CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application serial no. 102,127, filed September 28, 1987.

INTRODUCTION

Technical Field

The subject invention concerns coating of nucleating agents with nacre in vitro using mantle tisssue from mollusca.

Background

This invention relates to a methods and compositions of matter utilized therein, for culturing pearls, and in particular to a method of producing pearls by using an in vitro cell culture system to maintain biological and physiochemical activities of the mantle tissues found in pearl forming mollusks and gastropods.

A pearl is a gem most commonly formed by bivalve mollusks, particularly by several marine species known as pearl oysters. Pearls are formed as a protection against the irritation caused by foreign objects, generally parasites, grains of sand, or bits of gravel, which lodge inside the shell. A fold of soft tissue envelopes the foreign particle and deposits layer after layer of nacre on it, similar to the mother-of-pearl lining the shell. Any shelled mollusk or gastropod is theoretically capable of producing a pearl, but most pearls exhibiting gem quality are produced by a specific genus of pinctada, haliotis, and certain freshwater bivalves.

Techniques for culturing pearls by using live pearl oysters were developed in Japan during the turn of the century. And today, these methods are still employed in which various saltwater and freshwater bivalves are maintained alive during the pearl forming stages. Cultured pearls are generally produced by manually implanting a nucleus with small pieces of mantle tissue in an oyster for approximately two to three years.

Over the years changes in the ecological environment, such as an increase in water pollution and over harvesting of the pearl producing mollusks, have limited the potential productivity of the pearl industries. Furthermore, existing methods of culturing pearls have inherent limitations: 1) Large areas of land and sea must be designated and utilized; Pearl forming oysters must be reared for two to three years before a pearl nucleus can be inserted into the adult oyster; 3) It generally takes another two to three years to produce gem quality pearls themselves; 4) A large work force is needed in pearl producing farms. Consequently, current methods of pearl production have inherent production limitations with resulting limited diversity in the world market and continued high price levels.

SUMMARY OF THE INVENTION

The present invention circumvents the use of live oysters for culturing pearls. It is a process based on a cell culture system specifically designed and developed to provide sustenance and nourishment to pearl forming tissues kept in vitro. The process involves sustaining the viability of mantle tissue explants by providing necessary chemical and physical components in a culture medium. The composition of the medium to maintain and grow tissues includes a combination of nutrients associated with the naturally available medium in which the tissue grows, which medium may include inorganic salts, a source of amino acids, a sugar source, naturally occurring proteins, etc.

DETAILED DESCRIPTION OF THE INVENTION

The mantle tissues found in all shell forming of the phylum Mollusca, including bivalves, and certain gastropods, undergo physiological metabolism to produce intercellular and later to secrete extracellular matrix components responsible for the precipitation of nacreous (mother-of-pearl) material. In the present invention when mantle tissue is extracted from a mollusk and maintained under certain in vitro conditions, the tissue has physiological characteristics and functions duplicating those in normal biological conditions. The present invention uses complex growth media with the appropriate chemical and physical components necessary to simulate the ideal conditions in vitro for mantle tissues. By employing the media described in this invention the chemical and ultrastructural properties of the pearls produced by this invention are substantially identical to the naturally occurring or cultured pearls currently on the market, as has been demonstrated by a series of electron micrographs comparing the ultrastructure and chemical properties of conventionally cultured pearls with those of the pearls produced by the present invention. From transmission electron micrographs illustrating the surface structure of a conventionally produced cultured pearl presently found on the market and a pearl produced from the present invention, it could be seen that crystal size and shape of the nacre as well as general surface topography are substantially identical between the two pearls. Chemical identifications using X-ray diffraction of a conventionally produced cultured pearl and a pearl produced by the present invention showed that calcium carbonate (aragonite) is the predominate phase found in both pearls.

COMPLEX GROWTH MEDIUM

The culture media used in the process of this invention are complex growth media. The media employed may take many forms, normally providing an environment which to varying degrees approaches the natural fluids, cellular or extracellular. For the most part, the medium will include inorganic salts, a source of amino acids, a source of metabolic energy, normally a metabolizable or assimilable saccharide source, and desirably a source of hormones. For the purposes of the subject disclosure, the various components of the nutrient medium which may be involved may be separated generally into the following components: (i) inorganic salts, (ii) amino acids, (iii) vitamins, (iv) naturally occurring protein containing compositions, (v) antibiotics and antimycotics, (vi) assimilable energy source and (vii) CO₂, CO₃ and/or O₂.

The nutrient medium will have the inorganic salts necessary for growth of the tissue, approximating the natural medium. In addition, various other factors may be included, where some factors may be provided by the presence of other factors. In some instances, where naturally occurring materials are involved, the naturally occurring material may include the factors which are otherwise added to a synthetic medium. In other instances, the naturally occurring material may serve as an alternative to another component. Therefore, there may be wide variation in the media which are employed, and a number of different media may be employed, each with different degrees of success in the rapidity with which the pearls are obtained. For example, when using mollusk tissue as a component of the medium, the tissue may serve as a source of amino acids and hormones. Protein hydrolysates may serve as a source of amino acids, so that individual amino acids may not be included. Conditioned medium from the growth of mantle tissue may be employed, where the conditioned medium may be replenished to previous levels of certain components such as an energy source and undesirable factors reduced to a desirable level.

