WO2006104901A2 - Materials and methods for improved tissue engineering - Google Patents

Abstract

The subject invention provides materials and methods for improved tissue engineering. Specifically, the current invention pertains to the identification of advantageous extracellular matrix material scaffold coatings that can be used as described herein to modulate cell seeding, attachment, culturing, proliferation, and/or differentiation to a target cell. In a further embodiment, the scaffolds of the subject invention are used to modulate stem cells. In specific embodiments, a basement membrane preparation or gelatin can be used to coat polycaprolactone (PCL) scaffolds used for seeding, attaching, culturing, proliferating, and/or differentiating cells.

Description

DESCRIPTION

MATERIALS AND METHODS FOR IMPROVED TISSUE ENGINEERING

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of provisional patent application Serial No. 60/665,762, filed March 28, 2005, which is hereby incorporated by reference in its entirety, including any figures or tables.

BACKGROUND OF THE INVENTION

Tissue engineering aims to restore, maintain, and/or improve tissue function(s). The National Science Foundation defines tissue engineering as "the application of principles and methods of engineering and life sciences to obtain a fundamental understanding of structure- function relationships in novel and pathological mammalian tissues and the development of biological substitutes to restore, maintain, or improve [tissue] function." (Shalak, R. et al. 1988). In the last decade, over $3.5 billion dollars has been invested worldwide in tissue engineering research.

Several aspects of creating an engineered tissue make it a daunting task. One of the most difficult challenges is directing the behavior of specialized cells outside of the body to mimic the normal, endogenous phenotype those cells exhibit in vivo.

Biomaterial scaffolds are fundamental components of many tissue engineering strategies. The scaffold can serve as both a physical support and adhesive substrate for cells during in vitro culturing and subsequent implantation in vivo. Scaffolds are utilized to deliver cells to desired sites in the body, to define a potential space for engineered tissue, and to guide the process of tissue development. Cell transplantation on scaffolds has been explored for the regeneration of skin, heart, nerve, liver, pancreas, cartilage, and bone tissue using various biological and synthetic materials.

In order for an engineered tissue to be tolerated upon implantation, the material that provides the scaffolding for the cells must meet several important criteria. The material must be biocompatible, so as not to be toxic or injurious, and not cause immunological rejection. Because cells respond biologically to the substrate on which they adhere, the materials that provide the growth surface for engineered tissues must promote cell growth. Additionally, the material should allow cells to grow and function as they would in vivo.

Synthetic materials, such as polyester fiber (for example, DACRON) or polytetrafluoroethylene (PTFE) (for example, TEFLON), have been extensively used as implants to replace diseased or damaged body parts. However, these materials have enjoyed limited success because of their poor biocompatibility. Other synthetic materials that are more biodegradable than polyester fibers or PTFE have been used to fabricate tissue- engineering scaffolds such as polyglycolic acid (PGA) and polylactic acid (PLA). PGA, PLA and their copolymers are the most commonly used synthetic polymers in tissue engineering. Non-synthetic materials, such as animal materials, have also been used to produce tissue-regeneration/repair scaffolds. Another approach uses extracellular matrix (ECM) materials in processed or natural forms to regenerate tissue in vitro and in vivo. The interaction of cells with ECM in in vivo and in vitro environments is important in the organization, function and growth of all tissues and organs. Biochemical and biophysical signals between the cell and the ECM regulate fundamental cellular activities including adhesion, migration, proliferation, differential gene expression, and programmed cell death. Collagen-based gels have also been combined with specialized cells. This process depends upon interactions between the cells and collagen filaments in the gel so that the cells condense and organize. Synthetic biodegradable polymers over the years have gradually taken over the medical field, compared to natural polymers (Jalil R. et al. 1990; Wu X. 1995; Lewis D. 1990). Polycaprolactone (PCL), a polyester biodegradable polymer, is becoming one of these biomedical materials of interest (Armani D. et al. 2000; Kweon H. et al. 2003; Coombes A. et al. 2004). Polycaprolactone is synthesized by a ring opening polymerization of the ε- caprolactone monomer. Although not produced from renewable raw materials, it is fully biodegradable. It exhibits appropriate resistance to water, oil, solvent, and chlorine.

PCL is a semi-crystalline polymer that exhibits a low melting point (about 57 °C) and a low glass transition temperature (about -62 ⁰C) (Armani D. et al. 2000; Kweon H. et al. 2003; Coombes A. et al. 2004). It is considered a soft and hard-tissue biocompatible, non- toxic polymer (Armani D. et al. 2000; Kweon H. et al. 2003; Coombes A. et al. 2004). It is used mainly in thermoplastic polyurethanes, resins for surface coatings, adhesives and synthetic leather and fabrics. It also serves to make stiffeners for shoes and orthopedic splints, and folly biodegradable compostable bags, sutures, and fibers.

However, the rubbery characteristics of PCL have been utilized in low molecular weight drug delivery compositions, resorbable sutures, and bone graft substitutes. It has been found that PCL demonstrates a lower tensile modulus and strength than PLA, but higher extensibility, which is important in tissue scaffolding (Williamson M. et al. 2004).

Like other polyesters, PCL will undergo auto-catalyzed bulk hydrolysis degradation because of the susceptibility of its aliphatic ester linkage. However, the hydrophobic, semi- crystalline polymer retards degradation and resorption kinetics when compared to other aliphatic polyesters such as PLGA, which makes it more suitable for long term implantable devices (Armani D. et al. 2000; Kweon H. et al. 2003; Coombes A. et al 2004). Bulk hydrolysis breaks the ester linkage, which creates fragmentation and the release of oligomeric species. Low molecular-weight fragments are eventually engrossed by giant cells and macrophages. The byproduct e-hydroxycaproic acid is either metabolized via the tricarboxylic acid cycle or removed by direct renal secretion (Armani D. et al. 2000; Kweon H. et al. 2003). It is also possible for PCL to enzymatically degrade (enzymatic surface erosion) by lipases and esterases, though this is rare (Armani D. et al. 2000, Shimao M et al. 2001).

Embryonic stem (ES) cells, including human ES (hES) cells, are a promising source for cell transplantation due to their unique ability to give rise to all somatic cell lineages when they undergo differentiation. Differentiation of ES can be induced by removing the cells from their feeder layer and growing them in suspension, resulting in cellular aggregation and formation of embryoid bodies, in which successive differentiation steps occur. Several studies have shown that chemical cues provided directly by growth factors or indirectly by feeder cells can induce ES cell differentiation towards specific lineages. In some cell types, physical cues including surface interactions, shear stress and mechanical strain have induced differentiation.

Currently, attempts are being made to utilize degradable polymeric scaffolds loaded with embryonic stem (ES) cells to affect neo-tissue development both in vitro and in vivo (Levenberg S. et al. 2003; Gerecht-Nir S. et al. 2004). Unfortunately, specific scaffold factors influencing ES cell differentiation fate are not well known. Accordingly, there is a need for techniques to grow cells, including ES cells, and to differentiate ES cells to specific cells.

BRIEF SUMMARY OF THE INVENTION The subject invention provides materials and methods for improved tissue engineering. Specifically, the current invention pertains to the identification of advantageous extracellular matrix material scaffold coatings that can be used as described herein to modulate cell seeding, attachment, morphology, proliferation, and/or differentiation. In specific embodiments, MATRIGEL or gelatin can be used to coat polycaprolactone (PCL) scaffolds used for growing embryonic stem cells.

Another aspect of the subject invention is directed to bioreactors useful for modulating cells, including stem cells. The bioreactors may be used to clone a specific cell to proliferate cells, or to induce differentiation of stem cells into target cells.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication, with color drawing(s), will be provided by the Office upon request and payment of the necessary fee.

Figure IA shows a FESEM image at 10Ox magnification of a specific embodiment of the fibrous, phase separated scaffold. The scale marker is 100 μm.

Figure IB shows a FESEM image at 250Ox magnification of a specific embodiment of the fibrous, phase separated scaffold. The scale marker is 10 μm.

Figure 2A shows a FESEM image of a salt-leached PCL scaffold morphology. The scale marker bar is 100 μm. Figure 2B shows a FESEM image of a specific embodiment of a fibrous phase separated scaffold morphologies. The scale marker bar is 100 μm.

Figure 3 A is a FESEM image of a MATRIGEL coated, phase separated PCL scaffold of the subject invention.

Figure 3B is a FESEM image of a gelatin coated, phase separated PCL scaffold of the subject invention.

