Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-11-17
2002-02-26
Peselev, Elli (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S356000, C530S412000
Reexamination Certificate
active
06350732
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for extracting lipids from tissue samples for the purpose of storing and preserving the tissue samples and the product of that method. The method of the present invention is used to particular advantage as a storage solution prefatory to the cross-linking of the intact collagen fibrils of collagenous tissue samples. In more detail, the present invention relates to a high osmotic pressure (compared to physiologic) solution which has particular advantage when used as a storage medium for samples of collagenous tissue before cross-linking the collagen in the tissue with any number of methods including glutaraldehyde and/or in accordance with the method described in the above-referenced U.S. Pat. No. 5,147,514.
Photooxidative cross-linking of the collagen fibrils of tissue samples such as bovine pericardium in accordance with the method of U.S. Pat. No. 5,147,514 results in a product having physical and chemical properties which make that product particularly suitable for use as a biomaterial for use as an artificial tendon, heart valve, or pericardial patch. Such biomaterials are characterized by several properties which confer upon them significant advantages over previously available materials used as bioprosthetics. They are produced by harvesting a sample of such tissue, incubating the sample in an aqueous media solution of a photooxidative catalyst buffered to about physiological pH (6.8-8.6), and then irradiating with light to cross-link the collagen.
It was discovered that it was advantageous to “precondition” the tissue sample by incubation in a media solution which did not include the catalyst before transfer to the solution including the catalyst for irradiation. When preconditioned in this manner, the resulting product shows decreased susceptibility to proteolytic degradation. It was also discovered this high osmolality, first media solution not only gives desirable results when used to precondition the tissue sample before cross-linking by that photooxidative process but also when used to store the tissue sample before cross-linking with glutaraldehyde and other processes as known in the art. The method and product of the present invention appear to achieve this result by removing, or decreasing the content, of the non-collagenous components of the tissue sample, including, in particular, extracting lipids from the tissue sample. Because lipids, and particularly phospholipids, are the main component of the membranes of living cells, it appears that extraction of lipids has the desirable effect of devitalizing the tissue sample, thereby reducing the bioburden of the tissue sample. A reduction in bioburden is a strong indicator of a longer shelf life of the tissue sample, and for this reason, the high osmolality solution of the present invention is advantageously used as a storage medium for the collagenous tissue sample.
It has long been standard practice, for instance, in histological laboratories, to store tissue samples in alcohol at low temperature and to use freeze drying if the sample is to be preserved for longer periods of time. Standard practice for storage of samples for relatively short periods of time usually involves incubation in one of several known saline solutions, buffered to physiological pH, at low temperature. However, both methods jeopardize the maintenance of the native fibrillar structure of structural proteins such as collagen.
The maintenance of collagen fibrillar structure is of particular concern in light of experimental data indicating that the method described in U.S. Pat. No. 5,147,514 results in the cross-linking of the collagen fibrils in their true, native state, e.g., as intact collagen fibrils, and that this capability of that method appears to be responsible for the excellent mechanical properties of the resulting product and the ability of the product to resist in vivo degradation. It is, therefore, an object of the present invention to provide a process for extracting lipids from collagenous tissue samples before photooxidative cross-linking of the collagenous tissue sample. However, it is apparent from the results obtained when used in that process that the media solution and lipid extraction process of the present invention are useful in preserving tissues for other cross-linking processes, such as acyl azide, polyglycidylethers, carbodiimide, and glutaraldehyde cross-linking, and other cross-liking processes known in the art, for preservation of any proteinaceous material or tissue, and perhaps even more broadly, as a storage medium for many different types of tissues, biomaterials, and/or extracts or solutions of same and/or their component parts or molecules.
In a broad sense, therefore, it is an object of the present invention to provide a method for preserving biological specimens, tissues, extracts, biomolecules, and/or isolates which both maintains the native state of the sample and helps protect the sample from damage or degradation caused by harmful agents which may be found in the sample or opportunistic, invasive agents by extracting lipids from the sample.
It is another object to provide a method for lipid extraction having particular utility for storing tissue samples for longer periods of time than previously possible, e.g., for a period of several weeks, at room temperature.
Another object of the present invention is to provide a previously unknown product having decreased phospholipid content compared to fresh, or untreated, tissue samples, with a longer shelf life than previously possible.
These and other objects, as well as the several advantages of the invention, will be apparent to those skilled in the art upon reading the specification, the examples and the appended claims.
SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided a method, or treatment, for extracting lipids from a collagenous tissue sample comprising immersing the sample in a high osmolality aqueous solution of a salt and a sugar, the salt being a salt selected from the group of salts which are capable of penetrating the sample, the sugar functioning to maintain the high osmolality of the solution even as salt concentration in the solution decreases as the salt penetrates the tissue sample, to decrease the bioburden of the sample as compared to the bioburden of untreated tissue samples. In a preferred embodiment, the salt and sugar are utilized in proportions in which the salt is utilized in a weight to volume ratio of higher than about 11.7% and the sugar is utilized in a concentration of about 30 to about 80% (W/V).
Also provided is a tissue sample having improved shelf life when maintained in an aqueous solution which is produced by immersing the tissue sample in a storage medium having an osmolality higher than about 4500 mosm, the storage medium comprising water, sucrose, and a halide salt, wherein the tissue sample is characterized by a decreased bioburden and a decreased lipid content relative to tissue samples which are not produced in this manner. In a preferred embodiment, the storage medium comprises about 30 to about 80% (W/V) sucrose, and the halide salt is utilized in a concentration of higher than about 11.7% (W/V).
DETAILED DESCRIPTION OF THE INVENTION
Once removed from the animal, collagenous tissue can become hydrated and thicken. The thickening of the tissue is believed to be the result of the partial unwinding of the collagen fibrils, which makes the fibrils more susceptible to enzymatic degradation. The interaction of native helical collagen molecules inside the collagen fibrils must be kept intact in order to maintain the stability of the fibrils. To do so, it has been discovered that the ionic strength of the solution in which the sample is stored must be increased to such a point that the hydrophobic interaction between collagen molecules is maximized. This is accomplished by the use of a high salt concentration in the media solution of the present invention.
However, as the salt in the solution penetrates the tissue sa
Cheung David T.
Mcilroy Brian K.
Mechanic Gerald L.
Mechanic Richard
Moore Mark A.
Carbomedics Inc.
Mechanic Richard
Peselev Elli
Wisner Mark R.
LandOfFree
Method for extracting lipids from tissue samples using high... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for extracting lipids from tissue samples using high..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for extracting lipids from tissue samples using high... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2943187