The basic medium will have a salt medium approximating the natural medium of the tissue at an appropriate pH, a source of assimilable energy, a source of essential amino acids, and additional factors which promote growth. Such factors may include mollusca tissue, particularly the gonadal tissue components, which may also serve as the source of amino acids, fetal serum, a steriod source such as an androgen extract, yeast extract or the like.

The inorganic salts in the growth medium will usually contain all of the ions that are found in the mollusk mantle tissues themselves during their biological activities. In order to determine the chemical nature of the fluid surrounding the mantle tissue, the ionic concentrations of such fluid were investigated and determined. In Table I, the average concentrations of inorganic elements found are shown.

As a result of the above results, compositions of inorganic salts were formulated for use in the complex growth medium as found in Table II below.

The salt media proposed above are convenient, but other salt media may be formulated which would provide the desired ionic composition and normalities of the essential ions.

An amino acid analysis was also performed to identify and determine the concentrations of amino acids associated during pearl formation. Although concentrations of the amino acids varies from mollusk to mollusk, the consistency of the amino acid concentrations for a given type of mollusk was noted. The average composition and concentration of amino acids found are shown in Table III below.

While the mollusk can produce most of the amino acids, in order to enhance growth, it is desirable to provide a supply of those amino acids which enhance the growth of the cells. As a result, certain amino acid components in similar concentrations may be used in the complex growth medium and are shown in Table IV below.

Of particular interest as a source of individual amino acids are the amino acids serine, alanine, arginine, aspartic acid, glycine, histidine, leucine, methionine, and phenylalanine. In referring to amino acids, it is understood that the naturally occurring L-stereoisomer is intended. However, instead of individual amino acids, protein hydrolysates or extracts may be employed, by themselves or in conjunction with individual amino acids, where the protein hydrolysates or extracts may provide for a more economical source of amino acids.

Vitamins are optional. If included, the vitamins present in the complex growth medium are similar to those used for other cell and tissue cultures with some variations in concentration. Specific vitamins and concentrations are shown in Table V below. None some or all may actually be used in the complex growth medium.

The hormones found associated at or near the activities of the mantle tissues were determined and may be classified as corticosteroids, androgens or modified versions of estrogen. These hormonal components or metabolic intermediates were added to the medium to facilitate biosynthesis of these hormones. Androgen extract may be obtained from commercial sources and may be used in amounts varying from 0.005 to 0.2 mg/L. It has been observed that a growth medium containing serum extracts from the reproductive organs of the same mollusk was observed to yield the most success in proliferating the growth of the mantle tissues. The serum extracts were found to provide the levels of hormones necessary to sustain mantle tissue growth. Without the presence of this reproductive organ serum, however, the hormone additives, although not essential in maintaining tissue culture, were found to be desirable in enhancing tissue growth.

Of particular interest is to use tissue from the mantle explant source or other related species, particularly comprising gonadal tissue or lyophilized gonadal tissue. The tissue may be minced or ground either before or after lyophilization to provide the tissue in easily dispersible form, e.g. a powder. The tissue when employed will be composed of cells, fragments of the cells and other materials present in the tissue when removed from the source. The lyophilized tissue may be dispersed in the nutrient medium under mild conditions with agitation, and may be incorporated in biocompatible gels, such as agar or collagen gels. The amount of the lyophilized tissue may be varied widely and may be optimized in accordance with the other components of the nutrient medium. Beneficial results may be obtained by using a ratio of from about 0.1 to 2 of the volume of the tissue prior to lyophilization to the volume of medium.

Antibiotics and antimycotics such as penicillin and streptomycin may also be used during tissue extraction procedures and may also be used to control contamination levels during mantle tissue culture. The antibiotics and antimycotics used in the complex growth medium are listed below in Table VI.

Although growth and maintenance of the mantle tissues were successful without the addition of a source of energy, at least one saccharide was added to the growth medium to provide an additional source of energy to the cells. Saccharide sources include glucose, galactose, glycogen, sucrose, etc.

Other components which were employed in one or more formulated growth media are listed in Table VII below. The lactalbumin hydrolysate provided significant benefits in the absence of other protein sources.

The system is buffered to provide a physiologically acceptable pH. Sodium bicarbonate (inorganic salt) and phenol red were added to control and monitor the pH of the solution. The preferred pH range is 7.2 to 7.9. However, a pH as low as 6.5 and as high as 9.3 have been observed to sustain viability of the mantle tissues. Carbon dioxide enriched air or a carbonated source was added to the growth medium to keep the pH of the medium within the overall range of 6.6 to 8.9.