Figure 4 is a table of optical and fluorescent microscopic images on Day 9 of the mES cell study on phase separated, PCL scaffolds of the subject invention. Figure 5 is a table of optical and fluorescent microscopic images taken on Day 24 of the mES cell study on phase separated PCL scaffolds of the subject invention.

Figures 6A and 6B show a fluorescent microscopy showing cells on multiple layers of two scaffolds of the subject invention. The scale marker bar is 200 μm. Figure 7 shows a table providing a flow cytometry analysis at Day 24 of three different phase separated PCL scaffolds of the subject invention. One scaffold is uncoated, a second scaffold is coated with MATRIGEL, and a third scaffold is coated with gelatin.

Figure 8A shows a FESEM image of cellular attachment on a MATRIGEL coated phase separated PCL scaffold of the subject invention. The magnification is 2500 x, and the scale marker bar is 10 μm.

Figure 8B shows a FESEM image of cellular attachment in a MATRIGEL coated phase separated PCL scaffold of the subject invention. The magnification is 700Ox, and the scale marker bar is 1 μm.

Figure 9A shows a FESEM image of cellular attachment on a gelatin coated phase separated PCL scaffold of the subject invention. The magnification is 250Ox, and the scale marker bar is 10 μm.

Figure 9B shows a FESEM image of cellular attachment on a gelatin coated phase separated PCL scaffold of the subject invention. The magnification is 700Ox, and the scale marker bar is 1 μm. Figure 10 shows the RT-PCR analysis at Day 24 of a mES cell study conducted using phase separated PCL scaffolds of the subject invention.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides materials and methods for improved tissue engineering. Specifically, fibrous polycaprolactone (PCL) scaffolds optionally coated with different extracellular matrix materials create environments promoting cell seeding, attachment, culturing, and/or proliferation, and in an embodiment for embryonic stem (ES) cells, differentiation throughout the scaffold. In accordance with the subject invention, ES cell morphology and differentiation fate can be modulated via scaffold coating. Advantageously, the scaffolds of the subject invention are useful for generating cells, tissues, or organs in vivo or in vitro. The current invention also pertains to the identification of advantageous extracellular matrix (ECM) material scaffold coatings that can be used as described herein to modulate stem cell or cell morphology, proliferation, and/or differentiation.

The scaffolds of the subject invention comprise a three-dimensional porous matrix of PCL. In one embodiment, the three-dimensional porous matrix is provided by at least one layer of fibrous PCL. In one specific embodiment, the scaffold comprises only one layer of fibrous PCL. In a further embodiment, the scaffold comprises two layers of fibrous PCL. In yet another embodiment, the scaffold comprises three layers of fibrous PCL. hi a specific embodiment, the scaffolds of the subject invention have four or more layers of PCL. Each layer of the scaffolds of the subject invention increase the size of the scaffold, and as disclosed herein, the layers are fused together at the interface between each layer using phase-separation to form a single scaffold.

Advantageously, the layers of the PCL have an interconnected porous structure, which helps provide maximum surface area for cell seeding, attachment, culturing, proliferation, culturing, and/or differentiation. Consequently, the PCL scaffolds of the subject invention comprise a plurality of pores embedded within each fibrous PCL layer including the outer surfaces of the scaffolds, hi one embodiment, the pore size is asymmetric, and each pore may have a different shape and diameter than the other pores. For example, a pore shape may be circular, convex, concave, elliptical, or any shape that exposes surface area to a coating or a cell suspension. The pores may be distributed non-uniformly through the scaffold.

An embodiment of a scaffold of the subject invention comprises a plurality of pores, wherein each pore is about 10 μm to about 200 μm in diameter, or for non-spherical pores, equivalent diameter, hi further embodiments, the pore diameter is about 30 μm to about 50 μm or about 50 μm to about 100 μm, or about 100 μm to about 200 μm.

Although PCL scaffolds have been prepared using a salt-leaching technique, the resulting morphology differs from the morphology of the tissue scaffolds of the subject invention. (See Tollon, M.H. 2005). The salt leached scaffolds contain pockets of PCL material rather than the porous interconnected scaffolds of the subject invention. The porous three-dimensional matrix of the scaffolds of the subject invention are more suitable for cell seeding, attachment, culturing, proliferation, and/or differentiation than the pocketed salt- leached scaffolds. Although previously known tissue engineering scaffolds have disclosed the use of copolymers wherein one of the- monomers is ε-caprolactone and the other monomer is glycolide (See, for example, U.S. Patent No. 6,946,143), surprisingly, the subject invention's scaffolds are formed from the homopolymer polycaprolactone. There has never been a teaching about the phase separated PCL scaffolds' usefulness for seeding, attaching, culturing, proliferating, and/or differentiating cells.

IQ one embodiment, each layer of phase separated PCL scaffold weighs from about 125 mg to about 145 mg for a layer that is about 37 mm to about 53 mm in diameter. In a specific embodiment, each layer weighs about 136 mg, and the diameter of the layer is about 50 mm. In yet another embodiment, each layer is about 2 mm to about 6 mm in thickness. In further embodiments, each layer may be about 2 mm, about 3 mm, about 4 mm, about 5 mm, or about 6mm thick.

One embodiment comprises a scaffold of the subject invention coated with an extracellular matrix material useful for modulating cell morphology, proliferation, and/or differentiation. In one embodiment, the ECM coating is sufficiently thin and smooth to allow cells to adhere thereto and spread out across the scaffold.

In one specific embodiment, the PCL scaffolds of the subject invention are coated with MATRIGEL (BD Biosciences, Franklin Lakes, New Jersey), a solubilized basement membrane preparation extracted from the Englbreth-Holm-Swarm (EHS) mouse sarcoma. This coating solution contains an extract from sarcoma tumors that are rich in extracellular matrix proteins (Kleinman H. et al. 1982). Its main components are laminin, collagen IV, entactin, and heparan sulfate proteoglycan. It also contains TGF-β, fibroblast growth factor (FGF), tissue plasminogen activator (TPA), and other growth factors that are found naturally in EHS tumors (McGuire P. et al. 1989). At room temperature, the MATRIGEL matrix polymerizes to produce a biologically active matrix material that resembles the mammalian basement membrane. MATRIGEL is a viscous gel and must be kept frozen for storage purposes.

In yet another embodiment, the scaffolds of the subject invention are coated with a gelatin solution. Gelatin is a pure protein obtained by the partial hydrolysis of collagen. This collageneous material has been used from food substitutes to scientific studies. It also provides a basement membrane in cellular studies and can increase a surface's hydrophilicity (Hasirci V. et al. 2001). The gelatin solution has a viscosity close to water. The scaffolds of the subject invention are coated with collagen in another specific embodiment. Collagen is a structural protein, and the scaffolds of the subject invention may be coated with any of the types of collagen depending on the cell line targeted for implantation in the scaffolds of the subject invention. For example, Type I collagen is useful when the scaffold is utilized to seed, attach, culture, and/or proliferate cells commonly found in skin, tendon, or bone, or to seed, attach, proliferate, culture, and/or differentiate stem cells that are capable of differentiating into cells commonly found in skin, tendon, or bone.

In another embodiment, the scaffolds of the subject invention are coated with elastin, fibronectin, laminin, or proteoglycans. As described herein, flow cytometry analysis indicates a larger number of viable cells on both the coated scaffold types (MATRIGEL and gelatin) compared to uncoated scaffolds. Endodermal differentiation was also judged greater on coated scaffolds compared to uncoated controls illustrated by both fluorescence microscopy and flow cytometry analysis. RT-PCR data suggest that murine embryonic stem (mES) cells cultured on MATRIGEL coated PCL scaffolds possessed a more neural pattern of gene expression with high nestin transcript levels. In contrast, mES cells on gelatin coated PCL scaffolds had increased albumin transcript levels, indicative of a more hepatocyte-like expression profile.

The tissue engineering scaffolds of the subject invention may comprise at least one cell attached to the scaffold. A single cell may be seeded onto or attached to the scaffold for subsequent cloning into one or more cloned cells.

As discussed herein, FESEM images of the cells cultured on the different scaffolds also reveal an aggregate morphology on a MATRIGEL coated scaffold of the subject invention and a monolayer-like morphology on gelatin coated scaffolds of the subject invention. Without being limited by theory, the difference in mES cell expression profiles could, in part, be related to this morphologic difference.

