matrix, in which the myogenic cells reside in the secondary matrix, and (b) maintaining the device under conditions that induce differentiation of the myogenic cells. In particular embodiments, the differentiated cells fuse to form multinucleated myotubes within the secondary matrix. Typically, the myotubes are arranged according to the shape and size of the secondary matrix. Moreover, by having a plurality of regions that comprise myogenic cells within a secondary matrix, multiple regions of myotubes may be formed using a single device.

[0008] In other aspects, the invention relates to methods for evaluating the effect of a test agent on cell differentiation. In some embodiments, the methods involve culturing cells in a device comprising a primary matrix and one or more regions within the primary matrix that comprise a secondary matrix, in which the cells reside in the secondary matrix, and in which the stiffness of the secondary matrix is different than (greater than or less than) the stiffness of the primary matrix; contacting the cells with the test agent; and determining whether the cells differentiate or change (e.g., undergo a phenotypic change) within the secondary matrix in the presence of the test agent or after having been contacted with the test agent. In certain embodiments, the invention relates to methods for evaluating the effects of a test agent on myo genesis. For example, the methods may include culturing myogenic cells in a cell culture device of the invention; contacting the myogenic cells with the test agent; and determining whether the myogenic cells form myotubes within the device (e.g., within the secondary matrix of the device).

[0009] In other aspects, the invention relates to methods for evaluating the role of a gene product on cell differentiation. For example, the methods may involve culturing cells in a device comprising a primary matrix and one or more regions within the primary matrix that comprise a secondary matrix, in which the cells reside in the secondary matrix, and in which the stiffness of the secondary matrix is different than (greater than or less than) the stiffness of the primary matrix; contacting the cells with a test agent (e.g. , siRNA, antibody, etc.) that inhibits the gene product; and determining whether the cells differentiate within the secondary matrix. In certain embodiments, the invention relates to methods for evaluating the role of a gene product on myotube formation. For example, the methods may involve culturing myogenic cells in a cell culture device of the invention; contacting the myogenic cells with the test agent that inhibits the gene product; and determining whether the myogenic cells form myo tubes within the device (i.e., within the secondary matrix of the device).

[0010] The invention, in some aspects, relates to methods for evaluating the ability of a test cell to undergo myogenic differentiation. In certain embodiments, the methods involve culturing test cells (e.g., stem cells) in a device comprising a primary matrix and one or more regions within the primary matrix that comprise a secondary matrix, in which the test cells reside in the secondary matrix, and in which the stiffness of the secondary matrix is greater than the stiffness of the primary matrix; maintaining the cell culture device under conditions that induce the myogenic cells to form myotubes within the secondary matrix; and determining whether the test cells form myotubes within the secondary matrix.

[0011] In still further aspects, the invention relates to methods for producing a cell culture device. In some embodiments, the methods involve (a) producing a primary matrix that at least partially encompasses one or more solid objects; (b) removing one or more of the solid objects to produce one or more cavities; and (c) filling one or more of the cavities with a secondary matrix, in which the stiffness of the primary matrix produced is different than the stiffness of the secondary matrix. In certain embodiments, the secondary matrix includes cells (e.g., stem cells, pluripotent cells, myogenic cells). The secondary matrix may include other components such as extracellular matrix proteins (e.g. , collagen, fibronectin, etc.), growth factors, cytokines, drugs, or other components. In some aspects, the invention relates to a method for producing one or more matrices that have a defined shape. In some embodiments, the method involves producing a primary matrix that at least partially encompasses one or more solid objects; removing one or more of the solid objects to produce one or more cavities; and filling one or more of the cavities with a secondary matrix, in which the primary matrix has a melting temperature that is less than the secondary matrix. In some embodiments, the method also involves incubating the primary matrix having one or more cavities filled with the secondary matrix at a temperature sufficient to melt the primary matrix but not the secondary matrix, thereby releasing one or more secondary matrices having a shape defined by the geometry of the one or more cavities. The invention in some aspects, provides a cell culture device that comprises a primary matrix and one or more regions within the primary matrix that comprise a secondary matrix, in which the primary matrix has a melting temperature that is less than the secondary matrix. In one embodiment, the secondary matrix comprises cells. [0012] The invention also provides kits comprising a container or package housing any of the cell culture devices disclosed herein or one or more components for making the cell culture devices disclosed herein.

DEFINITIONS

[0013] As used herein, the term "appropriate standard" refers to a quantity indicative of a known outcome, status or result (e.g. , a known differentiation state).

[0014] As used herein, the term "agents" or "test agents" refers to peptides, polypeptides, proteins, small molecules, organic and/or inorganic compounds, polysaccharides, lipids, nucleic acids, particles, antibodies, ligands, or combinations thereof.

[0015] As used herein, the term "antibody" refers to an immunoglobulin, whether natural or wholly or partially synthetically produced. All derivatives thereof which maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly

synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG class, however, are preferred in the present invention.

[0016] As used herein, the term "antibody fragment" refers to any derivative of an antibody which is less than full-length. Preferably, the antibody fragment retains at least a significant portion of the full-length antibody' s specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')₂, scFv, Fv, dsFv diabody, and Fd fragments. The antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively, the antibody fragment may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages or other more stable linkages. The fragment may also optionally be a multimolecular complex.

[0017] As used herein, the term "approximately" or "about" in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0018] As used herein, the term "density" is used with its common technical meaning (e.g., mass per unit volume, weight per unit volume, etc.). In some cases, density may refer to a specific region within a matrix (e.g., density of polymers within a primary matrix, density of polymers within a secondary matrix, etc.). The density may be measured, for example, by taking the mass or weight divided by the geometric volume described by a shape.

[0019] As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism.

[0020] As used herein, the term "in vivo" refers to events that occur within an organism.

[0021] As used herein, the term "matrix" refers to a polymeric network (e.g., a cross- linked polymeric network). Polymers of the network may be natural, synthetic or a combination thereof. Any suitable chemical bonding may provide cross-links for a polymeric network including, for example, covalent bonds, ionic bonds, Van der Waals interactions, hydrogen bonds, hydrophobic interactions, etc. Cross-links may be formed by chemical reactions that are initiated by temperature changes, pressure changes, ionic changes, pH changes, or radiation, for example. A matrix may a porous solid or porous solid-like material. A matrix may be a gel, such as, for example, a hydrogel, organogel or xerogel.

[0022] As used herein, the term "nucleic acid" refers to a polymer of covalently linked nucleotide bases. A nucleic acid can be of biologic and/or synthetic origin. The nucleic acid may be in single-stranded or double- stranded form. Also included within the definition are nucleic acids having modified nucleotides. Other modifications may involve, for example, modifications of the backbone. The term nucleic acid embraces DNA, RNA, or PNA

(peptide nucleic acid), or a combination thereof.

[0023] As used herein, the term "peptide, " "polypeptide," or "protein" comprises a polymer of amino acid residues linked together by peptide (amide) bonds. The amino acid residue may be natural, unnatural, or a derivative thereof. The term(s), as used herein, refers to proteins, polypeptides, and peptide of any size, structure, or function.

[0024] As used herein, the term "primary matrix" refers to a polymeric network (e.g., a cross-linked polymeric network) that at least partially encompasses one or more regions that have a secondary matrix. A primary matrix may encompass multiple regions that have the same secondary matrix. A primary matrix may encompass regions that have different secondary matrices. The primary matrix may be more stiff or less stiff than the secondary matrix, in certain embodiments. A primary matrix may comprise one or more matrix-free channels. The primary matrix may have a permeability that permits the diffusion of growth factors, cytokines, carbon sources, nitrogen sources, vitamins, and other agents that influence cell function from one region within the matrix to another (e.g., from a region comprising a secondary matrix to another region comprising a secondary matrix, from a matrix-free channel to a region comprising a secondary matrix, etc.). The primary matrix may have a permeability that permits the removal (e.g., by diffusion) of cellular waste products from a secondary matrix. The primary matrix may have a structure that accommodates the migration of cells within it. The primary matrix may be biodegradable.

[0025] As used herein, the term "secondary matrix" refers to a polymeric network (e.g. , a cross-linked polymeric network) that is at least partially encompassed by a primary matrix (at least during its formation). In some embodiments, the secondary matrix has a different stiffness than the primary matrix. Typically, the secondary matrix provides a substrate for cell attachment. The secondary matrix may comprise growth factors, cytokines, carbon sources, nitrogen sources, vitamins, and other agents that influence cell function (e.g., growth, proliferation, differentiation, etc.). The secondary matrix may comprise cell culture media or a component thereof. The secondary matrix may be biodegradable.

[0026] As used herein, the term "short-interfering nucleic acid" refers to a small nucleic acid molecule (e.g. , 15 to 30 nucleotide, 19 to 23 nucleotides, a hairpin RNA, etc.) that inhibits the expression of a non-coding RNA or an mRNA. The small interfering nucleic acid may be a microRNA, siRNA, shRNA, antisense RNA, etc. The small interfering nucleic acid may inhibit transcription, translation and/or may result in degradation of a target nucleic acid (e.g. , of a target mRNA).

[0027] As used herein, the term "small molecule" is used to refer to molecules, whether naturally- occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Typically, a small molecule is an organic compound (i.e., it contains carbon). The small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g. , amines, hydroxyl, carbonyls, heterocyclic rings, etc.). In some embodiments, small molecules are monomeric and have a molecular weight of less than about 1500 g/mol. In certain embodiments, the molecular weight of the small molecule is less than about 1000 g/mol or less than about 500 g/mol. Preferred small molecules are biologically active in that they produce a biological effect in animals, preferably mammals, more preferably humans. Small molecules include, but are not limited to, radionuclides and imaging agents. In certain embodiments, the small molecule is a drug. Preferably, though not necessarily, the drug is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body. For example, drugs approved for human use are listed by the FDA under 21 C.F.R. §§ 330.5, 331 through 361, and 440 through 460, incorporated herein by reference; drugs for veterinary use are listed by the FDA under 21 C.F.R. §§ 500 through 589, incorporated herein by reference. All listed drugs are considered acceptable for use in accordance with the present invention.

[0028] As used herein, the term "stiffness" refers to a resistance to deformation.

Stiffness may refer to the resistance of an elastic, pseudoelastic or viscoelastic object to deformation. Stiffness may be isotropic or anisotropic. Stiffness may or may not depend on the shape, size or boundary conditions of the object. In some embodiments, stiffness is measured as a ratio of force and displacement. In some embodiments, stiffness is measured as a ratio of an applied moment and a rotation. In some embodiments, stiffness is measured as a ratio of applied shear force and shear deformation. In some embodiments, stiffness is measured as a ratio of applied torsion moment and angle of twist. In some embodiments, stiffness is measured as a ratio of stress and strain (i.e., Elastic modulus, Young's modulus or apparent Young' s modulus). Other appropriate measures of stiffness will be apparent to the skilled artisan.

[0029] As used herein, the term "subject" refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but are not so limited. Preferred subjects are human subjects. The human subject may be a pediatric, adult or a geriatric subject. The human subject may be of either sex.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 depicts a non-limiting example of a cross- section of primary matrix poured into the mold.

[0031] Figure 2 depicts a non-limiting example of primary matrix having a matrix-free cavity. [0032] Figure 3 depicts a non-limiting example of primary matrix having a region comprising a secondary matrix with cells.

[0033] Figure 4 depicts a non-limiting example of primary matrix having multiple regions comprising secondary matrix and cells.

[0034] Figure 5 depicts a non-limiting example of primary matrix having two parallel cylindrical regions, one comprising cells and secondary matrix, and the other being matrix- free.

