JPS60500014A - Chemical sterilization of biological tissue ready for transplantation - 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] Chemical sterilization of biological tissue ready for transplantation Background of the invention Preparation of biological tissues, such as heart valves, ligaments, legs, eardrums, etc., prior to transplantation into humans The chemical treatment of the tissue used in the Contains a series of procedures that ensure both structural and functional integrity. traditionally used These procedures include glutaraldehyde fixation (tanning) and formaldehyde fixation (tanning). There is sterilization. Glutaraldehyde fixation of tissue prior to transplantation renders the tissue biologically comparable. relatively inert and widely accepted as a necessary step in tissue processing. I came here. Similarly, formaldehyde sterilization of tissue prior to transplantation involves removal of the tissue and or effective in destroying numerous microorganisms in conjunction with tissue handling during subsequent processing. It was proven that. However, unfortunately, the currently used group Talaldehyde fixation and subsequent formaldehyde sterilization procedures are recommended for implantable living organisms. Physical tissues, especially biological prosthesis (bloprost, heilc) heart valves It has never been possible to effectively destroy all microorganisms associated with the tissue used for preparation. It was. Non-inventors have discovered something. Furthermore, the currently used form Aldehyde sterilization procedures quickly destroy many microorganisms associated with biological tissues capacity and require prolonged exposure to sterilant solutions.

Microas forceps/Nereus is a fungus from the Ascomycotidae family that has been biologically tested prior to transplantation. Glutaraldehyde and formaldehyde traditionally used for tissue processing Shows resistance to both extreme concentrations. Microas force Sunnereus spread throughout the world Common environmental organisms with geographical distribution that are associated with small skin lesions clinically identified.

According to the present invention, the inventors have developed a method for processing biological tissue prior to transplantation. However, this method uses formaldehyde sterilization6 and glutaraldehyde sterilization. Rice resistance sterilization agents. This method is The biochemical, structural, and This advantageously improves the efficiency of tissue sterilization methods while preserving functional integrity. Yet again , the inventors have already demonstrated susceptibility to formaldehyde or glutaraldehyde sterilization. Many microorganisms and organisms that have become sexually active can be treated using the present method and related compositions I unexpectedly discovered that it would be destroyed fairly quickly.

In accordance with the present invention, an improved method for processing biological tissue prior to transplantation to effect sterilization thereof will be announced. This method involves treating biological tissue under sterile conditions with formaldehyde or a sterile effective amount of a solution consisting of glutaraldehyde, alcohol and a surfactant; It consists of making contact.

detailed description of the invention According to the present invention, various types of implants derived from multiple animal sources and anatomical parts It is intended to facilitate the sterilization of biological tissues by conventional sterilization procedures. . Therefore, the organization may include cows, pigs, horses, sheep, chickens, or rabbits, but this It can be derived from a variety of sources including, but not limited to, elbows, ligaments, heart valves, etc. or the tissues used to construct the ICr visceral valve, such as the dura and epithelium. Including. Used for enlargement of skin pieces, epithelial pieces, aortic pieces, and tympanic membranes It is further contemplated that tissues are suitable for the present invention. Furthermore, Delastink, cloth or non-living items such as surgical instruments, medical devices, valve conduits, etc. made from metals. It is also contemplated that the ocular surface can be effectively treated in accordance with the present invention. of the present invention According to a particularly suitable embodiment, pigs used for the preparation of bioprosthetic valves The tissue flap and the ablated pericardial tissue are fixed with glutaraldehyde and the improved sterilization according to the present invention is performed. treated with drug solution and implanted subcutaneously into rabbits. Conventional sterilants, e.g. glutamate Loses its resistance to aldehyde and formaldehyde4 The processed valve tissue has improved valve tissue integrity compared to tissue processed using conventional methods. shows no significant difference.

