In vitro synthesis of a layered cell sorted tissue

Details In vitro synthesis of a layered cell sorted tissue In vitro synthesis of a layered cell sorted tissue

2001-12-07

2004-11-09

Lankford, Leon (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Two or more cell types, per se, in co-culture

C435S325000, C435S410000

Reexamination Certificate

active

06815202

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to a layered tissue cultivated in vitro, i.e., in tissue culture. In particular, the invention relates to a layered tissue that is generated by sorting of cells from a homogenous cell mixture into discrete layers by cell type that are connected by a basement membrane. Methods for forming and using the in vitro cell sorted layered tissue are also described.
BACKGROUND OF THE INVENTION
Advances in tissue culture technique have led to the development of artificial tissue and promising research for the development of artificial organs. However, artificial tissues are generally still inferior to their counterpart in vivo. For example, homeostatic regulation and many specific cell interactions that regulate cell proliferation and organization are lost so that in many structural aspects, the artificial and native tissues are not equivalent. The addition of various hormones and growth factors to culture media has helped to more closely simulate in vivo conditions, but remains an insufficient remedy.
The co-culturing of keratinocytes and fibroblasts, for example, has been documented as being “antagonistic”, resulting in an overgrowth of fibroblasts in Leigh et al. (1994).
The Keratinocyte Handbook,
Cambridge University Press, Great Britain. In one approach, researchers have attempted to restrict fibroblast growth in culture by using irradiated fibroblasts in feeder layers, but substantial improvement in epithelial structure and correct expression of differentiation markers was not found. Currently, cultured epithelia is being created using organotypic culture techniques (Hansbrough et al.,
JAMA
262:2125-2130 (1989); Cooper et al.,
Surgery,
109:198-207 (1991); and Boyce et al.,
Plast. Reconstr. Surg.,
91:632-641 (1993)) or methods that employ forming epidermal and dermal layers separately, and then sandwiching them together, or by relying on a synthetic support to generate the keratinocyte and/or fibroblast layer (Yannas et al.,
Science,
215:174 (1982)). In all these techniques, the epithelial tissues are formed using a preformed support.
Furthermore, products that employ artificial tissue are currently being marketed. For example, Dermagraft® (Advanced Tissue Sciences, La Jolla, Calif.) is a polylactic, polyglycol, or polygalactoside material upon which fibroblasts are cultivated. Autologous keratinocytes are then seeded onto these materials.
GraftSkin® (Organogenesis, Inc., Boston, Mass.) is a product where fibroblasts are cultivated on a collagen based substrate.
AlloDerm® (Life Cell Corp., The Woodlands, Tex.) constitutes human or pig's skin in which the basal membrane and dermal matrix remain intact. The tissue is stored at approximately −80° C. until ready to be used, then seeded with autologous fibroblasts and keratinocytes prior to application onto a patient.
However, these, and most other products in development do not allow the in vitro reconstruction of a functional epidermal-dermal junction or basement membrane. Thus, dissection or “blistering” between the keratinocyte and fibroblast layers tends to occur.
Another method described in U.S. patent application Ser. No. 09/037,191, which is herein incorporated by reference in its entirety, relates to the in vivo creation of an artificial layered tissue formed by sorting of keratinocytes and fibroblasts from a homogenous mixture of cells into discrete epidermal, dermal, and basal keratinocyte layers. However, the tissue generated in this manner could not be used in applications requiring sterility, i.e., where contamination by cells or factors from an intermediate host presents a problem for the final host receiving the graft.
Therefore, new artificial tissues that can be created in a sterile environment are needed. Furthermore, an artificial tissue that preserves native tissue architecture is needed, in particular, the formation of a proper epidermal-dermal junction, including a basement membrane is needed.
SUMMARY OF THE INVENTION
The present invention is a layered cell sorted tissue that includes a discrete first cell layer and a discrete second cell layer that are formed in vitro by the spontaneous sorting of cells from a homogenous cell mixture. The first cell layer may include any cell type, but preferably includes epithelial cells. In a preferred embodiment, the first cell layer includes keratinocytes. The keratinocytes are preferably obtained from an early passage number such as passage 2 or 3. Other cell types that may used in the first cell layer are CaCo2 cells, A431 cells, and HUC18 cells. The second cell layer may also include cells of any type, but preferably includes mesencyhmal cells. In a preferred embodiment, the second layer includes fibroblasts.
The number of cells used in the homogenous cell mixture is higher than that seen in the prior art, and is thought to contribute to the in vitro cell sorting process. For example, 4×10
6
cells of each cell type are generally used. In a preferred embodiment, 4×10
6
fibroblasts are added to the homogenous cell mixture.
Additionally, the layered cell sorted tissue possesses an epidermal-dermal junction that is substantially similar in structure and function to its native counterpart. That is, the tissue expresses the necessary integral proteins such as hemidesmosomes and collagen I, collagen IV, and collagen VII, to attach the epidermal and dermal layers with the proper basement membrane morphology. As used herein, the term “basement membrane” also refers to the basal lamina and may be used interchangeably with the term “basal lamina.” Therefore, the tendency of the layered cell sorted tissue to separate or “blister” is reduced in comparison to other tissue prototypes currently being marketed.
The layered cell sorted tissue may be formed to suit any particular arrangement of layers that is desired. In one embodiment, an epidermal layer is formed as the top layer of tissue. In another embodiment, the epidermal layer is the bottom layer of tissue.
The orientation of the cell layers is manipulated by contacting the homogenous mixture of cells with a connective tissue component during culturing. Connective tissue components that may be used are fibronectin, collagen IV, laminin, and mixtures thereof. Fibronectin is the preferred connective tissue component. The homogenous cell mixture sorts to form an epidermal layer that contacts the connective tissue component.
In one embodiment, the homogenous mixture of cells is added to a transwell membrane coated with fibronectin. The tissue that is formed is upside down, i.e., the epidermal layer is the bottom layer. In another embodiment, a solution of fibronectin is added to a homogenous mixture of cells. The tissue formed by this method has an epidermal layer that is on top.
In another embodiment, the layered cell sorted tissue may be used as a skin graft to treat traumatic wounds, burn injury, decubiti and other ulcerations due to such conditions as diabetes mellitus and chronic venous stasis. If used as a skin graft, the layered cell sorted tissue may be placed directly on the graft site. However, in another embodiment, a homogenous mixture of keratinocytes and fibroblasts may also be applied to the graft site and allowed to spontaneously sort into a discrete layer including keratinocytes and a discrete layer including fibroblasts.
In a further embodiment, the cell sorted tissue may be used in an in vitro assay to detect tissue responses to chemicals or drugs. Upon providing a cell sorted tissue, the tissue is contacted with the chemical or drug of interest. The response of the tissue to the chemical or drug is determined by comparing the effect of the chemical or drug on the phenotype, genotype, or both, between the tissue before and after contact with the chemical or drug.
In another embodiment, the layered cell sorted tissue is used as an in vitro assay for studying tumor cell metastasis, i.e., tumor cell invasion. Upon providing the layered cell sorted tissue on a light blocking plate such as a BD BioCoat FluorBlok plate (BD Biosciences

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