[0035] Figure 6 depicts a non-limiting example of primary matrix having two parallel cylindrical regions, one comprising endothelial cells and a secondary matrix, and the other comprising muscle cells and a secondary matrix.

[0036] Figure 7 depicts a non-limiting example of bundling multiple regions of differentiated muscle cells.

[0037] Figure 8 depicts a non-limiting example of fibrin gel contraction following cell seeding of three dimensional tissue constructs.

[0038] Figure 9A and 9B depict a non-limiting example of myotubes produced in a three dimensional tissue construct. Fibrous structures are F-actin. Nuclei are also stained.

[0039] Figure 10A and 10B depict non-limiting examples of fascicle-like structures produced in a three-dimensional tissue construct. Solid bodies were calculated from a stack of 2D microscopic images of F-actin staining and were displayed using Imaris, which is an imaging software.

[0040] Figure 11 depicts a non-limiting example of a procedure for producing gel casting molds.

[0041] Figure 12A depicts a non-limiting example of a gel casting mold mounted to a glass substrate with a wire passing through cell entry ports.

[0042] Figure 12B depicts a non-limiting example of a gel casting mold mounted to a glass substrate.

[0043] Figure 12C depicts non-limiting examples of gel casting molds for single seeding and dual seeding wells, and for creating multiple tissue constructs per well.

[0044] Figure 13 depicts a non-limiting example of a method for producing three- dimensional tissue constructs using sacrificial gels. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

[0045] Cell culture devices are provided herein that are useful for evaluating and characterizing cell growth and differentiation and preparing 3-D constructs. The devices are particularly useful for creating tissue-like structures, including components of muscle tissue (e.g., myotubes). The devices may be used for testing cell-cell interactions, cell-matrix interactions, or autocrine or paracrine signaling. The devices may be used for growing artificial organs (e.g., muscles), growing tissues, growing tissues with associated vasculature, etc. The devices provide a basis for evaluating the effects of test agents (e.g., therapeutic candidates) on cell function (e.g., growth and/or differentiation). The devices provide a three-dimensional context for characterizing the differentiation capacity of progenitor cells. The devices also provide a basis for identifying and characterizing genes that modulate cell function (e.g., genes that are involved in cell growth and/or differentiation). Thus, the devices provide a platform for biomarker discovery and therapeutic target identification.

Cell Culture Devices

[0046] The cell culture devices provided herein are composites that typically comprise a primary matrix and one or more regions comprising a secondary matrix. The secondary matrix, is typically different than the primary matrix in terms of its chemical and/or structural characteristics (e.g., stiffness, permeability) and provides a substrate or moieties for cell attachment. The cells organize and arrange themselves within the secondary matrix as a result of the relative stiffness between the secondary matrix and primary matrix.

Accordingly, characteristics of the primary matrix and secondary matrix may be tuned to control the organization and arrangement of the cells within the device.

[0047] The difference in stiffness between the secondary matrix of each region and the primary matrix may be designed and constructed to direct growth and/or alignment of the cells in three-dimensions and/or to promote cell differentiation or change. Accordingly, the cell culture devices have stiffness gradients within a three-dimensional composite matrix. The gradient may be continuous, a step-change in stiffness, or a combination of both. For example, the boundary between a region of the primary matrix and a region of the secondary matrix may be a step-change in stiffness between the primary matrix and the secondary matrix. Alternatively, the boundary between a region of the primary matrix and a region of the secondary matrix may be a continuous gradient of change in stiffness between the primary matrix and the secondary matrix. Alternatively, the boundary between a region of the primary matrix and a region of the secondary matrix may define a combination of a step- change and a continuous gradient of change in stiffness between the primary matrix and the secondary matrix.

[0048] The stiffness of the primary matrix may be less than the stiffness of the secondary matrix or greater than the stiffness of the secondary matrix, depending on the cell type and process under evaluation. The primary matrix may have an elastic modulus in a range of approximately 0.1 kPa to approximately 1000 kPa, approximately 0.5 kPa to approximately 500 kPa, approximately 0.5 kPa to approximately 250 kPa, approximately 0.5 kPa to approximately 200 kPa, approximately 0.5 kPa to approximately 100 kPa, approximately 0.5 kPa to approximately 50 kPa, approximately 0.5 kPa to approximately 10 kPa, or

approximately 0.5 kPa to approximately 6 kPa. The primary matrix may have an elastic modulus of approximately 0.1 kPa, 0.5 kPa, 1 kPa, 2 kPa, 5 kPa, 10 kPa, 20 kPa, 50 kPa, 100 kPa, 200 kPa, 500 kPa, 1000 kPa, or more. The secondary matrix may have an elastic modulus in a range of 0.1 kPa to 1000 kPa, 0.5 kPa to 500 kPa, 0.5 kPa to 250 kPa, 0.5 kPa to 200 kPa, 0.5 kPa to 100 kPa, 0.5 kPa to 50 kPa, 0.5 kPa to 10 kPa, or 0.5 kPa to 6 kPa. The secondary matrix may have an elastic modulus of approximately 0.1 kPa, 0.5 kPa, 1 kPa, 2 kPa, 5 kPa, 10 kPa, 20 kPa, 50 kPa, 100 kPa, 200 kPa, 500 kPa, 1000 kPa, or more.

[0049] In some cases where the stiffness of the primary matrix is less than the stiffness of the secondary matrix, the primary matrix may have an elastic modulus of up to approximately 0.5 kPa, and the secondary matrix may have an elastic modulus of greater than 0.5 kPa (optionally up to 300 kPa, or optionally up to 1 MPa).

[0050] In some cases, the difference in stiffness between the primary matrix and secondary matrix is a difference in elastic modulus of above 0.1 kPa, above 0.5 kPa, above 1 kPa, above 5 kPa, above 10 kPa, above 20 kPa, above 50 kPa, above 100 kPa, above 200 kPa, or above 500 kPa.

[0051] The primary matrix and/or the secondary matrix may comprise a fibrin gel, an alginate gel, a collagen gel, an agarose gel, matrigel, gelatin, a biopolymer, biodegradable synthetic polymers such as polyglycolic acid (PGA), poly-L-lactic acid, poly(lactic acid) (PLA), polyhydroxyalkanoate, poly-4-hydroxybutyrate, polycaprolactone-co-polylactic acid, polyethylene glycol (PEG), poly(glycerol sebacate) (PGS), or any combination thereof. The primary matrix and/or secondary matrix may be biodegradable. Matrices are typically biocompatible. Where a primary matrix and secondary matrix consist of the same polymeric constituents, different types and/or concentrations of cross-linking agents may be used, in some cases, to achieve different structural characteristics (e.g., permeability, stiffness, etc.) in the primary matrix compared with the secondary matrix. For example, where a primary matrix and secondary matrix are both fibrin gels, different concentrations of fibrinogen and/or thrombin may be used in the two matrices to achieve different structural

characteristics. A primary matrix may also comprise different polymeric constituents than a secondary matrix to achieve different structural characteristics (e.g., permeability, stiffness, etc.) than the secondary matrix. For example, the primary matrix may be an agarose gel, and the secondary matrix may be a fibrin gel. The stiffness of the agarose gel may be tuned by controlling the concentration of agarose. The fibrin gel may be tuned by controlling the concentrations of fibrinogen and thrombin. Further examples of primary and secondary matrix compositions will be apparent to the skilled artisan and are disclosed elsewhere herein.

[0052] The primary matrix and/or secondary matrix may comprise any one or more of a variety of different polymers, including synthetic or natural polymers. The primary matrix and/or secondary matrix may comprise polyesters, polyethers, polyamides, polycarbonates, polyureas, polystyrenes, polypeptides, polysaccharides, polyacrylates, polyacrylamides, etc.

[0053] Typically, the primary matrix and secondary matrix comprise fibrinogen and thrombin, and the concentrations of fibrinogen and thrombin are tuned to control the stiffness of the gel. A non-limiting example of parameters for tuning the stiffness of a fibrin gel are disclosed in Duong et ah, Modulation of 3D Fibrin Matrix Stiffness by Intrinsic Fibrinogen- Thrombin Compositions and by Extrinsic Cellular Activity, Tissue Engineering; Part A Volume 15, Number 7, 2009, incorporated herein by reference.

[0054] The primary matrix and/or secondary matrix may comprise fibrinogen at a concentration of approximately 0.1 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, or more. The primary matrix and/or secondary matrix may comprise fibrinogen at a concentration in a range of approximately 0.1 mg/ml to 5 mg/ml, 0.5 mg/ml to 10 mg/ml, 1 mg/ml to 15 mg/ml, 1 mg/ml to 20 mg/ml, 5 mg/ml to 30 mg/ml, 10 mg/ml to 50 mg/ml or 0.1 mg/ml to 50 mg/ml. The primary matrix and/or secondary matrix may comprise thrombin at a

concentration in a range of approximately 0.1 NIH Units /ml to 100 NIH Units / ml, 0.1 NIH Units /ml to 50 NIH Units / ml, 0.1 NIH Units /ml to 20 NIH Units / ml, 0.1 NIH Units /ml to 10 NIH Units / ml, 0.1 NIH Units /ml to 5 NIH Units / ml. The primary matrix and/or secondary matrix may comprise thrombin at a concentration of 0.1 NIH Units /ml, 0.5 NIH Units / ml, 1 NIH Units / ml, 2 NIH Units / ml, 5 NIH Units / ml, 10 NIH Units / ml, 15 NIH Units / ml, 20 NIH Units / ml, 50 NIH Units / ml, 100 NIH Units / ml, or more. The primary matrix and/or secondary matrix may contain trace amounts of thrombin. The primary matrix and/or secondary matrix may be free of thrombin. The primary matrix and/or secondary matrix may comprise up to 1 NIH Unit/ml of thrombin.

[0055] In other embodiments, when the primary matrix and secondary matrix are both fibrin gels, the primary matrix and secondary matrix comprise thrombin and fibrinogen at different concentrations to achieve different stiffness properties. For example, the primary matrix may comprise 1 mg/ml to 10 mg/ml of fibrinogen, and 0.1 NIH Units /ml to 5 NIH Units / ml of thrombin, while the secondary matrix comprises above 10 mg/ml to 50 mg/ml of fibrinogen and above 5 NIH Units /ml to 100 NIH Units / ml of thrombin. The primary matrix may comprise about 10 mg/ml of fibrinogen and about 1 NIH Units / ml of thrombin, while the secondary matrix comprises about 10 mg/ml of fibrinogen and no thrombin. The primary matrix may comprise about 1% to 5% agarose (weight to volume). The primary matrix may comprise about 0.1% to 10% agarose (weight to volume). The primary matrix may comprise about 1% to 5% alginate (weight to volume). The primary matrix may comprise about 0.1% to 10% alginate (weight to volume). The primary matrix may comprise about 1 mg/ml to 5 mg/ml collagen Type-I. The primary matrix may comprise about 0.1 mg/ml to 10 mg/ml collagen Type-I. The secondary matrix may comprise about 1 mg/ml to 5 mg/ml collagen Type-I. The secondary matrix may comprise about 0.1 mg/ml to 10 mg/ml collagen Type-I.