According to one embodiment of the invention, the implantable biological compound luminaldehyde Iij' is It makes an organism susceptible to sterilization, ie, destruction of the organism. Shi, Shi, Inventor From the research results of It is also contemplated that it will be useful in the destruction of sexual organisms. The inventors have Sterilant compositions according to the It was unexpectedly discovered that it is more effective against living organisms than in bath liquid. Ta. The reason for this phenomenon is currently unknown in detail, but the inventors have Sterilization containing maldehyde and alcohol will destroy the fruit, but will not be effective on the tissue. It was found that it takes much longer time (days). However, surfactants The presence of the solution makes the solution effective in a shorter time frame (hours).

The inventors have demonstrated that conventional formaldehyde or glutaraldehyde sterilization Yet another group of urinary important microorganisms that are previously difficult to destroy are uninvented. It has been discovered that the method becomes more susceptible to destruction. The present inventors, etc. before improved sterilization] The assembled fabric is 518JUN GO-500014 (3) Advantageously, it has now been discovered that it is effective against living organisms.

According to the invention, the improved sterilant component is daltaraldehyde or formaldehyde. or as a mixture with a conventional sterilant solution containing may be formulated into a composition. Usually, Glutarua up to about 1 candy such as fJo, Nito S, etc. Use a solution of aldehyde solution rL or a solution of about 6 to about 5 parts. Sterilize biological tissue prior to transplantation. A particularly suitable sterilant concentration is glutaralde 0.625% hydride, or about 4 to about 5% formaldehyde.

According to the present invention, tissue is maintained within a tissue stabilizing PH range, i.e., which is harmful to tissue components. It is preferable to process the tissue within a pH range that is clear. The effectiveness of the present invention is about 4.5 Although it has been demonstrated in the pH range from Choose based on compatibility with weaving. Particularly suitable pH ranges are from about 7.0 to about 7.6, and A more preferred pH range is from about 7.1 to about 7.4. Most according to the invention The preferred pH is 7.5.

The present inventors believe that the sterilization method according to the present invention can be performed using phosphate buffer, HEPES buffer, or Although we have found that it works well in unbuffered solutions, it has been found that it works well in unbuffered solutions; The sterilant composition used is preferably stable and does not interact with the sterilization method, and have sufficient buffering capacity to maintain a PI-1 that the fabric can accept. Good.

The selection of a suitable buffer and its concentration will depend on the specific tissue preparation conditions and its variation. Several shapes have been introduced by several manufacturers. Shock-absorbing conventional glue 9 mouths 1-0 ．． 02% (phosphate buffered saline (PBS) or phosphate deficient solution, e.g. 01-0.02M Contains less phosphate than PBS solution. Preferably, anything containing less than about 0.002M phosphate. But that's fine. Special IC common buffers according to the invention include borates, carbonates, bicarbonates. , cacodylate (found to be ffi in animals), and other compounds. synthetic, artificial, or organic buffers, such as HEPgS, N-2-hydroxyethyl Chilpiperazine-N'-2-ethanesulfonic acid, MOPS, 2-(N-morphol) d) Soropanosulponoic acid, and PIPES, 1,4-ebendi/bis(E) Contains tanosulfonic acid).

Preferably, the buffered or unbuffered solution used in accordance with the invention Agents such as dehyde or formaldehyde may interfere with the tissue sterilization process. should not be harmed. That is, they may react with drugs or cause tissue damage. 3) Do not interfere with sterilization. Typical examples of this are first class and Buffers containing secondary amines, such as tris(hydroxymethyl)aminomethano( Trls), which is an acetate of glutaraldehyde or formaldehyde. It is known to react with aldehyde groups and thus interfere with normal tissue sterilization processes.

The present invention allows tissue to be stored and processed under normal, well-known conditions; And as mentioned above, either sulfate buffer solution or phosphate-free buffer solution Fix as usual with 0.625% glutaraldehyde (tannin (g) can be done. Handling tissues outside of conventional sterilization methods terms and conditions are not considered part of this invention.