Another aspect of the subject invention pertains to novel phase separation methods for building the PCL scaffolds of the subject invention. In one embodiment, the technique of phase separation includes providing a PCL solution, wherein PCL is the solute and a ketone is the solvent, and contacting the PCL solution with a substrate. An exemplary ketone is acetone. A sufficient amount of solvent is utilized in the solution so that the PCL remains in solution until contacted with water. In one embodiment, the solution comprises about 5% (wt/vol) of PCL. In a further embodiment, the solution comprises about 1% (wt/vol) to about 50% (wt/vol) PCL. Other suitable ranges of PCL concentration include about 1% to about 10%, about 10% to about 25%, and about 25% to about 50%. In a further embodiment, the concentration of the PCL in solution may be from about 2% to about 8% or about 4% about 6%. The subject invention also contemplates providing a concentrated solution having more than 50% (wt/vol) of PCL. The concentration PCL solution may be diluted with a sufficient amount of solvent.

Advantageously, once the PCL solution is distributed atop a substrate such as a mold or vessel, the PCL phase separates from the solvent when contacted with a sufficient amount of water to induce the phase separation. In one embodiment, the water is applied in a spray. In yet another embodiment, the water is applied in steady stream. In a third embodiment, the water is applied dropwise. Advantageously, when the PCL separates from the solvent, it forms a single fibrous web layer of PCL. Advantageously, the providing and contacting steps may be repeated until the desired number of layers are prepared.

The methods of the subject invention further include optionally coating the scaffolds with an extracellular matrix material. The extracellular matrix material promotes cell seeding, attachment, proliferation, culturing, and/or differentiation to a target cell. Exemplary materials include MATRIGEL, gelatin, collagen, elastin, fibronectin, lamininin, and proteoglycans. Optionally, the ECM material may be added to a cell culture media before the scaffolds are coated. Suitable cell culture media includes, for example and without limitation, IMDM, penicillin, streptomycin, monothiglycerol, leukemia inhibitory factor (LIF); animal serum such as bovine serum, heat inactivated bovine serum, donor bovine serum, donor bovine serum with iron, fetal bovine serum, heat inactivated fetal bovine serum, charcoal stripped fetal bovine serum, ES-cell qualified fetal bovine serum, ultra low IgG serum, horse serum, heat inactivated horse serum, newborn bovine calf serum. Heat inactivated newborn bovine calf serum, chicken serum, goat serum, lamb serum, porcine serum, and rabbit serum; and basal media such as basal media eagle, BGJb medium, Brinster's BMOC-3 medium, CMRL medium, CO₂-independent medium, Dulbecco's Modified Eagle Media, Glasgow minimum essential media, Grace's insect cell culture media, improved MEM Zn option, IPL-41 insect media, Iscove's Modified Dulbecco's media, Leibovitz's L- 15 media, McCoy's 5 A modified media, MCDB 131 medium, media 199, medium NCTC- 109, RPMI media 1640, Schneider's Drosophila medium, Waymouth's MB 752/1 media, and Williams media E. However, a variety of culture media can be utilized to culture stem cells according to the methods of the subject invention. For example, if low calcium conditions are desired, Minimum Essential Medium (MEM), Joklik modification for suspension culture, with L-Glutamine, without calcium chloride and sodium bicarbonate (SIGMA, St. Louis, MO; Product No. M0518), or other low calcium media can be used (Eagle, H. et al, J, Biol. Chem., 214:845-847, 1956; Eagle, H., Media for Animal Cell Culture, Tissue Culture Association Manual, 3:517-520, 1976; Eagle, H., Science, 130:432- 437, 1959; Eagle, H., Science, 122:501, 1955). In a further embodiment, the ECM material may be diluted with water, preferably deionized water.

The coating step comprises any appropriate technique for contacting the coating with the three-dimensional porous matrix of the PCL scaffolds of the subject invention. For example, one suitable technique is submerging a scaffold of the invention in an excess of coating material. In one embodiment, the scaffolds are submerged for a sufficient period of time for the coating to adhere to the scaffold. Advantageously, this time is often relatively brief. For example, submersion for about two minutes is sufficient. However, the skilled artisan can modify the time period of the submerging depending on the concentration of the active ingredient of the coating or the thickness, depth, and length of the scaffolds of the subject invention. In another embodiment, the scaffolds are coated at least two times. The PCL scaffolds of the subject invention are coated three or more times with the extracellular matrix in a further embodiment.

The subject invention also contemplates other methods of coating the PCL scaffolds including spraying the PCL scaffolds with the coating or pouring the coating over the scaffolds.

Any excess coating material may be removed from the scaffolds of the subject invention using suitable techniques known in the art, for example, siphoning of the excess coating material from the scaffold. In a further embodiment, the coated scaffolds of the subject invention are incubated to maintain an environment suitable for cell seeding, attachment, proliferation, culture, and/or differentiation. The coated scaffolds may be incubated following each coating step if the scaffold has more than one layer of coating. The coated scaffolds may be incubated following the final coating step, hi a further embodiment, the incubator temperature is maintained at a suitable temperature, for example, about 37 °C, for cell seeding, attachment, proliferation, culturing, and/or differentiation. Each individual layer may be coated with an extracellular matrix material or in a further embodiment, the scaffold may be coated after the layers are fused together.

The methods of the subject invention also comprise fusing each layer of the PCL fibrous web together. Water, preferably de-ionized water, is applied to the interface between each web, wherein additional phase separation causes the individual webs to bond together. The water may be applied in a spray, dropwise, or pouring a steady stream onto the interface.

The methods of scaffold preparation of the subject invention also comprise optionally removing any excess solvent and water after the scaffold is prepared. In one embodiment, excess solvent and water is removed after each layer is formed. Moreover, the scaffolds of the subject invention or each individual layer may be bathed in a methanol bath and subsequently dried in one embodiment. In a further embodiment, the scaffolds or layers are dried under a vacuum.

Another aspect of the subject invention pertains to providing methods for utilizing the scaffolds of the subject invention as platforms for seeding, attaching, proliferating, culturing, and/or differentiating cells. Cells can be seeded onto and/or placed into contact with the porous structure of the scaffolds of the subject invention.

The cells that can be utilized with the subject invention include those cells arising from the ectoderm, mesoderm, or endoderm germ cell layers. Examples of cells include, but are not limited to, pancreatic islet cells, bone cells (such as osteoclasts, osteoblasts, chondrocytes, and osteocytes), blood cells, marrow cells, epithelial cells, neural cells (for example, neurons, astrocytes, and oligodendrocytes), muscle cells, adipocytes, tendon cells, ligament cells, dermal cells, fibroblasts, and dental cells (odontoblasts and ameloblasts). Seeded cells can be autogenic, allogenic, or xenogenic to the patient in which the scaffold is implanted. Seeded cells can be encapsulated or non-encapsulated. The cells can be stem cells or progenitor cells (such as stem cells or progenitor cells of any of the aforementioned differentiated cell types), or mature, differentiated cells. For example, the cells seeded onto and/or within the PCL scaffolds can be hematopoietic stem cells, mesenchymal stem cells, neural stem cells, or others. The stem cells, progenitor cells, or mature cells can be genetically modified or non-genetically modified. As used herein, the term "cell" is intended to include primary cells, cells of cell culture, and cells of cell lines (for example, cells of tumors or immortalized cells), unless otherwise specified. As will be understood by one of skill in the art, there are over 200 cell types in the human body. The methods and compositions of the subject invention may utilize any of these cell types, singly or in combination. Other cells suitable for use with the compositions and methods of the subject invention include those disclosed by Spier R.E. et al. Alberts B. et ah, which are incorporated herein by reference in their entireties.

In an advantageous use, the scaffolds of the subject invention are used for culturing and/or proliferating stem cells and for controlling any differentiation of the stem cells. Advantageously, the unique architecture of the subject invention's scaffolds provides an environment wherein stem cells can be differentiated into specific target cells in vitro or in vivo. In one embodiment, the target cell is a cell line. Specifically, the interconnected porous fibrous structures of the subject scaffolds are ideal for seeding with stem cells. However, the scaffolds of the subject invention can also be used for the attachment, proliferation, and/or culturing of any type of cell as disclosed herein.

hi one embodiment, the scaffolds of the subject invention are used to proliferate and/or culture cells in vitro. Advantageously, in one embodiment, the unique architecture of the scaffolds of the subject invention provide an environment wherein stem cells can be seeded in an undifferentiated state and can be differentiated into a target cell, hi a specific embodiment, the choice of the extracellular matrix coating facilitates the differentiation to the target cell. However, any type of cell can be seeded, attached, culture, and/or proliferated in the scaffolds of the subject invention.

hi yet another embodiment, the scaffolds of the subject invention are used to grow cells in vivo. The scaffolds can be used to generate tissue or organs for transplantation in an animal, hi one embodiment, the scaffolds are coated with an extracellular matrix as disclosed herein. The scaffolds can be used to promote cell and tissue growth within an animal by implanting a scaffold within the animal. For example, a therapeutically effective amount of the scaffold can be implanted, applied, or otherwise administered at a target site within a patient, hi one embodiment, the implanted, applied, or administered scaffold is not seeded with a cell before implantation, application, or administration. Cells from the target site provide the requisite cells for seeding, attachment, proliferation, culturing, and/or differentiation, hi a further embodiment, the scaffolds of the subject invention comprise the target cells before implantation, application, or administration. The amount to be implanted, applied, or administered will depend upon the particular medical application, and the clinical outcome that is sought. According to this embodiment of a method of the subject invention, the scaffold can be applied so that it directly contacts existing tissue adjacent to, or defining, the site of a defect or discontinuity, or the scaffold can be contacting another implant, or both.

