JPH02501441A - How to cultivate pearls - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] How to cultivate pearls [Cross reference 3 to related applications This application was filed on July 28, 1987 This is a partial continuation of Application No. 102 and No. 127 filed with the above.

Preface 〔Technical field〕 The present invention relates to a method for cultivating pearls and a composition used in the method. Biological and biochemical activities of mantle tissue found in pearl-producing mollusk gastropods. This invention relates to a method for producing pearls using an in vitro culture system that maintains their sex.

Most commonly, pearls are produced by shellfish, especially the variety of marine species known as pearl oysters. It is formed into a gemstone. Pearls are made of foreign matter that has entered the body of the oyster, generally a parasite, or a grain of sand. Or, it is formed as a defense against irritation caused by jellyfish particles. Foreign matter encased in soft tissue layers and then its surface similar to the parallel arrangement of nacre in the shell. Nacre is arranged on the surface. All crusted molluscs or gastropods theoretically produce pearls. However, most pearls of gem quality are produced in pearl oysters, ear oysters, and some types of pearls. produced by a specific genus of freshwater bispods.

Pearl farming techniques using living pearl oysters were developed in Japan at the turn of the century. . And today, we continue to utilize a variety of saline and freshwater bishells through the pearl-forming stage. Those methods are still in use today. Cultured pearls generally stay true for about 2 to 3 years. It is produced by manually implanting a nucleus with a small piece of mantle tissue into a pearl oyster.

Ecological environmental changes such as the expansion of water pollution and overfishing of pearl-producing molluscs in recent years The production capacity of the pearl industry has decreased due to the In addition, existing pearl cultivation methods (1) Vast areas of land and sea must be selected and utilized; Pearl-producing oysters must be cultured for two to three years before the pearl nucleus can be inserted into an adult oyster. (3) generally until they produce gem-quality pearls; It will take another 2-3 years; (4) a huge amount of labor is required in the pearl production factory; Which has inherent limitations. Therefore, current pearl production methods are There are substantial production limits that limit product availability and maintain high price levels.

[Summary of the invention]

The present invention avoids the use of live pearl oysters for cultured pearls.

Specifically, we aim to provide nutrients and nutrients that sustain pearl-producing tissues in vitro. This method is based on a cell culture system developed as a target. This method requires Proliferative persistence of mantle tissue explants by providing appropriate chemical and physical components Regarding. The culture medium composition for sustained tissue growth includes natural ingredients that grow such tissue. available medium (this medium contains inorganic salts, amino acid sources, sugar sources, naturally occurring protein ).

[Detailed statement of the invention]

Mantle tissue is produced between cells and is produced in nacre ( cells that cause the deposition of nacreous or mother-of-peal) All shell-forming soft body movements that undergo physiological metabolism that secrete IJx found in things. In the present invention, mantle tissue is extracted from a mollusk and Physiological to reproduce them under standard biological conditions when maintained under external conditions Has characteristics and functions. The present invention provides for mantle tissue under ideal in vitro conditions. A complex compound with the appropriate chemical and physical components necessary to simulate Use a culture medium. The ultra-fine morphology and chemical properties of ordinary cultured pearls have been incorporated into the present invention. As shown by a series of electron micrographs comparing with that of pearls produced by produced according to the present invention by utilizing the medium disclosed in the present invention. The chemical and microscopic forms of pearls are naturally occurring or cultured pearls on the market today. is substantially the same as Cultured pearls produced by conventional methods currently on the market and the present invention Transmission electron micrographs showing the surface morphology of pearls produced from Crystal size and shape and general surface topology are virtually identical between both pearls. It is. Chemistry using X-ray diffraction of conventional cultured pearls and pearls produced by the present invention The basic identification is that calcium carbonate (Koiseki) is the main phase found in both pearls. and

complex growth medium The medium used in the method of the invention is a complex growth medium.