According to the invention, alcohol is used to make resistant microorganisms more susceptible to sterilization. includes aliphatic as well as aromatic alcohols, and preferably from 1 to about Lower aliphatic alcohols containing 5 carbon atoms are preferred. Methano as aliphatic alcohol alcohol, ethanol, n-propanol, inzolopanol, n-butanol, butanol, 5ec-butanol, t-butanol, cyclohexanol, n -Includes, but is not limited to, octatool, allyl alcohol, etc. Yoshi Aromatic alcohols include be/zyl alcohol, cresol, carbinol, etc. It will be done. Lower aliphatic alcohols according to the invention include methanol, ethanol, and propane. This includes tools, sitanol, inpropatool, and pentanol. There are no restrictions on these. Alcohol according to the invention is ethanol and propatool. is more preferable. The concentration of alcohol to be used in the present invention is limited to The upper limit depends on the impact on tissue integrity, depending on its effectiveness in destroying objects. However, alcohol falls within a range limited by the effect and the alcohol used. It may depend to some extent on the type of The inventors, from about 10 sections Ethanol 6% in the range of up to about 3D% is effective against these microorganisms. Therefore, it is described as a particularly suitable range. More preferred alcohol The concentration ranges from about 20% ethanol to 25% ethanol.

The most preferred alcohol concentration is 22.5% ethanol, which contains cations and non-ion surfactants, and their salts. Characteristics of the surfactant in the present invention Suitable salts include sodium and potassium. Anionic surface activity according to the present invention The agent contains both aliphatic and aromatic groups attached to negatively charged ionic groups, and Those with a relatively thick hydrophobic portion of hydrocarbon residues, including combinations of be. Aliphatic residues can be branched, straight chain, cyclic, heterocyclic, saturated or unsaturated. But that's fine. These hydrophobic residues are carboxylates, sulfates, or salts. Whether directly bonded to ionic groups such as sulfonates, or esters and amino acids, via intermediate linkages such as hydrogen, sulfonamide, ether, or aryl groups. may be connected. Examples of the anionic surfactant as a specific example of the present invention include to or in the alkyl side chain of the steroid, for example in bile acids. It has a carboxylate bonded via a amino acid. Resident according to the invention Bile acids include deoxycholic acid, cholic acid, lithocholic acid, taurocholic acid, and glycocholic acid, and their salts, but are not limited to these. stomach. Anionic surfactants according to the invention preferably include from about 8 to about 20 carbon atoms. with a carboxylate group attached to a straight chain aliphatic group, e.g. aliphatic Also included are sodium salts of acids. An anion containing four carboxylic groups according to the invention On-surfactants include, for example, N-arkylamino acids and N-arkylated amino acids. As in amino acids, it is possible to Also included are those having a carboxylate group attached to the aqueous moiety. N-a A representative example of a lucanoyl amino acid is the formula RICONR2CHR,C02 (wherein R is Preferably an aliphatic group having from about 8 to about 18 carbon atoms, R2 being hydrogen or methyl and R3 is a normal amino acid side chain); There are no restrictions on these. Typical side chains include alanine, leu7ine, isoloinone, Nonpolar aliphatic side chains of valine and zoroline; phenyl, alanine and triply Aromatic ring of tophan; polar side chains such as glycerin, serine, threonine, and insulin ; and charged polar groups such as aspartic acid, glutamic acid, and lysine. . Particularly suitable carboxylates containing surfactants according to this embodiment of the invention are It contains an amide bond such as N-lauroylzurukoshi/.

Anionic surfactants according to other embodiments of the invention include aliphatic alcohols. sulfates, such as alkyl sulfates having from about 6 to 18 carbon atoms, Ethylene oxide modified sulfate of aliphatic alcohol, sulfated ethanolamide or alkylphenol, 'IAI't hasulfonated alkylphenyl ester Includes tel. Other anionic surfactants include alkanosulfonic acids and Contains alkylarylsulfonate.

A typical example of a sulfate of a fatty M alcohol is sodium dodenyl sulfate. Book Alkanosulfonic acids according to the invention have sulfur attached directly to the hydrophobic residue. , e.g. 1-decanosulfo/acid, or ester, amide, or ether. Included are those with sulfur attached through, such as toametertaurino. a Rukyl 71J-Rusulfonate is a compound that can be used for aromatic rings such as phenyl or naphthyl. the aromatic ring preferably has about 8 to about 18 carbon atoms It is bonded to a hydrophobic residue with . A typical example of the latter type of surfactant is Decylbenzenesulfonic acid.