The scaffolds of the subject invention may be any shape suitable for the particular in vitro or in vivo application. The suitable shape can be produced utilizing freeze-drying techniques. In one embodiment, the cross-sections may be round, elliptical, or irregularly polygonal, depending on the application. Scaffolds of the subject invention may be used for nerve, lung, bone, cartilage, and/or soft tissue repair in an embodiment. Scaffolds of the subject invention may be used in virtually all instances when it is desirable to provide a substrate for the growth of cells onto or into a tissue replaceable matrix, either in vitro or in vivo. The scaffold itself may be molded by the selection of a suitable substrate in the methods of preparation of the subject invention or cut into a specific shape that is applicable for its end usage.

In a specific embodiment, the methods of the subject invention include providing a scaffold of the subject invention and contacting the scaffolds with a suspension of cells. In one embodiment, the cells are a plurality of embryonic stems cells suspended in a solution of cell culture media. For example, the media may contain a mixture of Iscove's Modified Dulbecco's Medium (IMDM) (Hyclone, Logan, Utah), penicillin or streptomycin, and/or monothiglycerol. Other cell culture media useful for the methods of the subject invention can include leukemia inhibitory factor (LIF); animal serum such as bovine serum, heat inactivated bovine serum, donor bovine serum, donor bovine serum with iron, fetal bovine serum, heat inactivated fetal bovine serum, charcoal stripped fetal bovine serum, ES-cell qualified fetal bovine serum, ultra low IgG serum, horse serum, heat inactivated horse serum, newborn bovine calf serum, heat inactivated newborn bovine calf serum, chicken serum, goat serum, lamb serum, porcine serum, and rabbit serum; and basal media such as basal media eagle, BGJb medium, Brinster's BMOC-3 medium, CMRL medium, CO₂-independent medium, Dulbecco's Modified Eagle Media, Glasgow minimum essential media, Grace's insect cell culture media, improved MEM Zn^+"1" option, IPL-41 insect media, Iscove's Modified Dulbecco's media, Leibovitz's L-15 media, McCoy's 5A modified media, MCDB 131 medium, media 199, medium NCTC- 109, RPMI media 1640, Schneider's Drosophila medium, Waymouth's MB 752/1 media, and Williams media E. However, a variety of culture media can be utilized to culture stem cells according to the methods of the subject invention. For example, if low calcium conditions are desired, Minimum Essential Medium (MEM), Joklik modification for suspension culture, with L-Glutamine, without calcium chloride and sodium bicarbonate (SIGMA, St. Louis, MO; Product No. M0518), or other low calcium media can be used (Eagle, H. et al, J. Biol. Chem., 214:845-847, 1956; Eagle, H., Media for Animal Cell Culture, Tissue Culture Association Manual, 3:517-520, 1976; Eagle, H., Science, 130:432-437, 1959; Eagle, H., Science, 122:501, 1955).

In addition to the interaction between the embryonic stem cells and the uncoated or coated scaffolds that promote cell differentiation, cells can also be stimulated to differentiate by contact with one or more agents (for example, trophic factors, hormonal supplements) that induce full or partial differentiation, including forskolin, retinoic acid, putrescin-transferrin, cholera toxin, insulin-like growth factor (IGF), transforming growth factor (for example, TGF-α, TGF-β), tumor necrosis factor (TNF), fibroblast growth factor (FGF), epidermal growth factor (EGF), granulocyte macrophage-colony stimulating factor (GM-CSF), hepatocyte growth factor (HGF), sonic hedgehog protein, vascular endothelial growth factor (VEGF), thyrotropin releasing hormone (TRH), platelet derived growth factor (PDGF), sodium butyrate, butyric acid, cyclic adenosine monophosphate (cAMP), cAMP derivatives (for example, dibutyryl cAMP, 8-bromo-cAMP), phosphodiesterase inhibitors, adenylate cyclase activators, prostaglandins, ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophin 3, neurotrophin 4, interleukins (for example, IL-4), interferons (for example, interferon-gamma), potassium, amphiregulin, dexamethasone (glucocorticoid hormone), isobutyl 3-methyulxanthine, somatostatin, lithium, and growth hormone. The methods optionally include incubating the scaffolds and/or adding additional suspensions of cells as needed. If the scaffolds are incubated after seeding with the cell suspension, then a specific incubation temperature may be about 37 ⁰C.

Another aspect of the subject invention pertains to a bioreactor for tissue engineering. In one embodiment, the bioreactor comprises a housing with at least one chamber useful for holding a tissue engineering scaffold, and at least one tissue engineering scaffold of the subject invention located within the chamber. Each chamber may have a single scaffold, two scaffolds, three scaffolds, or four or more scaffolds. When a chamber house has more than one scaffold, the scaffolds may be located adjacent to one another, stacked on top of each other, or positioned apart from one another so that there is no contact between the scaffolds. In a further embodiment, the bioreactor of the subject invention further comprises at least one conduit for supplying a fluid media for contact with the scaffold. A single conduit is operatively interfaced with each chamber for each type of fluid that is added to the chamber. In one embodiment, the bioreactor comprises at least one reservoir for holding the fluid media. At least one conduit operatively connects each reservoir with each chamber. A reservoir may supply fluid media to more than one chamber. Thus more than one conduit may be operatively connected to a single reservoir. In another embodiment, any fluid media is stored externally to the housing of the bioreactor and is transferred to the bioreactor with the use of, for example, a pump or gravity, to an inlet of at least one conduit. In a further embodiment, fluid media is added directly to the chamber, for example, by pipetting fluid media into a well holding the scaffold.

In another embodiment, the fluid comprises a cell suspended in a fluid that is suitable for seeding, attaching, proliferating, culturing, and/or differentiating to the scaffolds of the subject invention. A single cell may be utilized to clone that cell using the bioreactors of the subject invention. In a further embodiment, more than one cell is suspended in a fluid. More than one cell may comprise a plurality of cells. The cell suspension may flow across a tissue engineering scaffold invention that is housed in the bioreactors of the subject invention so that the cell or cells are in contact with the pores and surfaces of the scaffold and at least one of the cells seed the pores and/or surfaces. The cell suspension may flow continuously across the scaffold while the bioreactor is in use. In a further embodiment, cell suspension flow is periodic. For example, flow may occur on a specified cycle. Alternatively, flow may occur as needed.

In a specific embodiment, the fluid media is normal saline. In further embodiments, the fluid media is phosphate buffer solution, Dulbecco's phosphate buffer solution, Earle's balanced salt solution, or Hank's buffered salt solution.

In a further embodiment, the fluid media is a cell culture media that facilitates the growth of cells and/or tissue. For example, the media may contain a mixture of IMDM, penicillin or streptomycin, and/or monothiglycerol. Other cell culture media useful for the bioreactors of the subject invention can include leukemia inhibitory factor (LIF); animal serum such as bovine serum, heat inactivated bovine serum, donor bovine serum, donor bovine serum with iron, fetal bovine serum, heat inactivated fetal bovine serum, charcoal stripped fetal bovine serum, ES-cell qualified fetal bovine serum, ultra low IgG serum, horse serum, heat inactivated horse serum, newborn bovine calf serum, heat inactivated newborn bovine calf serum, chicken serum, goat serum, lamb serum, porcine serum, and rabbit serum; and basal media such as basal media eagle, BGJb medium, Brinster's BMOC-3 medium, CMRL medium, CO₂-independent medium, Dulbecco's Modified Eagle Media, Glasgow minimum essential media, Grace's insect cell culture media, improved MEM Zn⁺⁺ option, EPL-41 insect media, Iscove's Modified Dulbecco's media, Leibovitz's L-15 media, McCoy's 5A modified media, MCDB 131 medium, media 199, medium NCTC-109, RPMI media 1640, Schneider's Drosophila medium, Waymouth's MB 752/1 media, and Williams media E. However, a variety of culture media can be utilized to culture stem cells according to the methods of the subject invention. For example, if low calcium conditions are desired, Minimum Essential Medium (MEM), Joklik modification for suspension culture, with L- Glutamine, without calcium chloride and sodium bicarbonate (SIGMA, St. Louis, MO; Product No. M0518), or other low calcium media can be used (Eagle, H. et al, J. Biol. Chem., 214:845-847, 1956; Eagle, H., Media for Animal Cell Culture, Tissue Culture Association Manual, 3:517-520, 1976; Eagle, H., Science, 130:432-437, 1959; Eagle, H., Science, 122:501, 1955).

