Cells or tissues with increased protein factors and methods...

Details Cells or tissues with increased protein factors and methods... Cells or tissues with increased protein factors and methods...

1998-08-21

2001-09-18

Clark, Deborah J. R. (Department: 1633)

Chemistry: molecular biology and microbiology

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

Solid support and method of culturing cells on said solid...

C435S001300, C435S325000, C435S347000, C435S373000, C435S402000, C435S455000

Reexamination Certificate

active

06291240

ABSTRACT:

INTRODUCTION
The invention relates to cells or tissues having an increased amount of at least one regulatory protein, and methods of producing and using those cells or tissues. More specifically, the invention relates to the induced production of regulatory proteins, such as cytokines, growth factors, angiogenic factors, inflammatory mediators and/or stress proteins, in cells or tissue constructs following cryopreservation and subsequent thawing of the cells or tissues. The compositions and methods of this invention are useful for the treatment of wound healing and the repair and/or regeneration of other tissue defects including those of skin, cartilage, bone, neurons, astrocytes, and vascular tissue as well as for enhancing the culture and/or differentiation of cells and tissues in vitro.
1. BACKGROUND
The ability to induce regulatory proteins such as growth factors, cytokines, and stress proteins in cells or tissues opens new possibilities in the preparation of tissue-engineered products for use in tissue repair, e.g., in the treatment of wounds and other tissue defects. For example, growth factors are known to play an important role in the wound healing process and evidence indicates that some stress proteins may also be important in this process. In general, it is thought desirable in the treatment of wounds to enhance the supply of growth factors by direct addition or, in the case of tissue-engineering, by enhancing cellular production.
Cellular cytokines and growth factors are involved in a number of critical cellular processes including cell proliferation, adhesion, morphologic appearance, differentiation, migration, inflammatory responses, angiogenesis, and cell death. Many of these cytokines and factors are induced as a result of stress placed upon cells. For example, studies have demonstrated that hypoxic stress and injury to cells induce responses including increased levels of mRNA and proteins corresponding to growth factors such as PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor), FGF (fibroblast growth factor), and IGF (insulin-like growth factor) (Gonzalez-Rubio, M. et al., 1996, Kidney Int. 50(1):164-73; Abramovitch, R. et al., 1997, Int J. Exp. Pathol. 78(2):57-70; Stein, I. et al., 1995, Mol Cell Biol. 15(10):5363-8; Yang, W. et al., 1997, FEBS Lett. 403(2):139-42; West, N. R. et al., 1995, J. Neurosci. Res. 40(5):647-59). Many of these responses include a cascade type reaction via tyrosine phosphorylation or the induction of stress proteins.
Two known stress proteins are GRP78 and HSP90. These proteins stabilize cellular structures and render the cells resistant to adverse conditions. Some stress proteins have also been found to aid in wound healing through the induction of growth factors, such as transforming growth factor-&bgr;, also known in the art as TGF-&bgr;. This family of dimeric proteins includes TGF-&bgr;1, TGF-&bgr;2, and TGF-&bgr;3 and regulates the growth and differentiation of many cell types. Furthermore, this family of proteins exhibits a range of biological effects, stimulating the growth of some cell types (Noda et al., 1989, Endocrinology 124:2991-2995) and inhibiting the growth of other cell types (Goey et al., 1989, J. Immunol. 143:877-880; Pietenpol et al., 1990, Proc. Natl. Acad. Sci. USA 87:3758-37662). TGF-&bgr; has also been shown to increase the expression of extracellular matrix proteins including collagen and fibronectin (Ignotz et al., 1986, J. Biol. Chem. 261:4337-4345) and to accelerate the healing of wounds (Mustoe et al., 1987, Science 237:1333-1335).