[0056] In some embodiments, the primary matrix is produced such that it solidifies at a relatively low temperature (e.g. , at or below room temperature) and melts at a relatively high temperature (e.g. , at a temperature suitable for cell growth, e.g., about 37 °C). In one embodiment, the primary matrix is maintained at a relatively low temperature (e.g., 4°C to 26 °C) while the secondary matrix is introduced (typically together with a cell suspension) into a cavity within the primary matrix. The secondary matrix solidifies at the relatively low temperature, forming a solid region within the cavity of the primary matrix that conforms to the dimensions of the cavity, and does not melt at the temperature at which the primary matrix melts (e.g. , the secondary matrix melts at a temperature above that at which the primary matrix melts). In one embodiment, the device is exposed (e.g. , by placing the device in an incubator) to a relatively high temperature (e.g., 37 °C) to melt the primary matrix and release the secondary matrix. This methodology may be used to produce a matrix having cells distributed within it and having a defined geometry. Thus, in some embodiments, the primary matrix functions as a sacrificial mold for making one or more matrices that have defined geometries. In some embodiments, this methodology is used to produce one or more matrices that have a defined shape (e.g., a muscle fascicle-like shape) and within which cells (e.g., myogenic cells) are distributed.

[0057] In some embodiments, the primary matrix comprises gelatin and the secondary matrix comprises a fibrin gel within which cells (e.g. , myogenic cells, e.g. , myoblasts) are distributed. In some embodiments, the primary matrix comprises gelatin at a concentration of about 10 w/v and the secondary matrix comprises a fibrin gel with a fibrinogen

concentration of about 10 mg/ml and thrombin concentration of about 5 NIH Units/ml within which cells (e.g. , myogenic cells, e.g. , myoblasts) are distributed. In one embodiment, the gelatin that makes up the primary matrix solidifies at a relatively low temperature (e.g. , at or below room temperature, e.g., 4°C to 26 °C) and melts at a relatively high temperature (e.g. , a temperature suitable for cell growth, e.g. , 37 °C). Thus, in some embodiments, a primary matrix comprising gelatin is used as a sacrificial mold. The primary gelatin matrix may be prepared using methods well known in the art. For example, the primary matrix may be prepared by dissolving gelatin in water at a relatively high temperature, and subsequently exposing the dissolved gelatin to a relatively low temperature to solidify the gelatin. The primary matrix may be produced by dissolving gelatin in water at a concentration of about 1% weight by volume (w/v), about 5% w/v, about 10% w/v, about 15% w/v, about 20% w/v, about 25% w/v, about 30% w/v, about 35% w/v, about 40% w/v, about 45% w/v, about 50% w/v, about 55% w/v, about 60 % w/v or more. The primary matrix may be produced by dissolving gelatin in water at a concentration in a range of 1 % w/v to 5 % w/v, 1 % w/v to 10 % w/v, 5 % w/v to 25 % w/v, 10 % w/v to 25 % w/v, 15 % w/v to 40 % w/v, 20 % w/v to 50 % w/v, or 40 % w/v to 60 % w/v. Other suitable concentrations of gelatin will be apparent to the skilled artisan.

[0058] The shape of each region may be designed and constructed to direct alignment of the cells in three-dimensions and/or to promote cell differentiation. Accordingly, the regions within the primary matrix may have any of a variety of shapes. The regions may have a polyhedron-like shape, a cylindrical shape, a torus-like shape , spherical shape, or a ellipsoidal shape. A plurality of differently shaped regions may be present in the primary matrix. Typically, the regions have an elongated shape. The elongated shape may have a length in a range of approximately 1 mm to approximately 1500 mm, 1 mm to 200 mm, 1 mm to 100 mm, 5 mm to 50 mm, or 5 mm to 10 mm. The elongated shape may have a length of up to 1 mm, 2 mm, 2.5 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 50 mm, 100 mm, 150 mm, 200 mm, 250 mm, 500 mm, 1000 mm, 1500 mm, or more. The elongated shape may have an average width (e.g. , diameter) in a range of 10 μπι to 5000 μπι, 10 μπι to 2500 μπι, 10 μπι to 2000 μπι, 10 μπι to 1500 μπι_^ 10 μπι to 1000 μπι_^ 10 μπι to 500 μπι, 10 μπι to 250 μπι, 10 μπι to 200 μπι, 10 μπι to 150 μπι, or 100 μπι to 1000 μπι. The elongated shape may have an average width (e.g. , diameter) of up to 10 μπι, 20 μπι, 25 μπι, 50 μπι, 100 μπι, 200 μπι, 250 μπι, 500 μπι, 1000 μm, or more. The elongated shape may have an average width (e.g. , diameter) of less than 1600 μπι. One or more of the secondary matrix regions may extend through the primary matrix.

[0059] The cell culture device often comprises a support structure (e.g. , mold) that interfaces with the primary matrix. The support structure may, among other things, serve to immobilize the primary matrix, provide a mold for casting the primary matrix and/or secondary matrix, and provide one or more conduits for circulating a fluid (e.g. , a cell culture media), test agents and other components to and/or from the device (e.g., circulating a fluid through matrix-free channels of the primary matrix). The support structure comprises a polymer, metal, ceramic, glass, Velcro or a combination thereof. The support structure may comprise, for example, a polymeric organosilicon compound or an acrylic compound. A non- limiting example of a polymeric organosilicon compound is polydimethylsiloxane (PDMS). A non-limiting example of an acrylic compound is polymethylmethacrylate (PMMA). A non-limiting example of a metal is aluminum. The support structure may comprise a coating (e.g. , a conformal coating, Teflon coating, etc.) to prevent sticking of the matrix.

[0060] The primary matrix may comprise one or more matrix-free channels through which a fluid, such as, for example, a cell culture medium may be circulated to provide nutrients and other factors to the cells in the device and to remove waste from the device. The matrix-free channels, like the one or more regions, may have an elongated shaped that passes through the primary matrix. For example, the matrix-free channels may have a length in a range of approximately 1 mm to 1500 mm, 1 mm to 200 mm, 1 mm to 100 mm, 5 mm to 50 mm, or 5 mm to 10 mm. The matrix-free channels may have a length of up to 1 mm, 2 mm, 2.5 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 50 mm, 100 mm, 150 mm, 200 mm, 250 mm, 500 mm, 1000 mm, 1500 mm, or more. The matrix-free channels may have an average width (e.g. , diameter) in a range of 10 μπι to 5000 μπι, 10 μπι to 2500 μπι, 10 μπι to 2000 μπι, 10 μπι to 1500 μπι_^ 10 μπι to 1000 μπι_^ 10 μπι to 500 μπι, 10 μπι to 250 μπι, 10 μπι to 200 μπι, 10 μπι to 150 μπι, or 100 μπι to 1000 μπι. The elongated shape may have an average width (e.g., diameter) of up to 10 μιη, 20 μιη, 25 μιη, 50 μιη, 100 μιη, 200 μιη, 250 μm, 500 μιη, 1000 μm, or more. Distances between matrix free channels and secondary matrix regions may be in a range of 100 μιη to 5000 μιη, for example.

[0061] The matrix-free channels are typically designed and constructed to accommodate flow of a fluid. The support structure may comprise one or more fluid inlets that are fluidically connected with one or more of the matrix-free channels. The support structure may also comprises one or more fluid outlets that are fluidically connected with one or more of the matrix-free channels. A reservoir may be fluidically connected with one or more of the matrix-free channels, e.g. , via the fluid inlets of the support structure. A pump may be installed to perfuse fluid from a reservoir through one or more of the matrix-free channels. A suitable controller may be connected to the pump to control the fluid flow rate through the device. The fluid flow rate may be controlled to ensure a proper circulation of nutrients and removal of waste to ensure cell homeostasis in the device.

[0062] Fluid circulating through the device may comprise one or more nutrients, (e.g., carbon source, 0₂, nitrogen source, minerals, vitamins), growth factors, cytokines, or other molecules that diffuse through the primary matrix into one or more of the regions that comprise the secondary matrix. These growth factors, cytokines, or other molecules may, for example, influence the growth, migration and/or differentiation of the cells in the secondary matrix. Fluid circulating through the device may also, or alternatively, comprises a test agent or other agent that diffuses through the primary matrix and contacts cells in the secondary matrix.

[0063] The matrix-free channels may contain cells that may interact with cells of the same or different cell type in adjacent secondary matrix regions. Cells in the matrix-free channels may include endothelial cells, neurons, fibroblasts, stem cells, muscle cells, and cancerous cells. Flow properties through the matrix-free channels may be used to influence the cells in the matrix-free channels, such as inducing shear stress on endothelial cells. Cells may be introduced to the matrix-free channels before or after the secondary matrix regions have been filled with secondary gel with or without cells. As a non-limiting example, endothelial cells may be seeded into a matrix-free channel, and induced to form a monolayer along the channel-primary matrix interface, followed by seeding of muscle precursor cells into an adjacent secondary matrix region.

[0064] The cell culture device may also be configured such that a microscope may be arranged to permit observation within one or more of the regions. Microscopic observation provides a basis for evaluating the phenotype of cells present or growing within the device. Any of a variety of microscopic techniques may be used to evaluate the cells including, for example, phase contrast, fluorescence, or confocal microscopy.

[0065] The cell culture device may also comprise a heat transfer element, which may be used to maintain cells at a predetermined temperature. The predetermined temperature may be in a range of approximately 30 °C to approximately 45 °C. The predetermined temperature is typically about 37 °C. The heat transfer device may also be used to melt the primary matrix and release cells (e.g., differentiated cells, e.g., myotubes) from the device.

[0066] The cell culture device may also comprise a force-transducer configured and arranged to measure the force of contraction produced by cells in the one or more regions, and may comprise a strain gauge configured and arranged to measure the extent of contraction produced by cells in the one or more regions.

Methods for Culturing Cells

[0067] The invention also provides methods for culturing cells using in any of the cell culture devices disclosed herein. Accordingly, the cell culture methods typically involve the use of a cell culture device that includes a primary matrix and one or more regions within the primary matrix that comprise cells within a secondary matrix. The cell culture device is typically maintained under conditions that support viability of the cells. For example, the device may be maintained in vitro or may be implanted in a subject and maintained in vivo to support viability of the cells.

[0068] The secondary matrix of one or more of the regions typically comprises cells. The cells may be added after the matrix has been cast or polymerized. Alternatively, the cells may be added before casting or polymerization of the gel. The cells may be any mammalian cells. The cells may be any human cells. The cells may be of mesenchymal, ectodermal, and endodermal origin. The cells may be selected from the group consisting of cord-blood cells, stem cells, embryonic stem cells, adult stem cells, progenitor cells, induced progenitor cells, autologous cells, heterologous cells, isograft cells, allograft cells, xenograft cells, and genetically engineered cells.

[0069] The cells may be selected from the group consisting of lymphocytes, B cells, T cells, cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells, myeloid cells, granulocytes, basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes, hypersegmented neutrophils, monocytes, macrophages, reticulocytes, platelets, mast cells, thrombocytes, megakaryocytes, dendritic cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, oxyphil cells, adrenal cells, chromaffin cells, pineal cells, pinealocytes, glial cells, glioblasts, astrocytes,

oligodendrocytes, microglial cells, magnocellular neurosecretory cells, stellate cells, boettcher cells; pituitary cells, gonadotropes, corticotropes, thyrotropes, somatotrope, lactotrophs, pneumocyte, type I pneumocytes, type II pneumocytes, Clara cells, goblet cells, alveolar macrophages, myocardiocytes, pericytes, gastric cells, gastric chief cells, parietal cells, goblet cells, paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells, enteroendocrine cells, enterochromaffin cells, APUD cell, liver cells, hepatocytes, Kupffer cells, bone cells, osteoblasts, osteocytes, osteoclast, odontoblasts, cementoblasts, ameloblasts, cartilage cells, chondroblasts, chondrocytes, skin cells, hair cells, trichocytes, keratinocytes, melanocytes, nevus cells, muscle cells, myocytes, myoblasts, myotubes, adipocyte, fibroblasts, tendon cells, podocytes, juxtaglomerular cells, intraglomerular mesangial cells, extraglomerular mesangial cells, kidney cells, kidney cells, macula densa cells, spermatozoa, Sertoli cells, Leydig cells, oocytes, and mixtures thereof.