The media used can take on a wide variety of conditions and are generally natural fluids, cellular or Provides an improved environment to approximate the extracellular environment. The medium contains the main part and mineral salts, amino acid sources, metabolizable energy sources, normal metabolizable or assimilable may include a capable sugar source as well as a preferred hormone source. The purpose of the invention is generally to: Discloses various nutrient medium components that may be divided into components. That is, (i) Inorganic salt, (ii) Amino acid, (1i) Vitamin:/, (1v) Naturally produced protein Ingredients, (v) Antibiotics and antifungal agents, (vi) Assimilated energy sources, etc. and (vli) CO2, CD3 and/or 02.

The nutrient medium contains the inorganic salts necessary for the growth of the tissue to mimic the natural medium. moreover, When a factor is supplied by the presence of another factor, it includes various other factors. But that's fine. For example, natural raw materials containing factors are added separately to synthetic media. It is a case of meaning. Another example is the use of natural ingredients to replace other ingredients. You may also use the fee. Therefore, there can be a wide variety of media used; A wide variety of media are available, each with different stages of rapid pearling results. It is. For example, if mollusk tissue is used as a media component, this tissue Serves as a source of amino acids and hormones.

Protein thaw products can serve as a source of amino acids, so they do not contain individual amino acids. Good too. Media prepared from growth mantle tissue can also be used; A prepared medium containing a given amount of energy source and desirable levels of undesirable factors. If it is reduced, certain ingredients can be supplemented. The basal medium is suitable for tissue culture at an appropriate pH. A salt medium that mimics the natural medium of It can have a promoting factor. Such factors include molluscan tissues, especially reproductive tissues. These may also include amino acid sources, fetal serum, androgen extracts or or can serve as a source of steroids, such as yeast extract.

Inorganic salts in growth media generally inhibit the biological activity of molluscs, mantle tissue. They can include all ions found in themselves. surround the mantle tissue The ion concentrations of these fluids are investigated and measured to determine their chemical properties. Ta. Table 1 shows the average concentrations of inorganic components measured.

HC0,0,223 As a result of the above, the inorganic salt composition used in complex growth media can be found in the following table. It will be something like that.

Table ■ NaC1! 16.0 -35.0 22.5KIJ! 0.4 - 1.2 0.76CaC120,81,41,08 MgC12・6) 120 0.7-14 10.3Na2SO,2,9-4,9 4,05 KHC0,0,25-0,750,358a) I2PO,・t120 0.15 - 0.50 0.25 NaHCOs 0.1 0.2 0.12 Salt medium as described above is preferred, but different salt cultures that provide the preferred ionic composition and normal essential ions are used. You may also prepare a base.

We also carried out amino acid analysis to identify and identify the amino acids involved during pearl formation. The concentration of was measured. Although amino acid concentrations vary between molluscs, the use Consistency in amino acid concentrations was observed for the different mollusk species. Measured a The average composition and concentration of amino acids are shown in Table 1 below.

Aspartic acid 132 Phenylalanine 31.9 Glutamic acid 33.5 Molluscs can provide most of the amino acids, but to increase proliferation it is necessary to increase cell proliferation. It is preferable to supply the amino acids that they provide. As a result, similar Specific amino acid components with specific concentrations can be used in complex growth media and are listed in the following table. vinegar.