Another such growth factor is PDGF. PDGF was originally found to be a potent mitogen for mesenchymal-derived cells (Ross R. et al., 1974, Proc. Natl. Acad. Sci. USA 71(4):1207-1210; Kohler N. et al., 1974, Exp. Cell Res. 87:297-301). Further studies have shown that PDGF increases the rate of cellularity and granulation in tissue formation. Wounds treated with PDGF have the appearance of an early stage inflammatory response including an increase in neutrophils and macrophage cell types at the wound site. These wounds also show enhanced fibroblast function (Pierce, G. F. et al., 1988, J. Exp. Med. 167:974-987). Both PDGF and TGF-&bgr; have been shown to increase collagen formation, DNA content, and protein levels in animal studies (Grotendorst, G. R. et al., 1985, J. Clin. Invest. 76:2323-2329; Sporn, M. B. et al., 1983, Science (Wash D.C.) 219:1329). The effect of PDGF in wound healing has been shown to be effective in human wounds. In human wounds, PDGF-AA expression is increased within pressure ulcers undergoing healing. The increase of PDGF-AA corresponds to an increase in activated fibroblasts, extracellular matrix deposition, and active vascularization of the wound. Furthermore, such an increase in PDGF-AA is not seen in chronic non-healing wounds (Principles of Tissue Engineering, R. Lanza et al. (eds.), pp. 133-141 (R.G. Landes Co. TX 1997). A number of other growth factors having the ability to induce angiogenesis and wound healing include VEGF, KGF and basic FGF.
A number of attempts have been made at increasing wound healing through the use of topical gels, ointments, and lotions containing angiogenic or other growth factors. These attempts have incorporated various growth factors and cytokines or combinations thereof. Some success has been recognized; however, problems exist with regard to the quantities, application, and responsiveness to these ointments. Such problems include the need for recurrent applications over a period of time, varying concentrations or application times, and adverse reactions to exogenous factors.
There are currently no simple effective methods or compositions for application of cytokines, growth factors or other regulatory proteins for the repair and regeneration of tissue defects, whereby the amount and duration of such proteins is determined by a naturally-occurring physiological response.
2. SUMMARY OF THE INVENTION
The present invention relates to cells or tissues containing an increased amount of at least one regulatory protein, and methods of producing and using those cells or tissues. More specifically, the present invention relates to the induction of regulatory proteins such as cytokines, growth factors, angiogenic factors, inflammatory mediators and/or stress proteins, in cells or tissues by cryopreservation of the cells or tissues, followed by thawing. The cryopreservation/thaw cycle induces the expression in the cells or tissues of one or more genes that encodes a regulatory protein such as a cytokine, growth factor, angiogenic factor, inflammatory mediator and/or stress protein, thus providing the cells or tissues with an increased amount of one or more of these factors.
According to the present invention, the cells or tissues are cryopreserved or frozen to below −50° C.; more preferably, to below −65 to −70° C. However, the cells or tissues may be frozen to temperatures as low as −150 to −180° C. The cryopreserved cells or tissues can be stored for a period of years, although they should be checked periodically for maintenance of viability. In order to induce the production of the regulatory proteins in accordance with this invention, the cryopreserved cells or tissues are thawed rapidly, preferably in a water bath at 34-37° C., to avoid damage to the cells.
According to one embodiment of the invention, cells are grown in two-dimensional culture, e.g., on a monolayer, and subjected to cryopreservation, followed by thawing.
According to another embodiment, cells are grown in three-dimensional culture and subjected to cryopreservation and subsequent thawing. According to a further preferred embodiment, the cells are grown on a non-living three-dimensional support, framework or scaffold to form a tissue construct that is subjected to cryopreservation and subsequent thawing for the enhanced production of regulatory proteins.
Thus, a preferred embodiment of the invention is a tissue construct comprising cells attached to a non-living three-dimensional framework, the tis

Affiliated with

Also associated with

Rating

Cells or tissues with increased protein factors and methods... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Cells or tissues with increased protein factors and methods..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cells or tissues with increased protein factors and methods... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2455440