The bioreactors of the subject invention may also contain at least one conduit to supply one or more agents that, in conjunction with the scaffolds and/or the scaffolds' coatings, induce full or partial differentiation of a stem cell to a target cell. In one embodiment, the one or more agents induce full or partial differentiation to a target cell line. Suitable agents include, without limitation, forskolin, retinoic acid, putrescin-transferrin, cholera toxin, insulin-like growth factor (IGF), transforming growth factor (for example, TGF-α, TGF-β), tumor necrosis factor (TNF), fibroblast growth factor (FGF), epidermal growth factor (EGF), granulocyte macrophage-colony stimulating factor (GM-CSF), hepatocyte growth factor (HGF), sonic hedgehog protein, vascular endothelial growth factor (VEGF), thyrotropin releasing hormone (TRH), platelet derived growth factor (PDGF), sodium butyrate, butyric acid, cyclic adenosine monophosphate (cAMP), cAMP derivatives (for example, dibutyryl cAMP, 8-bromo-cAMP), phosphodiesterase inhibitors, adenylate cyclase activators, prostaglandins, ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophin 3, neurotrophin 4, interleukins (for example, IL-4), interferons (for example, interferon-gamma), potassium, amphiregulin, dexamethasone (glucocorticoid hormone), isobutyl 3-methyulxanthine, somatostatin, lithium, and growth hormone. In another embodiment, the one or more agents may be added directly to the chamber housing the scaffold, for example, by pipeting the agent into the chamber.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a scaffold" includes more than one such scaffold, or a reference to "the cell" includes more than one such cell.

As used herein, the term "culture", or grammatical variations thereof, is intended to denote the maintenance or cultivation of cells in vitro, including the culture of single cells. Cultures can be cell, tissue, or organ cultures, depending upon the extent of organization.

As used herein, the term "differentiated", or grammatical variations thereof, refers to those cells that maintain in culture all, or a substantial amount of, their specialized structure and function typical of the cell type in vivo. Partially differentiated cells maintain less than a substantial amount of their full complement of specialized structure and/or function. For example, the methods of the subject invention advantageously permit the culture of stem cells in an undifferentiated state, not developing any, or a substantial amount of, their full complement of specialized structure and/or functions.

As used herein, the terms "host" and "patient" are used interchangeably and intended to include humans and non-human animals. Accordingly, cells cultured according to the method of the subject invention can be utilized for veterinary purposes. The transplanted cells can be allografts, autografts, or xenografts, for example. As used herein, the terms "cultured," "culturing," or "culture" are used to refer to cells that are in a maintenance stationary phase and although alive, are no longer proliferating.

As used herein, the terms "proliferate" and "propagate" are used interchangeably to refer to cell division. As used herein, the term "stem cell" is an unspecialized cell that is capable of replicating or self renewal and developing or giving rise to specialized cells of a variety of cell types. The product of a stem cell undergoing division is at least one additional stem cell that has the same capabilities of the originating cell. For example, under appropriate conditions, a hematopoietic stem cell can produce a second generation stem cell and a neuron. Stem cells include embryonic stem cells (for example, those stem cells originating from the inner cells mass of the blastocyst) and adult stem cells (which can be found throughout the more mature animal, including humans). As used herein, stem cells are intended to include those stem cells found in animals that have matured beyond the embryonic stage (for example, fetus, infant, adolescent, juvenile, adult, etc.). The list of tissues reported to contain stem cells is growing and includes, for example, bone marrow, peripheral blood, brain, spinal cord, umbilical cord blood, amniotic fluid, placenta, dental pulp, blood vessels, skeletal muscle, epithelia of the skin and digestive system, cornea, retina, liver, and pancreas.

MATERIALS AND METHODS Poly(ε-caprolactone) (PCL) (M_w about 120,000) was obtained from Sigma Aldrich.

Field emission scanning electron microscopy (FESEM) was performed with a JEOL 6335F at 1OkV. All fluorescence microscopy was conducted using an Olympus LX70. Flow cytometry was performed with a FACS Sort instrument equipped with CellQuest Acquisition software (BD Biosciences).

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. AU percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 — Scaffold Coatings

A. Scaffold Synthesis

Briefly, a 5% (wt/vol) PCL solution was mixed with deionized (DI) water to generate a fibrous web. Four webs were then layered to create a 3-D scaffold. Scaffolds were sectioned into 2.5cm x 2.5cm x 4mm blocks and sterilized in 70/30 ethanol solution. The scaffolds were coated three times in solutions of either 10% (vol/vol) MATRIGEL matrix

(BD Bioscience) mixed with serum-free Dulbecco's Modified Eagle Media (D-MEM/F-12) or

0.1% gelatin solution (Specialty Media). B. Seeding/Culture

The scaffolds were seeded with a cell suspension (2.17x10⁶ cells/mL) of mES cells with an α-fetoprotein/green fluorescent protein (Afp/GFP) marker system. The cells were cultured for 24 days in mES Differentiated Media at 37⁰C.

C. Fluorescence Microscopy

A series of three fluorescent micrographs were taken for each scaffold type:

* Afp/GFP - green fluorescent - cells that have differentiated towards endoderm

*Hoechst 33342 (Sigma) - blue fluorescent - nucleus of cells, both dead and alive

*Propidium iodide (PI) (Sigma) - red fluorescent - dead cells

D. Flow Cytometry

Cells were dissociated with 0.25% trypsin/EDTA after day 24 of the experiment. The flow cytometry was performed on FACS Sort (BD Biosciences) on a data set of 30,000 cells, which was then recorded using CellQuest Acquisition software (BD Biosciences). Flow cytometry helped illustrate the separation of GFP -positive and negative cells and the cell's size.

E. FESEM

The cells were fixed in a 2.5% glutaraldehyde (Electron Microscopy Sciences)/PBS

(Gibco) fixation. The scaffolds were rinsed with DI water and then dehydrated with ten minute baths of 70%, 80%, 95%, and 100% (three times) of ethanol/DI water solutions and then imaged.

F. Reverse Transcriptase-Polymerase chain reaction (RT-PCR)

Total RNA was extracted by using RNA aqueous kit (Ambion). Then cDNA was synthesized by using Superscript II first-strand synthesis system with oligo(dT) (Gibco).

PCR was performed by using Taq DNA polymerase (Eppendorf). For each gene, the DNA primers were originated from different exons to ensure that the PCR product represents the specific mRNA species and not geomic DNA. Gene markers were then compared to determine possible cells lineages.

EXAMPLE 2- Fibrous Phase Separated Scaffold The polymer used for this scaffold fabrication was polycaprolactone (PCL) (M_w approximately 120,000) provided by Sigma Aldrich. The polymer was first dissolved using histological grade acetone (Fisher) to get the desired 5% weight/volume. The solution was then refrigerated until needed.

First, 1 mL of a 5% (wt/vol) PCL solution was pipetted onto a 75mm diameter watch glass. The watch glass was slowly rotated in a circular pattern during the step of pipetting by holding and rotating the glass in one hand. Next, 2mL of deionized (DI) water was sprayed at the glass approximately 4 to 6 inches away with a sterile syringe and needle (22 gauge) in a left to right fashion covering the entire watch glass. This generated a phase separation forcing the polymer out of the solution creating fibrous webbing. Once the phase separation starts to occur, the excess "cloudy" solution was poured into the waste storage. Once the webbing was formed, the fibrous web was thoroughly rinsed with DI water. The watch glass was rotated 90°, and the above steps were repeated on top of the newly formed fibrous web to create another polymer deposit. Once the second application has been applied, the web was soaked with methanol to help free it from the glass and then dried in a vacuum oven for 30 minutes. After drying, this web is now considered one layer of the final scaffold.

Multiple layers were attached together to create the full scaffold. Layers were stored under vacuum until needed for the layering process.