Glycine 0.75-5.5 4.5 L-Alanine 2.5 -4.6 2.8L-Arginine 0.8 -2.5 2.4L-asparagine 0.05-0.3 0.05L-aspartic acid 0 ．． 32-0.95 0.6L-cystine 0.1-0.15 0.1L-gluta Mic acid 0.6-1.2 0.8L-Glutamine 0.1-1.0 0.25 L-histidine 0.50-2.8 2.1 L-isoleucine 0.01-0. 075 0.05L-Leucine 0.01-0.1 0.05L-Resine 0.2-0.4 0.2L-Methionine 0.01-0.75 0.05L -Phenylalanine 0.05-0.2 0.1OL -Proline 0.1-1 ．． 5 0.25L-Serine 0.55-1.4 1.0L-Threonine 0.01-0.05 0.03L-)Liptophan 0.01-0.02 0 ．． 01L-Tyrosine 0.05-0.15 0.1 Especially as a source of individual amino acids Of interest are the amino acids serine, alanine, arginine, aspartic acid, and glycan. They are lysine, histidine, leucine, methionine and phenylalanine. a When referring to amino acids, it is understood that the naturally occurring monostereoisomer is intended. Ru. However, instead of individual amino acids, protein lysates or extracts can Can be used alone or with individual amino acids, in which case protein lysate Alternatively, extracts can be supplied as a more economical source of amino acids.

Vitamins are optional. If present, the vitamins present in the complex growth medium are those used in other cell and tissue cultures with some degree of variation It is similar to Specific vitamins and concentrations are shown in Table 7 below. complex growth medium In this case, some or all of them are not actually used.

Biotin 0.01-1 0.1 Riboflavin 0.01-1 0. , I■-Inositol 0.1-10 1. 0 Thiamine 0.1-10 1.0 Niacin 0.1-10 1.0 Choline hydrochloric acid 0.1-10 1.0 Folic acid 0.1-10 1.0 Ascorbic acid 0.1-1 0.1 Vitamin A (acetate) 0.1-1 0.1 Pyridoxine 0.1-10 1.0 Hormones found near or associated with mantle tissue are measured and are associated with corticosteroids. may be classified as roids, androgens or modified estrogens. These ho rumone components or metabolic intermediates are added to the culture medium to stimulate the biosynthesis of these hormones. advanced. Androgen extracts are available from commercial sources and range from 0.005 to 0.00. Can be used with each insertion between 2/1.

A growth medium containing serum extracts derived from renewable organs of the same mollusk was observed to give the best results in replicative growth of mantle tissue. Ta. This serum extract provides the hormone levels necessary to sustain growth of the mantle tissue. It was discovered that This regenerating organ has no serum present and can sustain tissue culture. However, the addition of hormones is preferable to enhance tissue growth. I understand.

Of particular interest are tissues derived from mantle explants or especially reproductive tissues or Another related species is to use lyophilized reproductive tissue. tissue frozen Finely chopped or ground, either before or after drying, for easy dispersion. It can be provided in the form that it is obtained, for example as a powder. The tissue used is made from cells. If so, cell fragments and other materials present in the tissue may be removed from the source material.

Lyophilized tissue is dispersed in a nutrient medium under mild conditions with agitation to ensure biocompatibility. Can be incorporated into gels, such as agar or collagen gels. freeze-dried group The amount of tissue can be varied over a wide range and can be optimized in conjunction with other components in the nutrient medium. It can be done. Advantageous results indicate that the volume before lyophilization is approximately 0.1 to 2 It can be obtained by using in proportion.

Antibiotics and antifungal agents, such as penicillin and streptomycin, also May be used during the extraction process and used to control contamination levels during mantle tissue culture You may. Antibiotics and antifungal agents used in complex growth media are listed in the following table. Listed in

Table ■ Streptomycin 50-200 t=t/ml Penicillin G, sodium 100-2.000 U/rrL1 The growth and maintenance of the mantle tissue requires energy Although success was achieved without the addition of sugars, at least one sugar provided an additional effect on the cells. added to the growth medium to provide an energy source. Glucose as a sugar source , galactose, glycogen, sucrose, etc.

Other ingredients used in one or more of the prepared growth media are listed in Table 1 below. other In the absence of protein sources, milk albumin thaw products showed significant benefits.