[0070] In some cases, one or more regions in a primary matrix comprise cells of a first- type and one or more of regions in the primary matrix comprise cells of a second-type. The one or more regions comprising cells of the first-type may be different than the one or more regions comprising cells of the second-type; alternatively, the one or more regions

comprising cells of the first-type may be the same as the one or more regions comprising cells of the second- type (i.e., cells of the first- type and second- type may be present in different regions or in the same region). The cells of the first- type may secrete a growth factor or cytokine that modulates growth and/or differentiation of the cells of the second-type. The growth factor or cytokine secreted by the cells of the first-type may diffuse through the primary matrix into one or more regions comprising cells of the second-type. Thus, the proximity of regions within the primary matrix comprising different cells types may be controlled to influence the exchange of small molecules, growth factors, and cytokines between the regions. Accordingly systems of paracrine and autocrine signaling may be designed into the device. In some cases, cells of the first-type migrate into and through the primary matrix, and may enter into a region comprising secondary matrix that supports growth of cells of the second type. The methods are not limited to only two types of cells, any number of different cell types may be used. [0071] Moreover, any combination of cells may be used in the device. The cells of a first-type for example may be endothelial cells, embryonic stem cells (ESC) and neurons (e.g., motorneurons). Cells of a second type may be myogenic cells, such as, for example, skeletal muscle cells, smooth muscle cells, and cardiomyocytes, or cancer cells. Cells of the first-type may be engineered cells that produce a growth factor or cytokine that modulates growth and/or differentiation of the cells of the second-type. For example, the growth factor may be selected from the group consisting of an Insulin-like Growth Factor-I (IGF-I), Insulin-like Growth Factor- II (IGF- II), Serum response factor (SRF), Hepatocyte Growth Factor, , Fibroblast Growth Factor- 1, Fibroblast Growth Factor-2, Fibroblast Growth Factor- 6, Basic Fibroblast Growth Factor, Wnt3a, Transforming Growth Factor beta (TGF-β), Angiopoietin, and Vascular Endothelial Growth Factor (VEGF). The cytokine may be Retinoic Acid (RA), interleukin-6 or Sonic Hedgehog Homolog (SHH).

[0072] The methods may involve influencing cells to modulate one or more phenotypic characteristics. The methods may involve, for example, inducing the cells to differentiate within the secondary matrix. The cells may be induced to differentiate by, for example, contacting the cells with a growth factor, cytokine or other agent that induces differentiation of the cells. In some cases, various parameters of the secondary matrix are tuned such that presence of the cells within the secondary matrix induces differentiation. For example, the chemical properties of the matrix can be selected to promote differentiation along one or another lineage. Extracellular matrix factors (natural or synthetic) that are known to induced or promote differentiation may be included in the matrix. The degree of cross-linking of the matrix can be controlled to achieve certain matrix stiffness properties or permeability.

Certain cells differentiate when attached to a relatively stiff substrate; others differentiate when attached to a relatively compliant substrate. Thus, the stiffness of the matrix may be controlled accordingly.

[0073] In some cases, the delivery of nutrients, growth factors, cytokines, or other molecules is important for inducing differentiation of the cells within the secondary matrix. These factors, as described above, can be delivered via diffusion through the primary matrix from a matrix-free perfusion channel. Additionally, a gradient of these factors may be generated and controlled within the primary matrix and secondary matrix regions by utilizing a plurality of matrix-free channels with different independent factor concentrations flowing through them with independently controlled flow rates. Depending on the magnitude of pressure within matrix-free channels, and porosity of primary and secondary matrix gels, these chemicals may travel due to hydraulic force as well as diffusion. The primary matrix may also, or alternatively, be incubated in bath containing a cell culture medium with the growth factors, cytokines and other molecules, such that the molecules diffuse through the primary matrix to contact cells in the secondary matrix. In some cases, where the secondary matrix extends through the primary matrix, the secondary matrix may be in direct contract with the bath or perfusion channel. In any event, the porosity of the primary matrix and/or secondary matrix may be tuned to ensure that the growth factors, cytokines and other molecules can adequately travel via diffusion or pressure driven flow through the matrices and contact the cells. Other parameters of the matrices may also be controlled including for example the pH of the matrix, the ionic strength in the matrix, the hydrophilicity or hydrophobicity of the matrix, the polarity of the matrix, etc.

[0074] In some cases, the methods for producing myotubes are provided. For example, methods are provided that involve (a) culturing myogenic cells in a device comprising a primary matrix and one or more regions within the primary matrix that comprise a secondary matrix, in which the myogenic cells reside in the secondary matrix, and (b) maintaining the device under conditions that induce differentiation of the myogenic cells. The myogenic cells may be mesenchymal stem cells, myogenic stem cells, satellite cells, myoblasts or fibroblasts. The myogenic cells may be identifiable by any of the following biomarkers: Pax 7+, CD34+, CD45-, and Seal-.

[0075] Typically, the differentiated myogenic cells fuse to form multinucleated myotubes within the secondary matrix. Typically, the myotubes are arranged according to the shape and size of the secondary matrix. The shape of each region and/or the difference in stiffness between the secondary matrix of each region and the primary matrix may be designed and constructed to promote differentiation of the myogenic cells. The shape of each region may be designed and constructed to direct alignment of the myogenic cells, or myotubes formed therefrom. The difference in stiffness between the secondary matrix of each region and the primary matrix may be designed and constructed to direct alignment of the myogenic cells, or myotubes formed therefrom.

[0076] Moreover, by having a plurality of regions that comprise myogenic cells within a secondary matrix, multiple myotubes may be formed using a single device. The multiple myotubes can be isolated from the primary matrix and bundled together in some cases. The bundled myotubes can be used in vitro, e.g., to study myotube function {e.g., contractility mechanisms) or implanted in vivo for therapeutic purposes. Methods for Evaluating Test Agents, Gene Function and Test Cells

[0077] The invention also provides methods for evaluating the effects of a test agent on cell function (e.g., growth, differentiation, etc.). The methods often involve culturing cells in a device comprising a primary matrix and one or more regions within the primary matrix that comprise a secondary matrix, in which the cells reside in the secondary matrix, and in which the stiffness of the secondary matrix is different (greater than or less than) the stiffness of the primary matrix. The cells residing within the secondary matrix are contacted with the test agent and the effects of the test agent on the cells are evaluated. For example, the effect that the test agent has on differentiation of the cells within the secondary matrix may be determined.

[0078] In some cases, if the cells do not differentiate within the secondary matrix, the test agent is identified as inhibiting differentiation of the cells. For example, if the cells are treated with a growth factor, cytokine or other molecule that is known to induce

differentiation and, in the presence of the test agent, do not differentiate within the secondary matrix, then the test agent is identified as inhibiting differentiation of the cells. In other cases, if the cells do differentiate within the secondary matrix, the test agent is identified as inducing differentiation of the cells. For example, if the cells are maintained under conditions that are known to not induce differentiation and, in the presence of the test agent, the cells do differentiate, then the test agent is identified as inducing differentiation of the cells. For example, if the cells are treated with a growth factor, cytokine or other molecule that is known to inhibit differentiation and, in the presence of the test agent, the cells do differentiate within the secondary matrix, then the test agent is identified as inducing differentiation of the cells.

[0079] Methods for evaluating the effect of a test agent on myogenesis are also provided. For example, the methods may include culturing myogenic cells in a cell culture device of the invention; contacting the myogenic cells with the test agent; and determining whether the myogenic cells form myotubes within device (e.g., within the secondary matrix of the device).

[0080] In some cases, if the cells do not undergo myogenesis, the test agent is identified as inhibiting myogenesis of the cells. For example, if the cells are treated with a growth factor, cytokine or other molecule (e.g., IGF-I) that is known to induce myogenesis and, in the presence of the test agent, do not undergo myogenesis, then the test agent is identified as inhibiting myogenesis of the cells. In other cases, if the cells do undergo myogenesis the test agent is identified as inducing myogenesis of the cells. For example, if the cells are maintained under conditions that are known to not induce myogenesis and, in the presence of the test agent, the cells do undergo myogenesis, then the test agent is identified as inducing myogenesis of the cells. For example, if the cells are treated with a growth factor, cytokine or other molecule that is known to inhibit myogenesis (e.g., TGF-βΙ) and, in the presence of the test agent, the cells do undergo myogenesis within the secondary matrix, then the test agent is identified as inducing myogenesis of the cells.

[0081] The invention also relates to methods for evaluating the role of a gene product on cell differentiation. For example, the methods may involve culturing cells in a device comprising a primary matrix and one or more regions within the primary matrix that comprise a secondary matrix, in which the cells reside in the secondary matrix, and in which the stiffness of the secondary matrix is different (greater than or less than) the stiffness of the primary matrix. The cells are typically contacted a test agent (e.g. , siRNA, antibody, etc.) that inhibits the gene product; and a determination is made as to whether the cells

differentiate within the secondary matrix. When the gene product is a protein, the test agent may be a short-interfering nucleic acid that specifically targets the mRNA encoding the protein. When the gene product is a cell-surface protein (e.g., a receptor), the test agent may be antibody or antigen-binding antibody fragment that specifically targets and inactivates (or activates) the protein. When the gene product is a non-coding RNA, the test agent may be a short-interfering nucleic acid that specifically targets the non-coding RNA.

[0082] In some cases, if the cells do not undergo myogenesis, the gene product is identified as inducing myogenesis of the cells. For example, if the cells are treated with a growth factor, cytokine or other molecule (e.g. , IGF-I) that is known to induce myogenesis and, in the presence of an agent that inhibits the gene product, do not undergo myogenesis, then the gene product is identified as inducing myogenesis of the cells. In other cases, if the cells do undergo myogenesis the gene product is identified as inhibiting myogenesis of the cells. For example, if the cells are maintained under conditions that are known to not induce myogenesis and, in the presence of the test agent that inhibits the gene product, the cells do undergo myogenesis, then the gene product is identified as inhibiting myogenesis of the cells.

[0083] The invention also provides methods for evaluating the ability of a test cell to undergo myogenic differentiation. The methods typically involve culturing test cells (e.g. , stem cells) in a device comprising a primary matrix and one or more regions within the primary matrix that comprise a secondary matrix, in which the test cells reside in the secondary matrix, and in which the stiffness of the secondary matrix is greater than the stiffness of the primary matrix. The cells are typically maintained in the cell culture device under conditions that induce the myogenic cells to form myotubes within the secondary matrix; and a determination is made as to whether the test cells form myotubes within the secondary matrix.

[0084] The effectiveness of a test agent, the role of the gene product or the extent of differentiation may be established by comparing assay results with an appropriate standard. An appropriate standard may be experimentally determined or may be pre-existing (e.g., a historical quantity, etc.) For example, an appropriate standard for a positive effect on differentiation can be established by evaluating the effects of an agent known to induce differentiation. An appropriate standard for a negative effect on differentiation can be established by evaluating the effects of an agent known to inhibit differentiation. In any case, the appropriate standard may be a value indicative of differentiation status. For example, in the case of myogenic differentiation, the appropriate standard may be a number of nuclei per myotube, length of myotubes, fraction of non-differentiated to differentiated cells in a microscopic image field or within a region of a primary matrix. An appropriate standard may also be an image or images, or descriptive and/or quantitative features thereof, that are characteristic of a differentiated state or non-differentiated state.