In the cationic interface according to the invention, the active agents include alkyl quaternary amines and their The halogenated salt is stored.

In the present invention, particularly suitable surfactants are bonded directly to hydrophobic residues, or Includes chlorine and bromine salts of tertiary amines linked through amide bonds. Ami Preferably, aromatic residues such as benozeno, zaridine or naphthylene are used. aliphatic chains that are branched, unbranched, cyclic, saturated, or unsaturated; or aromatic residues or directly binds to a relatively large hydrophobic moiety that has a combination of both aliphatic and aliphatic residues. It is better to have Typical alkyl quaternary ammonium surfactants include chloride Cetylpyridinium, cetyltrimethylammonium bromide, trimethylphenyl chloride nyl ammonium, thenutrimethylammonium bromide, hexadenortribromide Contains methylammonium.

Nonionic surfactants according to the invention include polyoxyalkylene ethers, poly Oquine alkylene alkylaryl ether, aliphatic ether, polyether, Polyoxyalkylene ester derivatives, sugar ester derivatives, and combinations thereof Contains falsification. Hydroxyl terminals connected by nonionic alkylene oxide residues It has the following. An example of a polyoxyalkyleph ether is polyoxyethyl Renlauryl ether (Br1j), l’)oxyethylene oleyl ether ether, polyoxyethylene cetyl ether, etc. nonionic polyoxia Lukire/, preferably polyoxyethylene, alkylaryl ether, comparison a large hydrophobic residue and an aryl, such as benzene or naphthalene and one or more It has a hydroxyl group terminal bonded by an alkylene oxide residue above. . Examples of polyoxy7 alkylene alkylaryl ethers include polyethylene glycol Cole p-yne octylphenyl ether, such as Trizon )　X　-100 etc. are included. Nonionic polyether has the formula CH3(CH2) 0-(C2H40)1.4 (wherein, N is about 11, M is about 2 3).

Nonionic aliphatic esters include aliphatic fatty acid esters and polypropylene glycol. Fatty acid esters, such as propylene glycol monostearate, and glycerol Included are lino fatty acid esters such as glycerin monostearate. aliphatic fat The fatty acid ester is 2R, C00R5 (wherein R4 is preferably about 8 to about 2o carbon and R5 is an aliphatic residue having about 1 to about 5 carbon atoms. is a group). Saccharide and polyoxy/alkylene ester derivatives The body relies on penta- or hexoses in the former, or polyoxycarbons in the latter. A lekylene chain is linked to a relatively long hydrophobic residue via an ester bond.

Typical saccharide derivatives include nlupitol, which combines with fatty acids to form surfactants; For example, sorbitan trioleate, nrubitan stearate, nrubitan monomer Includes reato etc. Polyoxygen/alkylene ester derivatives include / Contains ethylene monooleate, polyoxyetherenomonostearate, etc. Ru. Polyokine alkylene ether derivatives found to be useful in the present invention The combination of conductor and sorbitol ester derivative includes polyoxoethylene Sorbitan/fatty acid derivatives such as polyoxyethylene(2o)rubitan monomer Reate [Polysorbaze-80 (Polysorbaze-30), Towie Includes Tvaen-80, Difco CDIFCO) .

According to particularly suitable embodiments of the invention, the concentration of surfactant is from about 0.1 to about 10%. (w//v), and preferably from about 0.5 to about 5%. most preferred The surfactant concentration is from about 0.5 to about 1.5%.

According to a particularly suitable embodiment of the invention, tissue, surgical instrumentation, and medical devices can be Treated with an improved sterilant composition at temperatures ranging from 20°C to about 100°C. Understand.

More preferably, the temperature is maintained at about 20°C to about 40°C.