Table ■ Milk albumin thawed product 1.00-20. Og/j’ 5.00g/j2 soft body movement Materials/Gastropod blood 1.00-100% by volume 5% by volume Fetal bovine serum 0.01- 10% by volume 0.05% by volume Yeast extract 1.0 - 20. Og/　1　10 ．． 00g/1 galactose 0.05-0.7g/l, 0.05g/fg Lucose 1.00-5.0g/(12,00g/12 Phenol Red 5 ■/rn1 The system is buffered and brought to a physiologically acceptable pH.

Sodium bicarbonate (an inorganic salt) and phenol red were added to the control and the solution The pH of the sample is monitored. The preferred p)I range is 7.2-7.9. deer However, the activity of the mantle tissue remains even at pH levels as low as 6.5 and as high as 9.3. was observed to continue. Carbon dioxide-rich air or a carbonation source in the growth medium The pH of the medium was maintained within the range of pH 6.6 to 8.9 throughout the whole body. Ta.

Commercially available cell culture media (e.g., Gibco, Inc., New York) were modified for mantle tissue culture. The prepared modified products generally include (i) carbonic acid addition of sodium hydrogen and glucose; (ii) to obtain the correct osmolarity of the solution; (iii) adding an appropriate concentration of NaCl2, and (iii) often adjusting the pH of the solution to 7. IM Na leaf is added to make it 4 or more.

Various complex growth media compositions have been successfully used in pearl production. Concrete example are listed in Tables ■A, ■B, and Table 10 below.

Table ■A NaCA 17.3 g/ll CaC721,55g/(I Kl! 0.85 g/l MgCA2・6H2010, 45g/(INa)IPO-・)120 0.20 g / INaHCOlo, 12 g / A'aspartic acid 0.6 g /l Galactose 0.7 ■/l Glucose 1.0 mg/1 Fetal bovine serum (Gibco Lab, NY) 10% by volume Niacin 1 , Omg/j’ Penicillin 100 U/mf Phenol red 5.0 mg/ml Table ■B NaCl22,8 g/β KCl21.15 g/β Na2) IPO, -8200, 9g/llCaCf 2 1.5 g/A' Na) Ic0. 0.72 g/A'aspartic acid 0.5. g/β Methionine 0.05g/j? Table ■B (continued) Fetal bovine serum 2o volume% Ascorbic acid 0.1 ■/l Vitamin A (acetate) 0.1 ■/l Penicillin 100 U/il Streptomycin 5o /- Table ■C Commercial media [i.e., Medium 199: Gibc.

Laboratories, Inc., Grand Land, NY] Modified with the following additives: Yeast extract (Yeastolate: Gibco Lab) 10 g/1 Penicillin 100 U/mj2 Growth using the salt composition of medium A or distilled water with seawater and reproductive tract extracts A media formulation was prepared. This reproductive organ extract extracts reproductive organs from the entire mollusk body. By isolating the reproductive tissue, freeze-drying the reproductive tissue, and then grinding the tissue into a powder. Obtained by. Next, the powder was uniformly dispersed in seawater by stirring. reproductive group The amount of fabric is a ratio of 0.25 to 1 of the original capacity of the tissue for light and water. there were. Other formulations may combine the aforementioned formulations or substitute reproductive tissue. Androgen extract (Sigma) was used. This androgen extract contains 0 ．． 0.01 to 0.1 mg/1.

method of invention Genus of molluscs or gastropods that produce nacre in their shells for purposes of in vitro culture, e.g. For example, pearl oyster (P1 nctada) genus, white oyster (I sognomon) ) genus, Pteria genus, Pinna genus, ear shell (Ha) 1iot is), Atrima genus, etc. [for example, Vincta Pinctada martensii, Pinktada martensii 7 Ligrite Huera (Pinctadamargr it if era), Pi Pinctada 7kiss 7 (Pinctada maxima), Pinctada maxima Pinctada fucata, Butyria macrobutella (P teria macroptera), Atrim japonica (Atrim) japonica), Ostrea gigas, sea urchin Llnio margritifera, Chris Cristaria plicata,) Ridacna Gigas (Tridacna gigas), Haliotis gigantea (Hal) iotis gigantea), Hypriobsis 8 shillegeri (t(ypriop) sis schlegeli), Haliotis fulgens fulgens), Haliotis corru gata), Haliotis °cracheology (Haliotis crache) rodii), Haliotis discus haneyi (tlaliotis di scus hannai), Ligumia nasut a), x Ribthio-ml Muplanata (Ellipti.