Methods for Producing Cell Culture Devices

[0085] The invention also relates to methods for producing cell culture devices. The methods typically involve the production of a primary matrix that at least partially

encompasses one or more solid objects. The primary matrix is typically formed by adding monomers, polymers, cross-linking agents and/or combinations thereof into a support structure (e.g., casting mold) under conditions that result into formation of a cross-linked polymeric network. For example, a primary matrix comprising a fibrin gel can be created by combining fibrinogen and thrombin in a mold under appropriate conditions (e.g., appropriate temperature conditions) for forming a fibrin matrix.

[0086] The solid objects are used to create cavities in the primary matrix. The cavities may be filled with secondary matrix or left vacant to create matrix-free channels. To produce one or more cavities (or regions) having a particular shape (e.g., elongated shape) within the primary matrix, one or more of the solid objects having the desired shape are used. The cavities are produced by removing the solid objects. However, cavities may also be created by casting a primary matrix without solid objects and boring a hole into or through the solidified primary matrix. The cavities can then be filled with a secondary matrix that has the desired set of chemical and structural properties. Typically, the stiffness of the primary matrix produced is different than the stiffness of the secondary matrix produced within the cavities.

[0087] Matrix-free channels may be created by producing a primary matrix that partially encompasses one or more solid objects that extend through the primary matrix. The objects are then removed without being filled with secondary matrix, leaving a matrix free channel. Matrix-free channels may also be created by casting a primary matrix without solid objects and boring a channel (hole) through the solidified matrix.

[0088] In some cases, a stiffening agent is introduced into the cavities either before or after they are filled with the secondary matrix. The stiffening agent diffuses into the primary matrix and produces in the primary matrix a gradient of increasing stiffness directed toward the one or more cavities. When the stiffening agent is introduced after the secondary matrix is added a continuous stiffness gradient or a combination of continuous gradient and step- change in stiffness may be generated, depending on the composition of the primary and secondary matrices. When the stiffening agent is introduced after the secondary matrix is added a step-change in stiffness or a combination of continuous gradient and step-change in stiffness may be generated, depending on the composition of the primary and secondary matrices.

[0089] Often each cavity is filled with the secondary matrix as the solid object is being removed. This can serve in part to prevent collapse of the cavities. In cases where the solid objects extend through the primary matrix, the solid object can be extracted from one end, while a secondary matrix is delivered to the cavity at the other end. According to this method, the secondary matrix is drawn in to the cavity by a negative pressure created by extraction of the solid object.

[0090] As described above the primary matrix and/or the secondary matrix may comprise a fibrin gel, an alginate gel, a collagen gel, an agarose gel, matrigel, gelatin, a biopolymer, or any combination thereof. When the secondary matrix comprises a fibrin gel, filling the cavities may involve mixing a first solution comprising fibrinogen and a second solution comprising thrombin and injecting the mixture into the one or more of the cavities. The mixing and injecting may be accomplished using a device having a first solution comprising fibrinogen in a first syringe and a second solution comprising thrombin in a second syringe, in which the first syringe and the second syringe have tips forming a common port, and wherein mixing occurs as the first solution and second solution are ejected through the common port. Cells may be present as a suspension in the first solution and/or the second solution, and thus, may be delivered to the device concomitantly with the secondary matrix.

Kits

[0091] The cell culture devices described herein may, in some embodiments, be assembled into kits to facilitate their use in assays, diagnostics, biomarker development, research or other applications. Components for producing the cell culture devices may also be assembled into kits for the same purpose. A kit may include one or more containers housing the components of the invention and instructions for use. Specifically, such kits may include one or more cell culture devices described herein, along with instructions describing the intended application and the proper use of these devices. Kits are also provided that include one or more components for producing cell culture devices along with appropriate instructions. Kits may include, for example, matrix constituents, pre-cast matrices, molds for casting matrices, solid regions for producing cavities within primary matrices, cells, etc.

[0092] As used herein, "instructions" can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the invention. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual {e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions may be in a form prescribed by a

governmental agency regulating the manufacture, use or sale of biological products.

[0093] The kit may contain any one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit, e.g., to produce a primary matrix or a secondary matrix. The kit may include a container housing components described herein. The components may be in the form of a liquid, gel or solid (powder). The components may be prepared sterilely, packaged and shipped refrigerated or frozen. Alternatively the

components may be housed in a vial or other container for storage.

[0094] The kit may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag. The kit may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat

sterilization, or other sterilization methods known in the art. The kit may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration etc.

[0095] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

[0096] Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. [0097] Exemplary embodiments of the disclosure will be described in more detail by the following examples. These embodiments are exemplary of the disclosure, which one skilled in art will recognize is not limited to the exemplary embodiments.

EXAMPLES

Example 1: Apparatus and Method for Organizing 3 -Dimensional Cell Structure by Guiding Cells Using Gel Stiffness Gradient

[0098] A cell culture device is produced having a stiffness gradient within a 3D matrix. The gradient may be continuous, a sudden step change in stiffness, or a combination of both. The matrix may be made of various gels. Examples include fibrin gel, collagen gel, agarose, or gelatin. The stiffness gradient may be directed towards internal positions of the matrix. The shape these internal positions make up may be a line or a cylinder, for example, or may be other shapes including a ring, sphere, etc. There may also be a plurality of such shapes within a single device. Cells seeded within the matrix follow direction cues from the stiffness gradient and are directed to move and/or position themselves in the desired shape or shapes.

[0099] A primary matrix may be formed in a mold as shown in Figure 1. Figure 1 is a cross-section of the primary matrix poured into the mold. The primary matrix is removed leaving a cavity in the center as shown in Figure 2. A secondary matrix may then be filled into the cavity of the primary matrix. The secondary matrix may contain cells as shown in Figure 3. In this example, a step in stiffness exists at the interface between the primary and secondary matrices. Additionally, chemicals such as crosslinkers or ligands, may diffuse across the initial boundary of the secondary and primary matrices, generating continuous stiffness gradients. The cells may be directed to differentiate by controlling their position by this method. For example, if the cells are myoblasts, and the stiffness gradient directs them to organize into positions with a distinct local axis, they will differentiate into myotubes aligned with their local axis as shown in Figure 3.

[00100] For devices containing a plurality of distinctly guided shapes, the multiple distinct regions may be used for a number of purposes, and different cell type combinations may be used in different regions. For example, chemicals may transport from one region to another, and or, multiple regions may be bundled together after a differentiation process has occurred independently in each region. As an example, the directed regions may be two distinct shapes, and may be two parallel cylinders as shown in Figure 4. The cells may be designed to grow and develop independently, or co-dependently depending on spacing and matrix parameters. For closer cylinders, in more porous matrices, signaling chemicals will diffuse easily between regions causing co-dependent development. Conversely, for cylinders further from each other, with less porous matrices, chemicals will not diffuse easily between regions, and development within each region will be more independent.

[00101] Regions may be made to have no matrix. These regions may be used as channels to provide perfusion to other regions. For example, there may be two or more parallel cylinder regions, one with cell and a secondary matrix, the other with no matrix as shown in Figure 5. Flow may be generated in the region with no matrix to increase the effectiveness of the perfusion.

[00102] Region to region interaction may influence cell motility directed away from the original directed regions as well. For example, endothelial cells in one region may be directed to travel towards muscle cells in a neighboring region as shown in Figure 6. Such endothelial cell migration can lead to vascularization of the muscles cell providing channels to supply nutrients, and discard waste, in some cases. An example of bundling multiple regions after growth and development time is where multiple parallel regions of differentiated muscle cells are bundled together as shown in Figure 7. Such bundling of muscle cells produces 3D muscle structure similar to that found in vivo.

Example 2: Bioengineered fascicle-like skeletal muscle tissue constructs

Introduction

[00103] Tissue engineered skeletal muscle constructs are useful in treating and evaluating various muscle injuries and diseases. Existing methods of producing skeletal muscle constructs grown in vitro are often limited in that muscle cell density as a fraction of total volume or mass, is often significantly lower than muscle found in vivo. In this example methods are described for producing skeletal muscle constructs in three-dimensions that results in increased muscle cell density compared with existing methods.

[00104] Formation of skeletal muscle typically involves a proliferation and expansion of myoblasts (or any suitable myogenic cells) until a sufficient local cell density is achieved, at which point the cells begin to fuse and form multinucleated myo tubes. These myo tubes mature to form relatively long contractile muscle cells.

[00105] Alignment of force generating cells, such as myotubes, influences their contractile behavior and such alignment is typically observed when cells are grown in an environment with sufficient axial stress and/or geometric constraints. Cells grown under conditions in which cells are not sufficiently aligned typically do not produce axial force as efficiently or effectively

[00106] Myogenic cells grown on two-dimensional substrates (e.g., cell culture dishes) may produce a muscle-like tissue that is limited in thickness, and thus scale, compared with that produced by the same myogenic cells grown in three-dimensions. Aspects of this example are based on the recognition that traditional 3D culture systems for producing muscle tissue and components thereof (e.g., myotubes) often lack scalability due, at least in part, to diffusion limitations at higher scales. Centrally located cells in such systems may migrate to the edges of constructs, appear unhealthy, and/or die resulting in much lower cell density than that found in muscles grown in vivo.

[00107] Increasing cell density in three-dimensional tissue constructs may be advantageous. For example, having more cells per unit area to produce force, increases the net force per area, or stress, of the construct. Also, there may be less stiff material within a tissue culture construct to inhibit contraction of the construct, allowing for greater displacement per length, or strain.

[00108] In this example, muscle cell density is increased at least in part by growing cell constructs in a geometry that is similar to the naturally occurring hierarchical muscle level of the fascicle. A fascicle is a bundle of muscles cells that is generally small enough in diameter to allow for perfusion of nutrients and innervation. It may function as an building block that is robust against individual cell failure, and can be combined in a parallel arrangement with other fascicles thereby allowing for scalability.

[00109] The system described in this example is capable of growing numerous fascicle-like structures in a high throughput manner. Once the constructs are formed, they may be coated with an ECM-like connective tissue and bundled for scaling to larger dimensions. Thus, the example describes a geometrically fascicle-like muscle construct that provides a muscle cell density that is similar to that of muscle tissue grown in vivo.

Materials and methods

[00110] C2C12 mouse myoblasts (American Type Culture Collection) were cultured in growth medium (GM) containing DMEM (American Type Culture Collection), supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 1% penicillin- streptomycin 100X (Invitrogen), and 0.1 mg/ml Normacin (Invivogen). Confluence was kept below 70% and cells were seeded into the experimental device at the 5th passage. [00111] Cell/gel suspension was molded such that a uniform cylinder of controlled dimensions was suspended in medium anchored at both ends to the walls of a well. The cell/gel suspension contained GM with C2C12 cells at a concentration of 15e6 cells/ml, fibrinogen (Sigma- Aldrich) at a concentration of 5 mg/ml, and 6-aminocaproic acid (Sigma- Aldrich) at a concentration of 1 mg/ml. The initial fluid surrounding the construct contained thrombin (Sigma- Aldrich) at a concentration of 1 U/ml. The wells were 5 mm in diameter, with walls made of polydimethylsiloxane (PDMS, Dow Corning) plasma bonded to a glass coverslip (VWR) which constituted the bottom of the well. The length of the molded gels were nominally 5 mm, and the initial nominal diameters of the molded gels ranged from 250 μιη to 500 μιη.