The present inventors have demonstrated that at temperatures above room temperature (20°G) and in the range of about 60 to about 400C. It was unexpectedly discovered that sterilization is accelerated. The most suitable range is approximately 32°C to approx. Up to 68°C. Temperatures above this physiological temperature range are may be harmful to other surgical instruments or medical devices according to the invention. It would not be harmful.

According to the present invention, the amount of microorganisms present and The exposure time is related to the level of coverage desired. Therefore, the time varies according to demand. A vine. According to a particularly specific embodiment of the invention, this is given by way of example. However, tissues with a temperature of about 100 to 10-6 are at least 7°C at 35±3°C. Requires hours of exposure time. Due to the interaction between time and temperature, if the temperature This requires that the exposure time necessarily be reduced. Therefore, if If the temperature were to be lowered, the exposure time would have to be reduced dramatically.

According to the present invention, a sterilant composition, if it exhibits approximately a 90% reduction in test organisms. If so, it is considered potentially effective. Preferably, an effective solution will be accepted. test organisms (105 to 106 hours) within a period of time (about 7 to about 8 hours) ) should be shown as complete destruction. Furthermore, the sterilant composition may be It is believed to be particularly effective if it is on the entrance surface or on the tissue (substrate). Up As discussed in , the inventors have developed a surfactant containing formaldehyde and ethanol. A solution without agents may or may not be equally effective against the microorganisms in the solution. I discovered something.・The inventors exposed the surfactant and removed the surfactant by rinsing. removed and then exposed to sterilizing agents such as glutaraldehyde and alcohol. We further found that biological tissues (biological proteins) are not effective. These observations Unexpectedly, tissues may be treated with glutaraldehyde or formaldehyde for optimal effect. that it must be exposed to dehyde, alcohol, and surfactant simultaneously. It shows. According to the present invention, the tissue can be divided into these three components in separate steps or as a single component. Can be exposed during the process.

It is particularly suitable to expose the tissue to the improved sterilant composition of the present invention in a single step.

The invention is further illustrated by the following examples, which are not intended to be limiting. stomach.

Example 1 The isolated porcine aortic heart valve tissue was incubated at 0.02 MI at pH 7.3 and approximately 4°C. Isotonic (285 ± 15 mm) Rinse well and transport in an isotonic solution at room temperature pH 7, 4, fix with 0.625 weight factor glutaraldehyde.

Example 2 From the excised tissue of Phosphate Example 1 with a spore count of 105 to 106 pups/ml. The final inoculum concentration was 7.0% by injection into various parts of the prepared bioprosthetic heart valve. I X 203 spores/valve. This valve tissue was treated with formaldehyde 4±0.4%, 0.02M phosphate buffer bath containing 20% of ethanol (100% absolute ethanol) , 100 ml + [pH 7, 4, further exposed for 48 hours at 20-22 °C, this destruction The effect of fungal agent solution on the growth of Microascus cinereus was investigated by removing the valve from the solution. remove, place them in a liquid nutrient solution that supports fungal growth, and place them for 48 hours. Assess at regular intervals by visually measuring the viable spores remaining on the post-mortem valve. worth it. At the end of 48 hours, spore counts in any of the five valve tissue samples tested It was clear that there was no decrease in

Example Microass forces in phosphate buffer baths with spore counts 105 to 106 spores/me A suspension of Su. to give a final inoculum concentration of 5.6 x 103 spores/valve. this Valve tissue was treated with formaldehyde (bitan monooleate polyoxyethylene) 1% 0.02M phosphate buffer solution (pH 7,4) containing 20-22° Further exposure for 48 hours at C. Microascus of this sterilant solution / Nereu It is placed in a liquid nutrient medium that supports the growth and growth of the grass, and after 48 hours of treatment it is placed in a benshi. Evaluate at regular intervals by visually measuring remaining viable spores.

From this analysis, the inventors found that three of the five samples tested showed no growth of organisms after 48 hours. and reduce spore numbers by a factor of 10 (11 og reduction ) is approximately 578.9 minutes (9 hours and 39 minutes). Microprepared bioprosthetic hearts with spore counts from 5 to 106 pups/ml Various parts of the visceral valve are injected to give a final inoculum concentration of 1.6 x 105 spores/valve.