complanata), Strophytus undulatus (Strophitus undulatus) usundulatus), Anodonta ca. turacta) and Lamsilis radia explanting part or all of the mantle tissue from a mollusk or gastropod belonging to and produces nacre for pearl formation.

Several pieces of mantle tissue fragments are optionally treated with an antibiotic and/or antifungal agent, e.g. 00 units of penicillin G (sodium salt)/rrL1 and 10/mf Washed with seawater containing streptomycin. Seawater may be prepared in the laboratory; Generally it consists of water with inorganic salts in their concentrations as shown in Table 1 above. .

Part of the tissue or the complete mantle tissue contains inorganic salts, amino acids, vitamins and growth factors. children, hormones, antibiotics and antifungal agents, animal serum extracts and glucose. The cells are transplanted into a complex growth medium containing carbon dioxide or oxygen.

Biocompatible materials such as glass, calcite made of calcium carbonate and/or Old shells containing nepheline and/or sphere- or hemisphere-like phosphorus Calcium acid is introduced into the medium for nacre deposition. This material is pearl-forming The nucleus may have any shape, usually spherical or ellipsoidal. It works. In general, the smallest dimension of the material that will serve as the core is usually at least about One stroke is generally at least about 2M, but may be larger than 20mm .

For pearls, the core generally has a diameter of about 2-15 mm, preferably 7-10 mm. .

According to a preferred embodiment of the method of the invention, for example, a piece of mantle tissue of less than 1 mm3 is It is harvested by cutting from an adult mollusk. Each fragment was washed with seawater containing the aforementioned inorganic salt components. The cells were cleaned and placed in pre-sterilized culture dishes. 0.2> Mi! J pore filter ( Millipor.

Corp., Bedford, MA 01730). Synthetic growth medium was added directly to the container containing the tissue fragments. Then a small nucleus, e.g. For example, spheres made from glass peas or bivalve shells can be used to make physical contact with tissue. It was placed in a container for maximum contact. Cultures are placed in an incubation device. It was maintained at 14°C to 23°C in a darker place. However, experiments have shown that the method of the present invention was shown to be able to withstand a temperature range of 8°C to 29°C. air rich in carbon dioxide introducing and maintaining the medium pH substantially between 6.6 and 8.9 ensures satisfactory quality. I pulled it out.

When the pH indicator phenol red changes color, replenish the medium using a syringe. provided. As the pH increases from 7.4 to 8, the bright phenol red becomes blueish. Changes to a deep red color.

Bright phenol red turns dark pink when pH drops to 7.4-6.5 or turn yellow. However, the pH of this medium tends to increase.

Remove old media and replace with fresh media every 1-3 days to achieve desired quality and size. Continue until the pearl is achieved. In some cases, for 2 days or/when the medium po is 7.8 or higher. when rising to the top, whichever occurs first, depending on the ratio of medium content to tissue volume. The medium may be replaced accordingly. Accurate measurement of pH can be done with a pH measuring device. stomach. Once the pearl has reached the desired quality and/or size, remove it from the dish. The attached tissue was removed. The removed tissue is incised and the pieces are used for other pearl production. Used again.

It is understood that the foregoing aspects are merely illustrative of the present application. Therefore , other embodiments that can be easily derived by a person skilled in the art are included in the main body of the present invention, and those must be within the spirit and scope of. The appended claims cover any such changes and It is intended that variations thereof be within the true scope and spirit of the invention. Honmei Publicly known documents and patent applications cited in the specification are just individual public documents and patent applications. Is it specified that the patent application is specifically and individually cited as the content of this specification? are incorporated herein by reference.