[00112] After 24 hours, the initial fluid was changed to GM supplemented with 1 mg/ml 6-aminocaproic acid. This medium was replaced daily for 2 more days, then switched to differentiation medium, DM, containing DMEM supplemented with 10% horse serum (Sigma- Aldrich) 1% pen-strep, 0.1 mg/ml Normacin, and 1 mg/ml 6-aminocaproic acid. DM was changed daily until the termination of the experiment.

Results

[00113] Constructs were approximately 5 mm long and contracted radially and matured over multiple weeks in an axially uniform manner. After initial seeding, the fibrin gel contracted radially a substantial amount within the first 2 days, reducing the diameter by a factor of approximately 2, as shown in Figure 8. The initial gel contraction occurred with the presence of cells. After initial contraction, the diameter of the construct remained relatively constant for up to about 3 weeks. The initial cross section was approximately a uniform circle. As the construct matured, the thickness along the gravitational axis decreased, as shown in Figure 10A.

[00114] Upon seeding, the gel geometry and cell distribution were uniform along the axis of the construct, and this uniformity along the axis was maintained throughout the experiment, as shown in Figures. 8, 9 and 10B. For particular initial gel diameters, the construct geometric properties were uniform and repeatable.

[00115] As the construct matured, aligned myo tubes were formed and occupied a substantial fraction of the construct cross sectional area. Actin forms aligned with the axis of the construct, and the nuclei elongated along the same axis as seen in Figure 9. Myo tubes were more mature in cells along the bottom plane (Figure 9B) and in the center (Figure 9A) of the construct as opposed to less mature cells along the sides (Figure 9A) and top. The myotubes in the center and along the bottom extended on the order of millimeters in length (e.g., up to 5 mm) and evidence of sarcomeric striations were visible (Figure 9B).

Differences in cell maturity were apparently due to initial settling of cells in the gel, and migration. As the cell/gel solution stiffened, the cells tended to flow due to gravity. This created a concentration of cells close to the bottom of the gel. The cells proliferated and then some fuse and matured into myotubes, while others migrated along the surface of the gel forming a monolayer along the surface.

[00116] The cells broke down the fibrin gel over time. The gel along the bottom was consumed fastest, which was due in part to more cells being present for a longer period of time along the bottom of the construct. This resulted in a decrease in thickness. The tension in the gel and geometric alignment cues promoted alignment as the myoblasts fused and matured into myotubes. The myotubes along the surface combined with the myotubes deeper within the construct both contributed to increasing the myotube density. The cross sectional area occupied by cells was fairly consistently about 30% of the total cross sectional area along the length of the construct. This occupancy was observed in multiple constructs, and was much higher than the approximate <5% found in muscle constructs produced using other technology (M. T. Lam et ah, Biomaterials 2009; S. Hinds et ah, Biomaterials 2011).

Example 3: Protocols for preparing a three-dimensional tissue construct

[00117] Provided below are example protocols for preparing three-dimensional tissue

(e.g., fascicle-like) constructs.

Gel Mold Fabrication

Preparation of POMS molds

1. Start with an aluminum mold with a central pocket 3 mm deep with a smooth sanded bottom.

2. Thread wire through holes in the aluminum pocket walls such that they span the

pocket (Figure 11 top.)

a. The holes are 1.5 mm above the bottom of the pocket and 1.5 mm below the surface of the pocket

3. Pour PDMS a. PDMS may be poured until the surface is just noticeably covering the spanning wires, and using this top surface to bond to glass.

4. Cure in 80 °C oven

5. Remove wires leaving cavities in the PDMS slab

6. Remove PDMS slab from the aluminum mold (Figure 11, ,middle.)

7. Cut PDMS slab into numerous pieces

8. Use biopsy punch to punch holes (1 mm to 6 mm diameter) in PDMS slab pieces (Figure 11 bottom left.)

9. Punch from top of PDMS slab to bottom of PDMS slab

a. Punch through the perpendicular channel(s) created by wires

b. Punch two holes per gel casting mold (one is for a medium reservoir)

10. Bond to glass via plasma bonding ((Figure 11 bottom right.)

11. Re-insert wire into cavities in the PDMS so that the wire spans both punched holes in the gel casting mold. See Figure 12A, B, and C for examples of PDMS molds.

Procedure 1: Fibrin Primary Matrix

[00118] The following is an example of a procedure that may be used to produce fascicle constructs in the presence of a surrounding primary gel.

Casting fibrin hydro gel matrix

1. Add a predetermined quantity (e.g., at least 1 mg) of fibrinogen powder per gel casting mold (depending on the mold size) to a container (e.g., a 1.5 mL centrifuge tubes) (e.g., 6.5 mg/tube maximum)

a. Weigh container and record

b. Add fibrinogen in a sterile BSC (biosafety cabinet)

c. Weigh again. Mark difference between measurements on tube.

2. Add GM+ to make 5 mg/ml fibrinogen in GM+ (X mg fibrinogen * 200 μΐ/mg GM+ = 200 X GM+)

a. Tap tube on side to scatter fibrinogen along the side of the tube to ensure the powder is not compressed on the bottom

b. Add warm GM+ and quickly mix 3. Place tube(s) in 37°C water bath until the fibrinogen is dissolved (~1 minute). Vortex gently and tap upside down if fibrinogen is settling and solidifying on the bottom of the tube. Avoid gelation of the contents.

4. Prepare the BSC for adding the fibrinogen solution to the gel casting molds. The solution solidifies quickly at room temperature.

a. Position gel casting molds, e.g., 6 molds per fibrinogen solution tube. b. Set 1ml pipette to at least 100 μΐ per gel casting mold (150 μΐ per gel casting mold is safer) (900 μΐ for 6 gel casting molds).

c. Position tweezers and pipette tips near the gel casting molds.

5. Add fibrinogen solution to experiment well

a. Avoid getting solution foam on pipette tip when drawing solution b. Draw full volume of tube into pipette tip

c. Hold the gel casting mold with tweezers to prevent it from moving while

filling.

d. Place the tip of the pipette at the bottom of the experiment well, just underneath steel pin to ensure no air pockets are left.

6. Place gel casting molds in 37°C incubator for at least 30 minutes before seeding cell suspension, monitor durations approaching 4 hours to avoid drying up of the solution.

Seeding hydrogel cell suspension

1. Trypsinize cells, count, and spin

2. Add fibrinogen to 1.5 ml tubes

a. Weigh 1.5 ml centrifuge tube and record

b. Add fibrinogen in sterile BSC

c. Weigh again and mark difference between measurements on tube

3. Add medium to fibrinogen to make fibrinogen solution

a. Tap tube on side to scatter fibrinogen along the side of the tube to ensure the fibrinogen powder is not compressed on the bottom

b. Depending on desired fibrinogen concentration (2-10 mg/ml), add appropriate amount of GM+ to tube ( (mg of F) / (X mg/ml) x (1000 μ1/πύ)= μΐ GM+) c. Tap tube to ensure no air bubbles are in the tube

d. Place in 37 C water bath until fibrinogen is dissolved in fluid (e.g., not more than 2 minutes or contents will gel) e. Put tube on ice. Solution will gel in about 30 minutes

4. Add appropriate amount of fibrinogen solution to pellet to obtain desired cell

concentration and mix with pipette until cell suspension is homogeneous ((desired 10⁶ cells/ml) / (10⁶ cell in pellet) x (1000 μΐ/ml )= μΐ fibrinogen solution)

5. Seed cell suspension into gel casting mold

a. Use 100 or 200 μΐ pipette and draw up 20 μΐ of cell suspension with pipette hand

b. Pick up gel casting mold, e.g., between index finger and thumb

c. Remove needle with pipette hand with single smooth motion

d. Position pipette tip at conduit entrance and pipette cell suspension e. Stop when you see fluid entering medium reservoir (avoid drawing fluid back out of conduit).

f. Repeat a-e for each gel casting molds

6. Place seeded gel casting molds in incubator for 30 minutes, then fill medium reservoir with GM+

Continued Culture:

1. Change medium at regular intervals, e.g. , daily

2. After 3-5 days switch from using GM to DM.

Procedure 2: Sacrificial Primary Matrix

[00119] The following is an example of a procedure that may be used to produce fascicle constructs in the absence of a surrounding primary gel. A schematic of aspects of the procedure is provided in Figure 13.

Casting sacrificial gelatin hydrogel

Reagents (for 18 gel casting molds, and T150 flask of cells)

• Gelatin (3 40-65 mg tubes)

• Thrombin solution (100 NIH Units/ml) (36 μΐ)

• NaOH solution (0.5 M) (18 μΐ)

• GM+ (1800 μΐ for reservoir + 210 μΐ for gelatin + 1000 μΐ for fibrinogen < 4 ml )

• GM (18 ml for spinning) • Trypsin (6 ml)

• PBS (24 ml for trypsinizing)

• Fibrinogen (5 mg)

• Cells (T150 flask, with, e.g., cells that are less than 70% confluent)

• Ice

7. Add an appropriate quantity of gelatin powder (e.g. , at least 10 mg) per gel casting mold to an appropriate container (e.g., 1.5 mL tubes) (e.g. , 65 mg/tube maximum) a. Weigh container and record

b. Add gelatin in sterile BSC

c. Weigh again. Mark difference between measurements on tube.

8. Add GM+ to make 50 mg/μΐ gelatin in GM+ (X mg gelatin * 20 μΐ/mg GM+ = 20 X GM+)

a. Tap tube on side to scatter gelatin along the side of the tube to ensure the gelatin powder is not compressed on the bottom

b. Add GM+ and quickly vortex until gelatin particles are well distributed (~5 seconds)

9. Place tube(s) in 37°C water bath until gelatin particles are completely dissolved (~5 minutes). Vortex and tap up-side down if gelatin is settling and solidifying on the bottom of the tube.

10. Add 1% vol/vol NaOH solution to neutralize PH ((X GM+) (0.01) ).

11. Add 1% thrombin solution ((X GM+) (0.01) )

12. Vortex for 5 seconds and return to 37°C water bath until immediately before adding to gel casting molds

13. Prepare the BSC for adding gelatin solution to the gel casting molds. The gelatin solution solidifies quickly at room temperature.

a. Position gel casting molds, e.g., maximum of 6 per gelatin solution tube. b. Set 1ml pipette to, e.g., at least 100 μΐ per gel casting mold (150 μΐ per gel casting mold may be appropriate) (900 μΐ for 6 gel casting molds). c. Position tweezers and pipette tips near the gel casting molds.

14. Add gelatin solution to experiment well a. Avoid getting gelatin solution foam on pipette tip when drawing gelatin solution

b. Draw full gelatin volume of tube into pipette tip

c. Hold the gel casting mold with tweezers to prevent it from moving while

filling.

15. Place gel casting molds in 4 °C refrigerator for before seeding cell suspension;

duration in the refrigerator may vary (e.g., at least 30 minutes); monitor durations approaching 4 hours to avoid drying up of the solution.