This valve tissue is further processed as in Example 3. At the end of 24 hours, the tested valve No logarithmic reduction in spore numbers was evident in any of the five tissue samples.

Example 5 7 Nereus floats with a spore count of 105 or 106 spores/ml. The fluid was injected into various parts of the bioprosthetic heart valve prepared from the excised tissue of Example 1. to give a final inoculum concentration of 7.7×1 [1” spores/valve, then inoculate the valve tissue as an example. further processed for 24 hours at 32°C. Ku. At the end of 4 hours, only 2 of the 5 samples tested showed biological growth; After - hours, none of the five samples tested showed any growth of organisms. Furthermore Also, the time required to reduce the number of spores by a factor of 10 (approximately 105 to 1 A suspension of Bacillus demirus in phosphate buffer with a spore count of o6u spores/ml. Injecting the free fluid into various parts of the bioprosthetic valve prepared from the excised tissue of Example 1. The final inoculum concentration was 1.6 x 104 spores/valve and then the valve tissue was Further processing is performed as per Keroyo, except that the sashimi tissue is processed at 32°C. 0 minutes At the end of was not detected.

Example 7 Chaetomium in phosphate buffer solution with a spore count of 105-106 spores/me. The suspension of Globosum was applied to the biological prosthetic heart valve prepared from the excised tissue of Example 1. Inject into the glazed area and adjust the final inoculum concentration to 2.2 x 10' 118 i/valve. do. This valve tissue is then further processed as in the example, except that the tissue except that it is processed at 62°C. At the end of 30 minutes, test 5 specimens of valve tissue development. was not detected.

Example 8 The excised tissue of Example 1 was treated with 22.5% ethanol and 4.0±0% formaldehyde. 4%1. syoO sorbitan monooleate polyoxoethylene (Towie 7 -80) O6ro2 containing 1.2%:, tIJ acid buffered saline solution (pH 7,3 ) 10ml at 65°C, complete sterilization was obtained after 8 hours. This place The tissue is further analyzed to assess tissue integrity after sterilization.

The results of the inventor's analysis show that the difference bond stability indicated by the shrinkage temperature, the pronar Tissue stability shown by enzyme digestion, amino acid analysis, di/hydrin analysis; uronic acid Content, Hematoki/Rin-Nio 7, Aldehyde Fukunono, PAS/Arunamp Histological examination as shown by staining with Roux, and trichrome; foot strain electron microscope? There were no significant differences in the surface morphology determined by and transmission electron microscopy.

Perform subcutaneous transplantation of tissue in developing and adult rabbits and expose them to current sterilants. Collakene fiber degradation, Recipient cell invasion and degree of tissue canceration (an important concern in tissue flaps) was measured. Recipient cell invasion was similar for both groups, but The improved method is less effective at decomposing fibers. Tissue canceration also occurs when exposed to improved sterilants. Phosphoric acid with a spore count of 105-106 spores/me is lower for Salt-mild formaldehyde 4±-0.4%, ethanol 20%, and Tween- p in 0.0:lv + phosphate buffered sterile solution rL100mj containing 80 (1%) H7,51,20-2200 to give a final inoculum concentration of 3.3 x 10” spores/ Let it be ml. Incubate while stirring the inoculated sterilant solution. 1. Take IQmJ and filter through a 0.45μ filter. The spores are collected and placed in a solid medium containing nutrients that support the growth of the fungus. Therefore, it was treated at 33°C for 10 days, and the residual amount was determined by visual counting. Determine the number of spores present. After 8 hours of exposure to the sterilant solution, no more spores can be detected. The average time required to reduce the number of spores by a factor of 1o is approximately 114 minutes. It is.

Example-10 The experiment of Example 9 is repeated in all essential details, but with the exception of sterile spore-inoculum. except that the agent solution is processed at 63°C instead of 20-22°C. 4 o'clock in the sterilant bath solution After exposure for a period of time, no more spores are detected and the number of spores is reduced by a factor of 10. The average time required for this is approximately 61 minutes.

