Cellular attachment to trans-epithelial appliances

a technology of transepithelial appliances and cellular attachment, which is applied in the field of cellular attachment to transepithelial appliances, can solve the problems of many primary cells and tissues that are difficult to establish in vitro on normal tissue culture plastics, inflammation and/or infection, and remain elusive in the purification of adhesion-facilitating proteins

Inactive Publication Date: 2000-08-29
DESMOS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purification of adhesion-facilitating proteins has remained elusive.
Any medical device, including indwelling catheters and colostomy tubes, which breach the skin for an extended period of time will result in inflammation and / or infection.
However, many primary cells and tissues are difficult to establish in vitro on normal tissue culture plastic.
Although this problem is partially alleviated by the use of extracellular matrix-coated cell supports, this is only a temporary solution.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of 804G Matrix

804G rat bladder carcinoma cells were maintained at 37.degree. C. in Modified Eagle's Medium with Earle's salts supplemented with 50 U / ml penicillin, 50 .mu.g / ml streptomycin and 10% FCS (Gibco, Grand Island, N.Y.). The cells were grown to confluency on either plastic Petri dishes or glass coverslips. The culture medium was discarded and the cells were washed in sterile Phosphate Buffered Saline (PBS). The cells were separated from the deposited matrix by incubation for 5 min. in 20 mM NH.sub.4 OH, followed by three rapid washes with distilled water.

The remaining matrix was removed from the substrate by solubilization in 8M urea, 1% SDS in 10 mMTris-HCl, pH 6.8. The 804G matrix polypeptide profile was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using routine experimental methods known to those of skill in the art. Three major proteins were present in the matrix, ranging in size from 135-150 kD. A minor peptide of 85 kD was also present in ...

example 2

Production of Polyclonal Antibodies against the 804G Matrix

Antiserum was prepared by injecting urea / SDS solubilized cell matrix, as described in Example 1, into a rabbit by standard methods. Briefly, solubilized 804G matrix was mixed with Freund's adjuvant and injected into a rabbit. Serum was collected at three weekly intervals following one booster injection. The isolated polyclonal antiserum (J18) had antibodies recognizing four glycosylated 135-400 kD species as well as the 85 kD polypeptide.

Monoclonal antibodies against the 804G matrix were then produced as described below.

example 3

Production of Monoclonal Antibodies against the 804G Matrix

A mouse monoclonal IgG (5C5) against the 804G cell matrix was prepared by injecting a solubilized 804G cell matrix sample into several mice. At two and three weeks after the initial injection the mice were boosted with further 804G matrix injections. Five days following the final boost, their spleens were removed and isolated spleen cells were fused with the myeloma cell line Sp2 for the production of hybridomas using standard techniques (Galfre and Milsrein, 1981). Hybridoma cells producing antibodies against matrix elements were selected on the basis of their immunoblotting and immunofluorescence reactivities against matrix samples. Selected hybridoma cells were cloned twice by limited-dilution. The 5C5 antibody recognized only a 150 and 140 kD polypeptide in the matrix preparation. Epithelial cells were plated on the matrix and functionally assessed for attachment and hemidesmosome formation as described in the following ...

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Abstract

A trans-epithelial appliance having a hemidesmosome formation-inducing protein composition derived from rat bladder carcinoma cells deposited thereon. This composition stimulates cell attachment and may be either the cell matrix or a soluble factor isolated from the conditioned medium. The appliance will be useful for diminishing inflammation and / or infection at the site of entry of the appliance. The appliance may also be used to stimulate gum junctional epithelium adhesion in the treatment of gingivitis and periodontitis. The composition may be used to maintain tissues ex vivo.

Description

FIELD OF THE INVENTIONThe present invention relates to the attachment of cells to shaped articles. More specifically, the invention relates to the attachment of epithelial cells to biologically compatible implants and appliances.BACKGROUND OF THE INVENTIONWhen organs of the body are formed, they develop in neatly organized arrays. Often, cell types are separated by connective tissue called basement membranes. In skin, for instance, the superficial layer of epidermal cells adheres to the underlying basement membrane. This skin basement membrane acts as a barrier between the epidermal cells on the outside, and the dermal cells underneath. A similar arrangement of cells occurs in the lining of the gut and in the oral cavity.Basement membranes have been implicated in the growth, attachment, migration, repair and differentiation of their overlying cell populations. Three layers have been defined in basement membranes: a) the lamina lucida, an electronmicroscopically clear region in close...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A61L29/00A61L27/00A61L27/34A61L29/08A61L31/08A61L31/10C07K14/435C07K14/78C07K16/18C12N5/06A61K35/12C12N5/071
CPCA61L27/34A61L29/085A61L31/10C07K14/78C07K16/18C12N5/0621C12N5/0629C08L89/00A61K35/12C12N2502/253C12N2533/30C12N2533/40C12N2533/90
Inventor JONES, JONATHAN C. R.QUARANTA, VITOTAMURA, RICHARD
Owner DESMOS
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