Seeding hydrogel cell suspension

7. Trypsinize cells, count, and spin

8. Add fibrinogen to 1.5 ml tubes

a. Weigh 1.5 ml centrifuge tube and record

b. Add fibrinogen in sterile BSC

c. Weigh again and mark difference between measurements on tube

9. Add medium to fibrinogen to make fibrinogen solution

a. Tap tube on side to scatter gelatin along the side of the tube to ensure the fibrinogen powder is not compressed on the bottom

b. Depending on desired fibrinogen concentration (e.g., 2-10 mg/ml), add

appropriate amount of GM+ to tube ( (mg of F) / (X mg/ml) x (1000 μ1/ιη1)= μΐ GM+)

c. Tap tube to ensure no air bubbles are in the tube

d. Place in 37 °C water bath until fibrinogen is dissolved in fluid (typically not more than 2 minutes to avoid gelation)

e. Put tube on ice. Solution will gel in about 30 minutes

10. Add appropriate amount of fibrinogen solution to pellet to get desired cell

concentration and mix with pipette until cell suspension is homogeneous ((desired 10⁶ cells/ml) / (10⁶ cell in pellet) x (1000 μΐ/ml) = μΐ fibrinogen solution)

11. Seed cell suspension into gel casting mold (This is a two handed multistep operation) a. Use 100 or 200 μΐ pipette and draw up 20 μΐ of cell suspension with pipette hand b. Pick up gel casting mold, e.g., between index finger and thumb c. Remove needle with pipette hand with single smooth motion

d. Position pipette tip at conduit entrance and pipet in cell suspension e. Stop when you see fluid entering medium reservoir (avoid drawing fluid out from conduit.)

f. Repeat a-e for each gel casting mold

12. Place seeded gel casting molds in incubator for 30 minutes, then fill medium reservoir with GM+

Remove sacrificial gelatin hydrogel

1. Remove gelatin via careful pipetting while gel casting mold is still warm, and gelatin is liquid leaving the fibrin thread spanning the seeding hole

2. Add GM to medium reservoir and seeding hole such that the fibrin thread is totally submerged

Continued Culture:

3. Change medium at regular intervals, e.g., daily

a. Add -100 μΐ to the top of each hole (depending on culture size)

b. Remove -70 μΐ via pipette from the bottom (depending on culture size) c. It may be desirable to ensure the fascicle is constantly wet, and not strained by surface tension

4. After 3-5 days switch from using GM to DM.

Multiple Fascicle Bundling:

1. When changing medium, remove liquid (e.g., remove essentially all liquid) from the seeding hole

2. Allow surface tension to bring fascicles together

3. Add fresh medium to seeding hole

Materials for Protocols 1 and/or 2

Reagents

PDMS Mold fabrication

• PDMS • Curing agent

Hydro gel and cell culture

• NaOH

• Thrombin

• Fibrinogen

• DMEM

• Fetal Bovine Serum (FBS)

• Gelatin

• Penicillin- streptomycin

• Aminocaproic acid

• trypsin

Equipment

Hydro gel and cell culture

• 1.5 ml tubes

• Water bath

• BSC

• 1 ml and 200 μΐ pipette and tips

• Tweezers

• Refrigerator

• 37 °C Incubator

Reagent Setup

• Growth Medium Plus (GM+): DMEM from ATCC, with 10% fetal bovine serum, 1 mg ml^"1 aminocaproic acid, 100 U ml^"1 penicillin, 100 μg ml^"1 streptomycin.

[00120] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[00121] All references disclosed herein are incorporated by reference in their entirety.

Claims

What is claimed is:

1. A cell culture device comprising:

a primary matrix at least partially encompassing one or more regions that comprise a secondary matrix, wherein there is a continuous gradient of change in stiffness between the primary matrix and the secondary matrix.

2. The cell culture device of any one of claims 1, wherein the secondary matrix of one or more of the regions comprises cells.

3. The cell culture device of claim 2, wherein the cells are myogenic cells.

4. The cell culture device of claim 3, wherein the myogenic cells are C2C12 cells, mesenchymal stem cells, myogenic stem cells, satellite cells, myoblasts, cardiomyocytes, smooth muscle cells, fibroblasts, or neurons.

5. The cell culture device of claim 4, wherein the myogenic cells are Pax 7+, CD34+, CD45-, and/or Seal-.

7. The cell culture device of any one of claims 2 to 5, wherein the shape of each region is arranged to direct alignment of the myogenic cells, or myotubes formed therefrom.

8. The cell culture device of any one of claims 2 to 7, wherein the difference in stiffness between the secondary matrix and the primary matrix directs alignment of the myogenic cells, or myotubes formed therefrom.

9. The cell culture device of any one of claims 2 to 8, wherein the shape of each of the one or more regions and/or the difference in stiffness between the secondary matrix of each of the one or more regions and the primary matrix promote the formation of myotubes by the myogenic cells.

10. The cell culture device of any one of claims 2 to 9, wherein one or more of the regions comprise cells of a first-type and one or more of the regions comprise cells of a second-type.

11. The cell culture device of claim 10, wherein one or more of the regions comprising cells of the first-type are different than one or more of the regions comprising cells of the second-type.

12. The cell culture device of claim 11, wherein cells of the first-type secrete a growth factor or cytokine that modulates growth and/or differentiation of the cells of the second-type.

13. The cell culture device of claim 12, wherein the growth factor or cytokine secreted by the cells of the first-type diffuses through the primary matrix into one or more of the regions comprising the cells of the second-type.

14. The cell culture device of claim 12, wherein the primary matrix and secondary matrix are configured to permit the migration of cells of the first-type into the primary matrix from the secondary matrix.

15. The cell culture device of any one of claims 10 to 14, wherein cells of the first- type are endothelial cells.

16. The cell culture device of any one of claims 10 to 14, wherein cells of the first- type are engineered to produce a growth factor or cytokine that modulates growth and/or differentiation of the cells of the second-type.

17. The cell culture device of claim 12, 13 or 16, wherein the growth factor is an Insulinlike Growth Factor-I (IGF-I), Insulin-like Growth Factor- II (IGF- II), Serum response factor (SRF), Hepatocyte Growth Factor, Fibroblast Growth Factor- 1, Fibroblast Growth Factor-2, Fibroblast Growth Factor-6, Basic Fibroblast Growth Factor, Wnt3a, Transforming Growth Factor beta (TGF-β), Angiopoietin, or Vascular Endothelial Growth Factor (VEGF).

18. The cell culture device of claim 12, 13 or 16, wherein the cytokine is Retinoic Acid (RA), interleukin-6 or Sonic Hedgehog Homolog (SHH).

19. The cell culture device of any one of claims 10 to 18, wherein cells of the second- type are myogenic cells.

20. The cell culture device of any one of claims 1 to 19, wherein the stiffness of the primary matrix is less than the stiffness of the secondary matrix.

21. The cell culture device of any one of claims 1 to 20, wherein the secondary matrix has an elastic modulus of up to 1 MPa.

22. The cell culture device of any one of claims 1 to 21, wherein the secondary matrix has an elastic modulus in a range of 0.5 kPa to 6 kPa.

23. The cell culture device of any one of claims 1 to 21, wherein the primary matrix has an elastic modulus of up to 0.5 kPa.

24. The cell culture device of any one of claims 1 to 23, wherein the primary matrix comprises fibrinogen and thrombin.

25. The cell culture device of claim 24, wherein the primary matrix comprises 1 mg/ml to 100 mg/ml of fibrinogen and up to 100 NIH Units / ml of thrombin.

26. The cell culture device of claim 21, wherein the primary matrix comprises about 5 mg/ml of fibrinogen and about 1 NIH Units / ml of thrombin.

27. The cell culture device of any one of claims 1 to 26, wherein the secondary matrix comprises fibrinogen and thrombin at different concentrations than in the primary matrix.

28. The cell culture device of claim 21, wherein the secondary matrix comprises above 10 mg/ml to 50 mg/ml of fibrinogen and above 5 NIH Units /ml to 100 NIH Units / ml of thrombin.

29. The cell culture device of claim 21, wherein the secondary matrix comprises about 10 mg/ml of fibrinogen and about 1 NIH Units / ml of thrombin.

30. The cell culture device of any one of claims 1 to 29, wherein the primary matrix and/or the secondary matrix comprises a fibrin gel, an alginate gel, a collagen gel, an agarose gel, matrigel, gelatin, a biopolymer, biodegradable synthetic polymers such as polyglycolic acid (PGA), poly-L-lactic acid, poly(lactic acid) (PLA), polyhydroxyalkanoate, poly-4- hydroxybutyrate, polycaprolactone-co-polylactic acid, polyethylene glycol (PEG), poly(glycerol sebacate) (PGS) or any combination thereof.

31. The cell culture device of any one of claims 1 to 30, wherein the primary matrix and/or secondary matrix is biodegradable.

32. The cell culture device of any one of claims 1 to 31, wherein one or more of the regions have a polyhedron-like shape, a cylindrical shape, a torus-like shape or a ellipsoidal shape.

33. The cell culture device of any one of claims 1 to 32, wherein one or more of the regions have an elongated shape.

34. The cell culture device of claim 33, wherein the elongated shape has a length in a range of 1 mm to 200 mm.

35. The cell culture device of claim 34, wherein the elongated shape has a length in a range of 5 mm to 10 mm.

36. The cell culture device of claim 35, wherein elongated shape has an average width in a range of 100 μιη to 3000 μιη.

37. The cell culture device of claim 36, wherein elongated shape has an average width in a range of 100 μιη to 1000 μιη.

38. The cell culture device of any one of claims 1 to 37, wherein one or more of the regions extend through the primary matrix.

39. The cell culture device of any one of claims 1 to 38, wherein the primary matrix further comprises one or more matrix-free channels.

40. The cell culture device of claim 38, wherein one or more of the matrix-free channels have a length in a range of 1 mm to 200 mm.

41. The cell culture device of claim 39 or 40, wherein one or more of the matrix-free channels have a length in a range of 5 mm to 10 mm.

42. The cell culture device of any one of claims 39 to 41, wherein one or more of the matrix-free channels have an average width in a range of 100 μιη to 3000 μιη.

43. The cell culture device of any one of claims 40 to 42, wherein one or more of the matrix-free channels have an average width in a range of 100 μιη to 1000 μιη.

44. The cell culture device of any one of claims 40 to 43, wherein one or more of the matrix-free channels are designed to accommodate flow of a fluid.

45. The cell culture device of any one of claims 1 to 44, wherein the primary matrix surrounded at least in part by a support structure.

46. The cell culture device of claim 44, wherein the support structure comprises a polymer, metal, ceramic, glass, velcro or a combination thereof.

47. The cell culture device of claim 46, wherein the support structure comprises a polymeric organosilicon compound or an acrylic compound.

48. The cell culture device of claim 46, wherein the polymeric organosilicon compound is polydimethylsiloxane (PDMS).

49. The cell culture device of claim 50, wherein the acrylic compound is polymethylmethacrylate (PMMA).

50. The cell culture device of claim 33, wherein the metal is aluminum.

51. The cell culture device of any one of claims 45 to 50, wherein the support structure comprises one or more fluid inlets that are fluidically connected with one or more of the matrix-free channels.

52. The cell culture device of any one of claims 45 to 50, wherein the support structure comprises one or more fluid outlets that are fluidically connected with one or more of the matrix-free channels.

53. The cell culture device of any one of claims 45 to 50 further comprising a reservoir fluidically connected with one or more of the matrix-free channels.

54. The cell culture device of claim 53 further comprising a pump configured for perfusing fluid from the reservoir through one or more of the matrix-free channels.

55. The cell culture device of claim 54, wherein the fluid comprises one or more growth factors or cytokines that diffuse through the primary matrix into one or more of the regions that comprise the secondary matrix.

56. The cell culture device of any of claims 1 to 55 further comprising a microscope arranged to permit observation within one or more of the regions.

57. The cell culture device of any of claims 1 to 56 further comprising a heat transfer element configured and arranged to maintain the cell culture device at a predetermined temperature.

58. The cell culture device of claim 57, wherein the predetermined temperature is in a range of approximately 30 °C to approximately 45 °C.

59. The cell culture device of claim 58, wherein the predetermined temperature is approximately 37 °C.

60. The cell culture device of any one of claims 1 to 59 further comprising a force- transducer configured and arranged to measure the force of contraction produced by cells in the secondary matrix.