Although the invention has been described in particular detail and with reference to particularly suitable embodiments thereof, the main aspects of the invention It will be understood by those skilled in the art that modifications and changes may be made without departing from the spirit and scope. It should be understood.

Letter of Submission of Translation of Written Amendment (Article 184-7, Paragraph 1 of the Patent Office) Commissioner of the Patent Office 1. Indication of patent application: PCT/US831017022, title of invention: ported Chemical sterilization of biological tissues 3, patent applicant none Address: Shin-Otemachi Building 3, 2-2-1 Otemachi, Chiyo 1η-ku, Tokyo 100 31 5. Scope of claims amended on the 2nd date of submission of supplementary city report Received from the International Bureau on February 28, 1984. Paragraph 1 of the so-called scope is not amended. , Replacement of claims 2 to 4 with new claims 15 to 36 I got it. The scope of the new boast is as follows. )15 Transplant biological tissue into animals The tissue is heated at 4°C from about 300 to about 40°C in The aqueous solution is placed in an aqueous solution for a sufficient period of time to destroy a substantial amount of the microorganisms; a sterilizing effective amount of glutaraldehyde or formaldehyde; from about 0.1 to about 10% by weight; cationic, non-ionic, or anionic surfactants; ,and from about 10 to about 60% aromatic or aliphatic alcohol; The above method.

16. Claim 1, wherein the solution contains about 0.2 to about 1% glutaraldehyde The method described in Section 5.

17. The solution of claim 15, wherein the solution contains about 5% formaldehyde. How to put it on.

Contains 25% aliphatic alcohol.

The method described in Section 15 of the scope of Shishu.

19 The aliphatic alcohol has 1 to about 5 carbon atoms. Claim No. 18 How to put it on.

2ro 20. The alcohol is ethanol or inzolopanol. Claim No. ′ The method according to item 19.

21 The temperature is about 62° to about 38°G. The method according to claim 15.

22. The surfactant is about 0.5 to about 1.5 parts by weight.

The method according to claim 15.

23. The surfactant is cationic and contains an alkyl quaternary amine or its halide. It is a dianide salt. The method according to claim 15.

24 The alkyl quaternary amine surfactant contains aromatic residues, aliphatic residues, or aromatic residues. From a tertiary amine attached to any combination of both aromatic and aliphatic residues and the aromatic residue further consists of benzene, f+)dine, or naphthalene, Aliphatic residues are branched, unbranched, cyclic, saturated, or unsaturated. Claim 2 The method described in Section 3.

25. The surfactant is nonionic, and polyoxyalkylene ether, polyol Quinalkylene alkylaryl ether, aliphatic ester, polyether, polyether is a lyoxyalkylene diester, a saccharide ester, or a combination thereof . The method according to claim 15.

26 The surfactant is anionic and contains bile salts, from about 8 to about 20 carbon atoms. salts of fatty acids with N-alkanoyl amino acids (with aliphatic moieties ranging from about 8 to about 18 carbons); 2A Holding table Showa 60-500014 (8) having an elementary atom), N-amyl anhydride Zarphenol, an aliphatic alcohol having from about 6 to about 18 carbon atoms , alkali/sulfonoacids and alkylarylsulfonic acids. Claims No. The method according to item 15.

27.@Liquid contains about 6 to about 5% formaldehyde, about 20 to about 60% methane alcohol, ethanol, or inpropertool, about 0.5 to about 5% nonionic field. It consists of a surfactant and has a temperature of from 32°C to about 68°C.

The method according to claim 15.

28 The solution contains about 6 to about 5% formaldehyde, about 20 to about 25% formaldehyde an aliphatic alcohol having about 5 carbon atoms, a nonionic surfactant with a ratio of about 0.5 to about 5; The temperature ranges from about 62°C to about 68°C.

The method according to claim 15.

The 29 surfactant is sorbitano monooleate foralkyreno. The scope of the claims The method according to paragraph 27.