Commercially available cell culture media, e.g., Gibco, Inc., New York, were modified and tested for culturing mantle tissues. The modifications made were generally: (i) addition of sodium bicarbonate and glucose, (ii) adition of appropriate concentrations of NaCl to obtain the correct osmolality in the solutions, and (iii) 1 M NaOH solutions were often added to bring the pH in the solution to 7.4 or above.

Various complex growth media compositions were used successfully in producing pearls. Specific examples are listed below in Tables VIIIA, VIIIB, and VIIIC.

Formulations were prepared for growth media using distilled water containing the salt composition of Medium A or sea water and gonad organ extracts, where the gonad organ extracts were obtained by isolating the gonads from the body mass of the mollusc, lyophilizing the gonadal tissue and grinding the tissue to form a powder. The powder was then uniformly dispersed in the water by stirring. The amount of gonadal tissue was based on the original volume of the tissue and was in a volume ratio to water of 0.25 to 1. Other formulations combined the above formulation or used androgen extract (Sigma) in place of the gonadal tissue. The androgen extract ranged in the amount of 0.01 to 0.1mg/L.

Invention Process

A portion or all of a mantle tissue is explanted from a mollusk or gastropod which belong to genus thereof which produce mother-of-pearl material in their shells, such as the genera Pinctada, Isognomon, Pteria, Pinna, Haliotis, Atrima, etc., e.g., Pinctada martensii, Pinctada margritifera, Pinctada maxima, Pinctada fucata, Pteria macroptera, Atrima japonica, Ostrea gigas, Unio margritifera, Cristaria plicata, Tridacna gigas, Haliotis gigantea, Hypriopsis schlegeli, Haliotis fulgens, Haliotis corrugata, Haliotis cracherodii, Haliotis discus hannai, Ligumia nasuta, Elliptio complanata, Strophitus undulatus, Anodonta caturacta, and Lamsilis radiata, for the purpose of in vitro cultivation so as to produce nacreous material for pearl formation.

The pieces of the mantle tissue fragments are optionally washed in seawater containing antibiotics and/or antimycotics, e.g., 100 Units penicillin G (sodium salt)/ml and 100 µg/ml streptomycin. The seawater may be prepared in the laboratory and would consist generally of water with inorganic salts in concentrations thereof as indicated in Table II above.

The tissue part(s) or intact mantle tissue(s) are transferred into a carbon dioxide or oxygen enriched complex growth medium comprised of inorganic salts, amino acids, vitamins and growth factors, hormones, antibiotics and antimycotics, animal serum extracts and glucose.

Biocompatible material such as glass, porcelain, old shells containing calcite and/or the aragonite phase(s) of calcium carbonate, and/or calcium phosphates in the form of spheres or hemispheres are introduced into the medium for nacre deposition. The material which serves as a nucleating substrate may be of any shape, normally being round or ellipsoid for pearl formation. Usually, the smallest dimension of the material which serves as the substrate will be usually at least about 1mm, usually at least about 2mm and may be as large as 20mm or larger. For pearls, the substrate will usually have a diameter from about 2 to 15, usually 7 to 10mm.

In one successful embodiment of the invention process, small, i.e., less than 1 mm³ pieces of mantle tissue were sectioned and removed from a pearl forming adult mollusk. Each piece was washed in saltwater containing the inorganic salt components described above and placed in pre-sterilized culture dishes. Sterilized complex growth medium, filtered through a 0.22 µm millipore filter (Millipore Corp., Bedford, MA 01730) was immediately added to each dish containing the tissue fragments. Small nuclei, i.e., glass beads or spheres made from clam shells, were then placed in each dish and positioned so that maximal physical contact was made with the tissue. The cultures were maintained between 14°C and 23°C in the dark by incubation means. However, experimentation has shown that the invention process will withstand temperatures ranging from 8°C to 29°C. Carbon dioxide enriched air was introduced and withdrawn in sufficient quantities to effectively keep the pH of the medium in a range from 6.6 to 8.9. The medium was replenished using syringes when the pH indicating phenol red changed colors. The bright phenol red changes to a bluish red as the pH rises from 7.4 to 8. The bright phenol red changes shade toward pink or yellow as the pH drops from 7.4 to 6.5. The pH of this medium tended, however, to rise. The removal of old medium and substitution of fresh medium took place every one to three days and continued until production of a pearl of desired quality and size was achieved. Optionally, the medium is changed every two days or when the medium pH rises above 7.8, whichever occurs sooner and depending on tissue mass to medium content ratio. Precise measurement of pH may be done with a pH measuring apparatus. After the pearl reached its desired quality and/or size, it was removed from the dish and the attached tissue removed. The removed tissue was dissected and fragments thereof used again in the production of additional pearls.

It is understood that the above described embodiment is merely illustrative of the application. Other embodiments, therefore, may be readily devised by those skilled in the art which will embody the principles of the invention and fall within the spirit and scope thereof. The appended claims are intended to cover such modifications and variations which are within the true scope and spirit of this invention.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been de­ scribed in some detail by way of illustration and ex­ ample for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.