61. The cell culture device of any one of claims 1 to 60 further comprising a strain gauge configured and arranged to measure the extent of contraction produced by cells in the one or more regions.

62. A method of culturing cells, the method comprising

obtaining a cell culture device of any one of claims 2 to 61; and

maintaining the cell culture device under conditions that support viability of the cells.

63. A method for culturing cells in vitro, the method comprising:

culturing cells in a device comprising

a primary matrix that at least partially encompasses one or more regions that comprise a secondary matrix, wherein there is a continuous gradient of change in stiffness between the primary matrix and the secondary matrix.

64. A method for producing myotubes in vitro, the method comprising:

culturing myogenic cells in a device comprising a primary matrix that at least partially encompasses one or more regions that comprise a secondary matrix,, wherein the myogenic cells reside in the secondary matrix, and wherein the stiffness of the secondary matrix is greater than the stiffness of the primary matrix; and

maintaining the cell culture device under conditions that induce the myogenic cells to form myotubes within the secondary matrix.

65. A method for evaluating the effect of a test agent on cell differentiation, the method comprising:

culturing cells in a device comprising a primary matrix that at least partially encompasses one or more regions that comprise a secondary matrix,, wherein the cells reside in the secondary matrix, and wherein the stiffness of the secondary matrix is greater than the stiffness of the primary matrix;

contacting the cells with the test agent; and

determining whether the cells differentiate within the secondary matrix.

66. The method of claim 67, wherein if the cells do not differentiate within the secondary matrix then the test agent is identified as inhibiting differentiation of the cells.

67. The method of claim 65, wherein if the cells differentiate within the secondary matrix then the test agent is identified as inducing differentiation of the cells.

68. A method for evaluating the effect of a test agent on myogenesis, the method comprising:

culturing myogenic cells in a device comprising a primary matrix that at least partially encompasses one or more regions that comprise a secondary matrix,, wherein the myogenic cells reside in the secondary matrix, and wherein the stiffness of the secondary matrix is greater than the stiffness of the primary matrix;

contacting the myogenic cells with the test agent; and

determining whether the myogenic cells form myotubes within the secondary matrix.

69. The method of claim 68, wherein if the myogenic cells do not form myotubes within the secondary matrix then the test agent is identified as inhibiting myogenic differentiation of the cells.

70. The method of claim 68, wherein if the myogenic cells form myotubes within the secondary matrix then the test agent is identified as inducing myogenic differentiation of the cells.

71. A method for evaluating the role of a gene product on cell differentiation, the method comprising:

contacting the cells with the test agent that inhibits the gene product; and

determining whether the cells differentiate within the secondary matrix.

72. The method of claim 71, wherein if the cells do not differentiate within the secondary matrix then the gene product is identified as inhibiting differentiation of the cells.

73. The method of claim 71, wherein if the cells differentiate within the secondary matrix then the gene product is identified as inducing differentiation of the cells.

74. A method for evaluating the role of a gene product on myotube formation, the method comprising:

contacting the myogenic cells with the test agent that inhibits the gene product; and determining whether the myogenic cells form myotubes within the secondary matrix.

75. The method of claim 74, wherein if the myogenic cells do not form myotubes within the secondary matrix then the gene product is identified as inhibiting myogenic

differentiation of the cells.

76. The method of claim 74, wherein if the myogenic cells form myotubes within the secondary matrix then the gene product is identified as inducing myogenic differentiation of the cells.

77. The method of any one of claims 74 to 76, wherein the gene product is a protein and wherein the test agent is a short-interfering nucleic acid that specifically targets the mRNA encoding the protein.

78. The method of any one of claims 74 to 76, wherein the gene product is a non-coding RNA and wherein the test agent is a short-interfering nucleic acid that specifically targets the non-coding RNA.

79. The method of any one of claims 74 to 76, wherein the gene product is a cell-surface protein and wherein the test agent is an antibody or antigenic binding fragment thereof that specifically binds to the cell-surface protein.

80. A method for evaluating the ability of a test cell to undergo myogenic differentiation, the method comprising:

culturing test cells in a device comprising a primary matrix that at least partially encompasses one or more regions that comprise a secondary matrix,, wherein the test cells reside in the secondary matrix, and wherein the stiffness of the secondary matrix is greater than the stiffness of the primary matrix;

maintaining the cell culture device under conditions that would induce myogenic cells to form myotubes within the secondary matrix; and

determining whether the test cells form myotubes within the secondary matrix.

81. The method of claim 80, wherein if the test cells do not form myotubes within the secondary matrix then the test cells are identified as non-myogenic cells.

82. The method of claim 80, wherein if the test cells form myotubes within the secondary matrix then the test cells are identified as myogenic cells.

83. The method of claim 82, wherein the test cells are stem cells.

84. The method of claim 83, wherein the stem cells are pluripotent stem cells or induced pluripotent stem cells.

85. A method for producing a cell culture device, the method comprising:

(a) producing a primary matrix that at least partially encompasses one or more solid objects;

(b) removing one or more of the solid objects to produce one or more cavities; and

(c) filling one or more of the cavities with a secondary matrix, wherein the stiffness of the primary matrix produced in (a) is different than the stiffness of the secondary matrix.

86. The method of claim 85 further comprising introducing a stiffening agent into the one or more cavities filled or to be filled with the secondary matrix such that the stiffening agent diffuses into the primary matrix and produces in the primary matrix a gradient of increasing stiffness directed toward the one or more cavities.

87. The method of claim 86, wherein the stiffening agent is introduced before filling the one or more cavities with the secondary matrix in step (a).

88. The method of claim 86, wherein each cavity is filled with the secondary matrix as the solid object is being removed.

89. The method of any one of claims 85 to 88, wherein the primary matrix and/or the secondary matrix comprises a fibrin gel, an alginate gel, a collagen gel, an agarose gel, matrigel, gelatin, a biopolymer, or any combination thereof.

90. The method of any one of claims 85 to 89, wherein filling the one or more cavities with a secondary matrix, in step (a), comprises mixing a first solution comprising fibrinogen and a second solution comprising thrombin and injecting the mixture into the one or more of the cavities.

91. The method of claim 90, wherein mixing and injecting is accomplished using a device having a first solution comprising fibrinogen in a first syringe and a second solution comprising thrombin in a second syringe, wherein in the first syringe and the second syringe have tips forming a common port, and wherein mixing occurs as the first solution and second solution are ejected through the common port.

92. The method claim 90 or 91, wherein the first solution and/or the second solution comprises cells.

93. The method of any one of claims 85 to 88, wherein one or more of the solid objects have an elongated shape.

94. The method of claim 93, wherein the elongated shape is cylindrical or polyhedronlike.

95. The method of any one of claims 93 to 94, wherein one or more of the solid objects extends through the primary matrix.

96. The method of any one of claims 85 to 95, wherein the secondary matrix comprises cells.

97. The method of claim 96, wherein the cells are myogenic cells, optionally which are mesenchymal stem cells, myogenic stem cells, satellite cells, myoblasts or fibroblasts..

98. The method of any one of claims 85 to 104, wherein the stiffness of the primary matrix is less than the stiffness of the secondary matrix.

99. The method of any one of claims 85 to 98, wherein the primary matrix is a gel, and wherein producing the primary matrix in step (a.) comprises casting the gel in a support structure.

100. The method of any one of claims 85 to 99, wherein one or more of the cavities are matrix-free channels in the primary matrix.

101. A kit comprising a container housing the cell culture device of any one of claims 1 to 61 or claims 102 to 112.

102. A cell culture device comprising:

a primary matrix at least partially encompassing one or more regions that comprise a secondary matrix, wherein the primary matrix has a melting temperature that is less than the secondary matrix.

103. The cell culture device of claim 102, wherein the primary matrix melts at a temperature in a range of about 34 °C to 40 °C.

104. The cell culture device of claim 102 or 103, wherein the primary matrix melts at a temperature of about 37 °C.

105. The cell culture device of any one of claims 102 to 104, wherein the secondary matrix melts at a temperature above 40 °C, above 50 °C, above 60 °C, above 70 °C , above 80 °C, or above 90 °C.

106. The cell culture device of any one of claims 102 to 105, wherein the stiffness of the primary matrix is different than the stiffness of the secondary matrix.

107. The cell culture device of any one of claims 102 to 106, wherein the secondary matrix comprises cells.

108. The cell culture device of any one of claims 102 to 107, wherein the cells are myogenic cells.

109. The cell culture device of claim 108, wherein the myogenic cells are mesenchymal stem cells, myogenic stem cells, satellite cells, myoblasts, cardiomyocytes, smooth muscle cells, fibroblasts, or neurons.

110. The cell culture device of any one of claims 102 to 109, wherein the primary matrix and/or the secondary matrix comprises a fibrin gel, an alginate gel, a collagen gel, an agarose gel, matrigel, gelatin, a biopolymer, or any combination thereof.

111. The cell culture device of any one of claims 102 to 110, wherein the primary matrix comprises an alginate gel, a collagen gel, an agarose gel, matrigel, gelatin, a biopolymer, or any combination thereof.

112. The cell culture device of claim 111, wherein the secondary matrix comprises a fibrin gel.

113. A method comprising:

(c) filling the one or more cavities with a secondary matrix, wherein the primary matrix has a melting temperature that is less than the secondary matrix; and

(d) incubating the primary matrix having the one or more cavities filled with the secondary matrix at a first temperature sufficient to melt the primary matrix but not the secondary matrix, thereby releasing the one or more secondary matrices, wherein each of the released secondary matrices has a shape defined by the geometry of the cavity within which it was formed.

114. The method of claim 113, wherein the first temperature is in a range of 34 °C to °40 C.

115. The method of claim 113, wherein the first temperature is about 37 °C.

116. The method of claim 115, wherein the primary matrix comprises gelatin.

117. The method of claim 116, wherein producing the primary matrix comprises

(i.) dissolving gelatin in a solvent at a second temperature sufficient to dissolve the gelatin in the solvent at a desired concentration;

(ii.) pouring the gelatin solution into a mold such that the gelatin solution at least partially encompasses the one or more solid objects; and

(iii.) exposing the gelatin solution to a third temperature sufficient to solidify the gelatin, wherein the solidified gelatin is the primary matrix.

118. The method of any one of claims 113 to 117, wherein filling the one or more cavities with a secondary matrix, in step (a), comprises preparing a first solution comprising fibrinogen and injecting the first solution into the one or more cavities.

119. The method of any one of claims 113 to 117, wherein filling the one or more cavities with a secondary matrix, in step (a), comprises preparing a first solution comprising fibrinogen and a second solution comprising thrombin and injecting the first solution and second solution into the one or more cavities.

120. The method of claim 119, wherein injecting is accomplished using a device having the first solution comprising fibrinogen in a first syringe and the second solution comprising thrombin in a second syringe, wherein in the first syringe and the second syringe have tips forming a common port, and wherein mixing occurs as the first solution and second solution are ejected through the common port.

121. The method claim 117 or 120, wherein the first solution and/or the second solution comprises cells.

122. The method of any one of claims 113 to 121, wherein the secondary matrix comprises cells.

123. The method of claim 122, wherein the cells are myogenic cells.

124. The method of claim 123, wherein the myogenic cells are mesenchymal stem cells, myogenic stem cells, satellite cells, myoblasts or fibroblasts.

125. A secondary matrix produced by the method of any one of claims 113 to 124.

126. A method for producing myotubes in vitro, the method comprising producing a secondary matrix according to the method of claim 122 or 123, and culturing the myogenic cells in the secondary matrix for a period of time sufficient for the myogenic cells to differentiate and form myotubes.