Four layers were first placed on top of each other in a 75mm diameter watch glass. Next, the layers were united together by spraying ImL of the polymer solution with a sterile syringe and needle (22 gauge) in a left to right fashion covering the entire layer. Quickly spraying DI water over the layers caused a phase separation once again, which fastened the four layers together. Once the phase separation is visible, the "cloudy" solution was poured into waste storage and was discarded at a later date. The layers were thoroughly rinsed with DI water to clean out any excess solution. Next, the newly formed scaffold was flipped over and the above steps repeated to bond the layers again from the other side. After the layers were connected, the scaffold was soaked in methanol, removed from the methanol, and then dried in a vacuum oven for 30 minutes. The scaffolds were subsequently stored under vacuum until needed for future experiments.

After fabrication, the PCL scaffolds were weighed and observed for general handling properties. The weights of the scaffold layers were 136 ± 8.56 mg and demonstrated an elastic quality when handled in the lab. Once the layers were bonded together, the final scaffold's weight was 661 ± 8.55 mg. Also, the scaffold became more rigid after the final bonding of the layers was complete but still had elastic handling qualities. The scaffold's final dimensions were 50 ± 3mm in diameter with a thickness of 4 ± 2mm.

EXAMPLE 3- In Vitro Murine ES Cell Studies

The scaffolds prepared in Example 2 were sectioned into lin x lin samples (thickness was dependent on original scaffold) and placed in a bath overnight of 70/30 solution of ethanol and DI water, respectively. The scaffolds were dried, washed with phosphate buffer solution (PBS) twice, and then dried once again. The scaffolds were kept sterile until coated.

The two coating solutions were prepared as listed below (700μL):

• 10% (vol/vol) MATRIGEL matrix (BD Bioscience) mixed with D-MEM/F-12 serum free media

• 0.1% gelatin solution (Specialty Media) in ultra pure water The MATRIGEL matrix is a solubilized basement membrane preparation extracted from the Englbreth-Holm-Swarm (EHS) mouse sarcoma, which is a tumor rich in extracellular matrix proteins (Kleinman H. et al. 1982). Its main components are laminin, collagen IV, entactin, and heparan sulfate proteoglycan. The matrix also contains TGF-β, fibroblast growth factor (FGF), tissue plasminogen activator (TPA), and other growth factors that are found naturally in EHS tumors (McGuire P. et al. 1989). At room temperature, the MATRIGEL Matrix polymerizes to produce a biologically active matrix material that resembles the mammalian basement membrane. MATRIGEL is a viscous gel and must be kept frozen for storage purposes.

Gelatin is a pure protein obtained by the partial hydrolysis of collagen. This collageneous material has been used from food substitutes to scientific studies. It also provides a basement membrane in cellular studies and can increase a surface's hydrophilicity (Hasirci V. et al. 2001). The gelatin solution has a viscosity close to water and differs greatly from the MATRIGEL's hydrogel properties.

After the preparations have been completed, the coating procedure was started immediately. For each coating material, the same procedure is followed. The scaffold was first submerged in the coating solution for 2 minutes. Next, the scaffold was removed with excess coating solution being siphoned off. The scaffold was then incubated for 5 minutes at

37 °C. The process is repeated until 3 coatings have been applied and incubated, respectively.

The cell line used for the studies was murine Alpha fetoprotein/green fluorescent protein (Afp/GFP) ES stem cells. This cell line can be used to monitor primitive endoderm differentiation (Hamazaki T. et al. 2004). The mES cells were maintained prior to the study in an undifferentiated state on gelatin-coated dishes in Knock-out Dulbecco's Modified Eagle Medium (DMEM) (Gibco) containing 10% knockout serum replacement (Gibco), 1% fetal bovine serum (FBS) (Atlanta biologicals), 2mM L-glutamine, 100units/mL penicillin, lOOμg/mL streptomycin, 25mM HEPES (Gibco), 300μM monothioglyercol (Sigma), and 1000unit/mL recombinant mouse LIF media (Chemicon).

The cells were suspended before seeding them on the scaffold. The media was siphoned out of the culture dish trying not to disturb the cells. A wash of PBS was applied to the Petri dish and cells and then siphoned off. While rotating the dish in a circular pattern, ImL of 0.25% Trypsin/EDTA (Gibco) was added to dissociate the cells from the dish. The mES cells were then suspended in the mES Differentiated Media: 50OmL of IMDM (Gibco), 5mL of penicillin/streptomycin (Gibco), 16μL of monothiglycerol (Sigma).

Next, 10OmL were removed from the prepared solution and stored for later use. Finally, 10OmL of 20% FBS was added to the remaining solution. The mES Differentiated Media was then stored for future cell experiments. The sectioned and coated scaffolds were first placed in its individual 12 well culture dish. Next, lOOμL of mES cell suspension (2.17x106 cells/mL) were added in each well. The suspension was placed on top of the scaffold to help cellular adhesion. The culture wells were then incubated for 30 minutes at 37 ⁰C. Once removed from the incubator, ImL of mES Differentiated Media was added to each culture dishes. The scaffolds were incubated at 37 °C again until needed for observation. The scaffolds were observed with an optical fluorescent microscope on day 0, 2, 4, 7, 9, 21, 24 for cellular analysis. The cells were observed under a fluorescence microscope (OLYMPUS 1X70), and three fluorescent labels were used. The Afp/GFP cells expressed a green fluorescent protein after they have differentiated to visceral endoderm. Hoechst 33342 is a blue fluorescent stain used to mark the nucleus of cells, both dead and alive. Propidium iodide (PI), a red fluorescent stain, was also used because it is only permeable to non-living membranes and is used for detecting dead cells. By comparing GFP, Hoechst and PI presence, live cells were distinguished from dead ones and identified if the embryonic stem cells differentiated into the more mature endodermal cells (Hamazaki T. et al. 2004).

Figures 4 and 5 illustrate representative areas of the mES cell-scaffold interactions with the various scaffold and coating types.

On day 9, cells were mainly located on corners of the scaffolds. Figure 4 displays representative areas where cells were found on each specific scaffold coating. The uncoated scaffolds exhibited unattached embryoid bodies with the outer layers differentiating to endodermal cells. Some cells attached and had also differentiated into endoderm. By comparing the nuclei stains, most cells seemed dead on the scaffold.

The gelatin coated scaffolds did not seem to form embryoid bodies or differentiated endodermal cells. However, the cells that did attach seemed alive and spreading onto the scaffold.

The MATRIGEL coated scaffolds showed little evidence of embryoid bodies forming in the fluorescent microscopy. The mES cells seemed to have attached, spread, and differentiated endodermally in locations in the scaffold's interconnected structure. After monitoring the MATRIGEL coated scaffolds, cells seemed to be alive rather than dead.

Day 24: At Day 24, mES cells were discovered throughout the scaffolds and representative areas were selected for fluorescent microscopy seen in Figure 5. The uncoated PCL scaffolds had some cellular attachment and differentiation into endoderm. Some embryoid bodies were also visible in the culture well.

The gelatin coated scaffold displayed both cellular attachment and spreading. Some differentiations into endodermal cells were found, along with some embryoid bodies. Also, few dead cells were spotted using PI and fluorescent microscopy. The MATRIGEL coating also presented cellular attachment and spreading. Some differentiations into endoderm were spotted throughout the scaffold, hi both the MATRIGEL and gelatin coated scaffolds, cells were located on multiple layers of the scaffold as observed in Figures 6 A and 6B.

Cells were dissociated with 0.25% trypsin/EDTA after day 24 of the experiment. The flow cytometry was performed on FACS Sort (BD Biosciences) on a data set of 30,000 cells, which was then recorded using CellQuest Acquisition software (BD Biosciences). Flow cytometry helps illustrate the separation of GFP-positive and negative cells and the cell's size

(Hamazaki T. et al. 2004).

Figure 7 illustrates the differences between the uncoated scaffold and the coated scaffolds. The uncoated scaffold had a lower individual cell size and few GFP positive cells. Both the MATRIGEL and gelatin coated scaffold had a higher population of individual cells with larger cell sizes and coarser nuclear configurations than the uncoated scaffolds.

The scaffold's morphology and cellular attachment was examined by FESEM (JEOL-

6335F). Once the cell study was completed, the cells were fixed in a 2.5% glutaraldehyde/PBS fixation made from 50% glutaraldehyde (Electron Microscopy Sciences) and PBS (IX) with a pH of 7.4 (Gibco). The scaffolds were rinsed with DI water and then dehydrated with ten minute baths of 70%, 80%, 95%, and 100% (three times) of ethanol/DI water solutions. After the scaffolds were fixed and dehydrated, they were mounted on an aluminum stub, and a C evaporation coating was applied. The samples were imaged at 1OkV with a working distance of 15mm. Low acceleration voltages were used initially to ensure no beam damage. Images were taken at high and low magnifications and of both layers and completed scaffolds to identify the scaffold's microstructure and any possible cellular attachment.