The foregoing invention has been described in some detail by way of specificity and example for clarity of understanding. described herein, but without departing from the spirit or scope of the appended claims. Certain modifications and improvements will readily occur to those skilled in the art in light of the technology of the present invention. It will be obvious.

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Claims (13)

[Claims] 1. A method of coating biocompatible nuclei with nacre in an in vitro culture medium, the method comprising: Living mantle tissues of nacre-producing members of the phylum Mollusca are grown in a suitable nutrient medium. contacting the biocompatible nucleus within the tissue and coating the nucleus with nacre. incubating the tissue under conditions that sustain proliferation for a sufficient period of time; and said method comprising isolating the resulting nacre-coated nuclei. 2. The medium may contain mollusc serum, mollusk reproductive composition, steroid source, yeast extract. 2. The method of claim 1, comprising at least one of: 3. 2. The medium according to claim 1, wherein the medium contains assimilable sugars and/or amino acids. the method of. 4. 2. The medium has a salt composition that mimics the salt composition of the natural medium of the mollusc. 4. The method according to any one of 1 to 3. 5. A method of coating biocompatible nuclei with nacre in a culture medium, the method comprising: Live mantle tissue of nacre-producing molluscs or gastropods is grown in a suitable nutrient medium. The culture medium mimics the natural medium of the mollusk or gastropod. It contains salt, an amino acid source and/or an assimilable sugar source, and the core is coated with nacre. Incubating the tissue under conditions that sustain proliferation for a sufficient period of time for the tissue to cuveate, and said method comprising isolating the resulting nacre-coated nuclei. 6. 6. The method of claim 5, wherein said core consists of calcium carbonate. 7. The nutrient medium further comprises mollusk blood, mollusk reproductive composition, steroid source. or fetal serum. Method. 8. The mollusk is Pinctada martensii (Pinctadamart). ensii), Pinctada marigritifera (Pinctadamarg) ritifera), Pinctada maxima ), Pinctada fucata, Pteria ma Croptera (Pteriamacroptera), Atolyma japonica (A trjmjaponica), Ostrea gigas, Uniomargritifera, Crista Cristaria plicata, Tridacna gigas (Tridacnagigas), Haliotis gigantea (Haliotis) gigantea), Hypriopsis silegeri (Hypriopsissch) legeli), Haliotis fulgens , Haliotis corrugata, Haliotis corrugata Haliotis cracherodii, Haliotis ・Haliotis-discushannai, ligumi A nasuta (Ligumiana suta), Elliptio complanata (El liptiocomplanata), Strophytus undulatus (St. rophitusundulatus), Anodonta cutulacta (Anodo ntacaturacta) or Lamsilisr. 8. The method according to any one of claims 1 to 7, wherein 9. A nacre-coated core produced by the method according to any one of claims 1 to 8. 10. A nutrient medium for coating biocompatible nuclei with nacre using mantle tissue. So, Salts mimicking natural media associated with said mantle tissue, optionally amino acid sources or resources oxidative sugar sources, or mollusk blood, mollusk reproductive composition, steroid sources, yeast extracts, etc. said medium comprising at least one of fetal serum or fetal serum. 11. the amino acid source comprises at least one individual amino acid, and the assimilable sugar is containing at least one of glucose or galactose and further comprising mollusc reproduction The nutrient medium according to claim 10, comprising a tissue material. 12. the amino acid source comprises at least one individual amino acid, and the assimilable sugar is at least one glucose or galactose and further a steroid source The nutrient medium according to claim 10, comprising: 13. Living mantle tissue from nacreous molluscs, biocompatible nuclei and claims A composition comprising the nutrient medium according to any one of items 10 to 12.