The filter O3 solution contains about 0.2 to about 1% glutaraldehyde, about 20 to about 25% methanol, ethanol, or inpropertool, about 0.5 to about 5% on surfactant and at a temperature of about 32° to about 38°C. The scope of the claims The method according to paragraph 15.

31 The surfactant is norbitan monooleate polyalkylene. scope of claim The method described in Box No. 0.

32. Biological tissue consists of bones, ligaments, heart valves, dura mater, epithelium, tympanic membrane, and skin fragments. Ru. A method according to claim 28.

6. The biological tissue is the four heart valves, and the microorganisms are the microascus. neraeus, the alcohol concentration is about 20 to about 25%, and the surfactant the concentration is about 0.5 to 1.5% and the surfactant is sorbitan monooleate. Polyethylene. A method according to claim 28.

Ro4. Methods for sterilizing biological tissues prior to transplantation into animals At a temperature of about 32° to about 38°C, formaldehyde or glutamate Rudehyde Biological tissues such as cavities, ligaments, heart valves, dura mater, epithelium, tympanic membrane, and and said bathing liquid consists of About 2% to about 1% glutaraldehyde 3% to about 5% formaldehyde from about 0.1 to about 1.5 weight factors of cationic, nonionic, or anionic surfactants ,and About 20 to about 25 aromatic or aliphatic alcohols (1 or 1 have about 5 carbon atoms) The above method consists of:

6 65. Claim 64, in which the resistant microorganism is Microascus/I. reus Method described.

older brother. The claim is that the biological tissue is a microorganism and the surfactant is ionic. The Shira method described in Box 25.

Claims (1)

[Claims] 1. In a method of processing biological tissue prior to transplantation, this tissue is brought under sterile conditions. Below formaldehyde or glutaraldehyde, alcohol, and kaimonkatsu contact with a sterile effective amount of a solution comprising a sex agent. The above method. 2. Sterilization@The concentration of surfactant in the liquid is from about 0.1 to about 10 parts by weight. request The method described in item 1 of the scope of the request. Rofa5. The harshness of the surfactant in the liquid is from about 0.5 to about 5 Mfa%. The alcohol has an e-degree of about 10 to about 90 g by weight. The scope of the claims The method described in paragraph 1. 4. The a degree of alcohol in the sterile solution is from about 10 to about 30% by weight. Claim The method described in item 1. 5. Contacting the biological tissue with a sterile solution at a temperature of about 200 to about 40°C . The method according to claim 1. 6. Contacting the biological tissue with a sterile solution at a temperature of about 20' to about 40'C. . 3. The method described in item 6 of the scope of the request. 7. Contacting the biological tissue with a sterile solution at a temperature of about 600 to about 408 C de - The method described in claim 6. 8 Low 6 alcohol containing 1 to about 5 carbon atoms It is a class aliphatic alcohol. The method according to claim 1. 9 The alcohol is methanol, ethanol, or improper tool. request The method described in item 1 of the scope of the request. 10 The surfactant is sorbitan monomer ethyl +;') yrquinoethylene, Sodium dode/l sulfate, or boliletele/glycol p-in The method according to claim 1, which is a pyl ether. 11 Biological tissues were treated with about 4 to about 5% formaldehyde and lower aliphatic alcohols. and about 20 to about 60% surfactant and about 0.5 to about 5% surfactant. Contacting is carried out at a temperature of 30 to about 40°C. J. The method according to claim 1. 12 Biological tissues are used to prepare heart valves7, and lower fat Is flucol is used as a medicinal tanol, ethanol, or inpropertool, and the surfactant is sorbitol. /monooleate polyalkylene and has a temperature of about 62 to about 38°C. . The method according to claim 11. 16 Sterilizing effective amount of glutaraldehyde or formaldehyde, from about 10 to about Alcohol in an amount up to 60% by weight, surfactant in an amount from about 0.5 to about 5% by weight A composition consisting of an agent. 14 The alcohol is a lower aliphatic alcohol with a concentration of about 20% or about 30%. Ru. Claim 11] Satoru 16 Hanki 24 composition. 1　1MBaGO-500014(2)