Figures IA, IB, and 2B show an interconnected porous scaffold at 2 different levels of magnification. The open morphologies of the scaffolds created using the phase separation technique are evident. By way of comparison, PCL scaffolds created by the salt-leaching technique create "pockets" in the scaffolds, as illustrated in Figure 2 A.

Figures 3A and 3B display the effect of coatings on the fibrous phase separated PCL coated scaffolds. The MATRIGEL coated scaffold (Figure 3A) had a much rougher, bumpy surface after it had been dried and prepared for FESEM analysis. This is probably a result of fact that the MATRIGEL coating is much more viscous and globular then the gelatin coating.

The gelatin coating seen in Figure 3B looks more like thin sheet coating over the scaffold.

Without being limited by theory, it was hypothesized that the morphologies of these coatings may affect the cell's morphology on the coating surface. However, the true morphologies of these coatings cannot be represented using FESEM imagining and is believed that the coatings would be hydrated and swollen in solution.

The MATRIGEL coated scaffolds had cellular attachment throughout the samples, and the cells were mainly found clustered together. It also appears that the cytoplasm had collapsed in towards the nucleus as seen in Figures 8A and 8B. This effect is thought to be caused by how the cells adhered to the viscous MATRIGEL. As the cells grasped the scaffold's structure, they clustered together (Figure 8A) because of the hydrogel properties of the coating. Figures 9A and 9B display a representative area of a gelatin coated, phase separated

PCL scaffold that shows how the mES cells attach thereto. Once again, the nucleus and cytoplasm are visibly seen spread out on the surface of the scaffold, unlike the clusters formed with MATRIGEL. However, the cells did not collapse like the MATRIGEL coated scaffolds. The gelatin coating was more of a thin sheet coating the scaffold and allowed the cells to adhere and spread out instead of clustering.

Total RNA was extracted by using a RNA aqueous kit (Ambion). Then, cDNA was synthesized by using Superscript II first-strand synthesis system with oligo(dT) (Gibco). PCR was performed by using Taq DNA polymerase (Eppendorf). For each gene, the DNA primers were originated from different exons to ensure that the PCR product represents the specific mRNA species and not geomic DNA (Hamazaki T. et al. 2004). Gene markers were then compared to determine possible cells lineages.

As seen in Figure 10, expression of various differentiation protein markers were recorded after Day 24. These markers help determine the differentiation of the cells and their possible final cell line. The "housekeeping" gene, β-actin, is used to prove that cellular life exists and that the scaffolds did not create a toxic environment for the cells (Hamazaki T. et al. 2004; Hamazaki et al. 2003). Other forced expressions of transcription factors that were chosen to look for were specific linage differentiation markers, such as GATA6, hepatocyte nuclear (HNF4), and transthyretin (TTR) for primitive endoderm (Hamazaki T. et al. 2004; Hamazaki et al. 2003).The expression of Albumin (ALB) can imply the cells are becoming hepatic or visceral endoderm differentiated (Hamazaki T. et al. 2004; Hamazaki et al. 2003). Also, Nestin proteins were also examined as markers for neural differentiation. After observing the scaffolds, differences were observed between protein markers expressed in the RT-PCR data, β-actin was expressed for all the scaffolds proving that cellular adhesion existed on all the scaffolds. The gelatin coated scaffolds proved to be the most expressed scaffold of differentiation markers for hepatic/visceral endoderm differentiation showing GAT A6, HNF4, TTR, and ALB. Therefore, both gelatin coated and MATRIGEL coated scaffolds appear to further the ES cells to differentiate into a specific cell linage. MATRIGEL coated scaffolds showed some evidence of possible future neural differentiation with the expression of Nestin. The MATRIGEL coated scaffolds also have a slight possibility to differentiate into an early hepatocyte/visceral endoderm cell. The uncoated scaffolds showed no real assistance to help further the mES cell populations into the desired cell lines (that is, liver or nerve).

EXAMPLE 4— Target Cells

There are over 200 cell types in the human body and the methods, scaffolds, and bioreactors of the subject invention are useful in seeding, attaching, proliferating, culturing, and/or differentiating any of these cell types, therapeutic, manufacturing, or other purposes. Examples of cell types that can be seeded, attached, proliferated, cultured, and/or differentiated using the methods, scaffolds, and bioreactors of the subject invention are listed in the table below. Other examples of cell types that can be seeded, attached, proliferated, cultured, and/or differentiated are disclosed herein.

Table 1. Examples of Target Cells

Keratinizing Epithelial Cells keratinocyte of epidermis basal cell of epidermis keratinocyte of fingernails and toenails basal cell of nail bed hair shaft cells medullary cortical cuticular hair-root sheath cells cuticular of Huxley's layer of Henle's layer external hair matrix cell Table 1. Examples of Target Cells

Cells of Wet Stratified Barrier Epithelia surface epithelial cell of stratified squamous epithelium of cornea tongue, oral cavity, esophagus, anal canal, distal urethra, vagina basal cell of these epithelia cell of urinary epithelium

Epithelial Cells Specialized for Exocrine Secretion cells of salivary gland mucous cell serous cell cell of von Ebner's gland in tongue cell of mammary gland, secreting milk cell of lacrimal gland, secreting tears cell of ceruminous gland of ear, secreting wax cell of eccrine sweat gland, secreting glycoproteins cell of eccrine sweat gland, secreting small molecules cell of apocrine sweat gland cell of gland of Moll in eyelid cell of sebaceous gland, secreting lipid-rich sebum cell of Bowman's gland in nose cell of Brunner's gland in duodenum, secreting alkaline solution of mucus and enzymes cell of seminal vesicle, secreting components of seminal fluid, including fructose cell of prostate gland, secreting other components of seminal fluid cell of bulbourethral gland, secreting mucus cell of Bartholin's gland, secreting vaginal lubricant cell of gland of Littre, secreting mucus cell of endometrium of uterus, secreting mainly carbohydrates isolated goblet cell of respiratory and digestive tracts, secreting mucus mucous cell of lining of stomach zymogenic cell of gastric gland, secreting pepsinogen oxyntic cell of gastric gland, secreting HCl acinar cell of pancreas, secreting digestive enzymes and bicarbonate

Paneth cell of small intestine, secreting lysozyme type II pneumocyte of lung, secreting surfactant

Clara cell of lung

Cells Specialized for Secretion of Hormones cells of anterior pituitary, secreting growth hormone follicle-stimulating hormone luteinizing hormone prolactin adrenocorticotropic hormone thyroid-stimulating hormone cell of intermediate pituitary, secreting melanocyte-stimulating hormone cells of posterior pituitary, secreting oxytocin Table 1. Examples of Target Cells vasopressin cells of gut and respiratory tract, secreting serotonin endorphin somatostatin gastrin secretin cholecystokinin insulin glucagons bombesin cells of thyroid gland, secreting thyroid hormone calcitonin cells of parathyroid gland, secreting parathyroid hormone oxyphil cell cells of adrenal gland, secreting epinephrine norepinephrine steroid hormones mineralocorticoids glucocorticoids cells of gonads, secreting testosterone estrogen progesterone cells of juxtaglomerular apparatus of kidney juxtaglomerular cell macula densa cell peripolar cell mesangial cell

Epithelial Absorptive Cells in Gut, Exocrine Glands, and Urogenital Tract brush border cell of intestine striated duct cell of exocrine glands gall bladder epithelial cell brush border cell of proximal tubule of kidney distal tubule cell of kidney nonciliated cell of ductulus efferens epididymal principal cell epididymal basal cell

Cells Specialized for Metabolism and Storage hepatocyte fat cells (for example, adipocyte) white fat brown fat Table 1. Examples of Target Cells lipocyte of liver

Epithelial Cells Serving Primarily a Barrier Function, Lining the Lung, Gut, Exocrine Glands, and Urogenital Tract type I pneumocyte pancreatic duct cell nonstriated duct cell of sweat gland, salivary gland, mammary gland, etc. parietal cell of kidney glomerulus podocyte of kidney glomerulus cell of thin segment of loop of Henle collecting duct cell duct cell of seminal vesicle, prostate gland, etc.

Epithelial Cells Lining Closed Internal Body Cavities vascular endothelial cells of blood vessels and lymphatics {e.g., microvascular cell) fenestrated continuous splenic synovial cell serosal cell squamous cell lining perilymphatic space of ear cells lining endolymphatic space of ear squamous cell columnar cells of endolymphatic sac with microvilli without microvilli "dark" cell vestibular membrane cell stria vascularis basal cell stria vascularis marginal cell cell of Claudius cell of Boettcher choroid plexus cell squamous cell of pia- arachnoid cells of ciliary epithelium of eye pigmented nonpigmented corneal "endothelial" cell

Ciliated Cells with Propulsive Function of respiratory tract of oviduct and of endometrium of uterus of rete testis and ductulus efferens of central nervous system

Cells Specialized for Secretion of Extracellular Matrix epithelial: ameloblast planum semilunatum cell of vestibular apparatus of ear interdental cell of organ of Corti Table 1. Examples of Target Cells nonepithelial: fibroblasts pericyte of blood capillary (Rouget cell) nucleus pulposus cell of intervertebral disc cementoblast/cementocyte odontoblast/odontocyte chondrocytes of hyaline cartilage of fibrocartilage of elastic cartilage osteoblast/osteocyte osteoprogenitor cell hyalocyte of vitreous body of eye stellate cell of perilymphatic space of ear

Contractile Cells skeletal muscle cells red white intermediate muscle spindle-nuclear bag muscle spindle-nuclear chain satellite cell heart muscle cells ordinary nodal

Purkinje fiber Cardiac valve tissue smooth muscle cells myoepithelial cells: of iris of exocrine glands

Cells of Blood and Immune System red blood cell (erythrocyte) megakaryocyte macrophages monocyte connective tissue macrophage

Langerhan's cell osteoclast dendritic cell microglial cell neutrophil eosinophil basophil mast cell plasma cell

Table 1. Examples of Target Cells adrenergic peptidergic

)orting Cells of Sense Organs and of Peripheral Neurons supporting cells of organ of Corti inner pillar cell outer pillar cell inner phalangeal cell outer phalangeal cell border cell Hensen cell supporting cell of vestibular apparatus supporting cell of taste bud supporting cell of olfactory epithelium

Schwann cell satellite cell enteric glial cell

Neurons and Glial Cells of Central Nervous System neurons glial cells astrocyte oligodendrocyte

Lens Cells anterior lens epithelial cell lens fiber

Pigment Cells melanocyte retinal pigmented epithelial cell iris pigment epithelial cell

Germ Cells oogonium/oocyte spermatocyte

Spermatogonium blast cells fertilized ovum

Nurse Cells ovarian follicle cell

Sertoli cell thymus epithelial cell (e.g., reticular cell) placental cell

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

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Claims

CLAIMS We claim:

1. A tissue engineering scaffold comprising a three-dimensional porous matrix of polycaprolactone, wherein the matrix is provided by at least one fibrous web layer of polycaprolactone, wherein the matrix is optionally coated with at least one coating of an extracellular material selected to promote cell seeding, attachment, proliferation, culturing, or differentiation to a target cell, or a combination of any of the foregoing.

2. The scaffold according to claim 1, wherein the extracellular material is a solubilized basement membrane preparation.

3. The scaffold according to claim 1, wherein the extracellular material is gelatin.

4. The scaffold according to claim 1, wherein the extracellular material is collagen, elastin, laminin, fibrillin, fibronectin, or proteoglycan.

5. The scaffold according to claim 1, wherein the target cell is hepatocyte endoderm cells.

6. The scaffold according to claim 1, wherein the target cell is visceral endoderm cells.

7. The scaffold according to claim 1, further comprising one or more cells attached to and within the three-dimension matrix.

8. The scaffold according to claim 7, wherein one or more cells comprise a plurality of cells attached to and within the three-dimensional matrix.

9. The scaffold according to claim 7, wherein the one or more cells comprise one or more stem cells.

10. The scaffold according to claim 7, wherein the one or more cells comprise one or more cells of the target cell.

11. The scaffold according to claim 1, wherein the pores of the three-dimensional matrix are distributed non-uniformly throughout the scaffold.

12. The scaffold according to claim 1, wherein the pores are asymmetric.

13. The scaffold according to claim 1, wherein the pores are each about 10 μm to about 100 μm in length.

14. The scaffold according to claim 1, comprising at least two fibrous web layers, wherein each fibrous web layer is fused together by phase separation.

15. The scaffold according to claim 1, comprising two fibrous web layers, wherein each fibrous web layer is fused together by phase separation.

16. The scaffold according to claim 1, comprising three fibrous web layers, wherein each fibrous web layer is fused together by phase separation.

17. The scaffold according to claim 1, comprising four or more fibrous web layers, wherein each fibrous web layer is fused together by phase separation.

18. The scaffold according to claim 7, wherein the one or more cells comprise a target cell line.

19. The scaffold according to claim 7, wherein the one or more cells comprise more than one cell, and wherein the more than one cell comprises one or more stem cells and one or more target cells differentiated from a stem cell.

20. A method for preparing a scaffold useful for tissue engineering, said method comprising: a) providing a solution comprising polycaprolactone in a solvent, wherein the polycaprolactone comprises about 1% (wt/vol) to about 10% (wt/vol) of the solution; and b) contacting the solution with a sufficient amount of water to generate a phase separation, wherein the polycaprolactone separates from the solvent and forms a three- dimensional porous matrix comprising a layer of a fibrous web.

21. The method according to claim 20, further comprising: c) optionally, coating the matrix with an extracellular material selected to promote cell seeding, attachment, proliferation, culturing, or cell differentiation to a target cell line, or a combination of any of the foregoing.

22. The method according to claims 20 or 21, wherein the polycaprolactone comprises about 5% (wt/vol) of the solution of step a).

23. The method according to claims 20 or 21, wherein step a) and step b) are repeated at least once to form a scaffold with more than one layer.

24. The method according to claim 23, wherein each layer is fused together by placing a first layer in contact with a second layer to form an interface between the first layer and second layer, and applying water to the interface to generate a phase separation.

25. The method according to claim 23, wherein each layer is coated with an extracellular material selected to promote cell seeding, attachment, proliferation, culturing, or cell differentiation to a target cell line, or a combination of any of the foregoing.

26. The method according to claims 20 or 21, wherein the solvent is acetone.

27. The method according to claim 21, wherein the extracellular material is a solubilized basement membrane preparation.

28. The method according to claim 21, wherein the extracellular material is gelatin.

29. The method according to claim 21, wherein the extracellular material is collagen, elastin, laminin, fibrillin, fibronectin, or proteoglycan.

30. A method for using a tissue engineering scaffold as a platform useful for cell seeding, attaching, proliferation, culturing, or differentiating, wherein the method comprises: a) providing a scaffold according to any of claims 1-19; b) contacting the scaffold with a cell suspension; wherein the cell seeds and attaches to the three-dimensional porous matrix.

31. The method according to claim 30, wherein the contacting step comprises submerging the scaffold in the cell suspension.

32. The method according to claim 30, wherein the contacting step comprises passing the cell suspension through the scaffold.

33. The method according to claim 32, wherein the suspension is continuously passed through the scaffold.

34. The method according to claim 30, wherein the cell suspension comprises one or more stem cells.

35. The method according to claim 30, wherein a stem cell attaches to the three- dimensional porous matrix, and wherein the method further comprises differentiating the stem cell into a target cell.

36. The method according to claim 35, wherein the differentiating step further comprises contacting the scaffold with one or more agents that induce differentiation.

37. The method according to claim 30, wherein the cell suspension comprises one or more cells suspended in a fluid media.

38. A bioreactor comprising: a) a vessel comprising a housing having at least one chamber; and b) at least one scaffold of any one of claims 1-19; wherein each chamber houses at least one scaffold.

39. The bioreactor according to claim 38, further comprising c) one or more reservoirs, wherein each reservoir holds a fluid media; and d) one or more conduits operatively connecting each reservoir with each chamber.

40. The bioreactor according to claim 38, further comprising a plurality of conduits operatively connected to each chamber and interfaced with the housing.

41. The bioreactor according to claims 38, 39, or 40, wherein fluid media is in contact with each scaffold.

42. The bioreactor according to claim 41, wherein fluid media is passed through the scaffold.

43. The bioreactor according to claim 41, wherein fluid media is continuously passed though the scaffold.

44. The bioreactor according to claim 39, wherein one or more reservoirs contain a fluid media selected from the group consisting of saline, phosphate buffer solution, cell culture media, agents that induce cell differentiation, and suspensions of one or more cells; wherein the cells are selected for seeding, attaching, proliferation, culturing, or differentiation to a target cell, or combinations of any of the foregoing.

45. The use of the tissue engineering scaffold of any of the preceding claims as a cell or tissue culture.

46. The use of a tissue engineering scaffold of any of the preceding claims as an organ useful for transplantation.