TISSUE CRYOPRESERVATION METHOD

Abstract: A method for cryopreserving tissues begins with dissected tissues, which are divided into at least two groups having higher and lower levels of microbial contamination. The first group is treated with a first antimicrobial regimen that renders most or all of them substantially free of contamination while maintaining cell viability at a high level. The second group is subjected to a second antimicrobial regimen that minimally reduces cell viability and renders substantially all of the tissues free of microbial contamination. Both the first and second groups are cryopreserved, thereby maximizing yield. Preferably, the second antimicrobial regimen is conducted at 37 DEG C, and it utilizes a specific set of antimicrobial agents as described in the specification. Another method for cryopreserving tissues begins with dissected tissues. All the dissected tissues are treated with an antibiotic regimen which renders all of them substantially free of contamination while maintaining cell viability at a high level. Preferably, the regimen is conducted at 37 DEG C, utilizing a specific set of antimicrobial agents as described in the specification, and cryopreserved.

Description: TISSUE CRYOEKESERVAΠON METHOD Cross Reference to Related Applications This is a continuation-in-part of copending U.S. Patent Application Serial No. 07/645,979 filed January 24, 1991. Background of the Invention This invention relates to an improved method for cryopreserving tissues such as heart valves, veins, and musculoskeletal connective tissue, and in particular to such cryopreservation methods which include improved tissue decontamination steps. U.S. Patent 4,890,457, assigned to the assignee of the present invention, discloses a cryopreservation method suitable for use with heart valves. Heart valves cryopreserved as disclosed in this patent retain a high level of cell viability. High viability cryopreserved valves provide significant longevity when transplanted. Two trends have developed over the past several years which have resulted in an acute shortage of high viability cryopreserved human heart valves. The first trend is the ever increasing use of allograft tissue for cardiac valve replacement and outflow track reconstruction. The second trend is the change in criteria for whole heart transplant that has necessitated a change in source for allograft valve tissue from heart beating donors to asystolic tissue donors. Because of the extreme scarcity of allograft valves, there is an essential need to fully utilize heart valve tissue which has been donated by families and recovered by community service organ/tissue agencies. As the first trend developed, the use of heart valve allografts became the treatment of choice for many congenital heart reconstructions and for several indications in adult aortic valve replacement. This greatly increased the demand for heart allograft tissue such that the level of demand far exceeded the level of donation. The second trend necessitated that most of the allograft valves had to be recovered from asystolic tissue donors with a significantly higher incidence of bacterial contamination. While the number of available heart valves increased, these trends combined to decrease the percentage of tissue requests that could be filled with high viability cryopreserved valves. Accordingly, it is a primary object of this invention to provide alternative preservation methods that will expand the supply of tissue available for transplant, without substantially reducing the number of high viability cryopreserved tissues, or their level of viability. SUMMARY OF THE INVENTION According to a first aspect of this invention, a method is provided for cryopreserving tissues comprising the following steps: a. obtaining dissected tissues selected from the group consisting of heart valves, veins and musculoskeletal connective tissue; b. dividing the dissected tissues into at least two groups including a first group characterized by a lower level of microbial contamination and a second group characterized by a higher level of microbial contamination; c. subjecting at least the first group of dissected tissues to a first antimicrobial regimen effective to render at least some of the first group of dissected tissues substantially free of contamination and to maintain cell viability of a first group of tissues at a first level; d. cryopreserving the first group of dissected tissues after completion of step c; e. subjecting the second group of dissected tissues to a second antimicrobial regimen effective to render the second group substantially free of contamination and to minimally reduce cell viability below said first level; f. cryopreserving the second group of dissected tissues after step e. This method, when practiced as described below, increases the total yield of cryopreserved tissues without substantially decreasing the yield of high viability cryopreserved tissues. Preferably, the first antimicrobial regimen and the dividing step maintain high cell viability in the first group of tissues, and cryopreservation of the first group of dissected tissues is not delayed excessively. Because the second antimicrobial regimen is preferably conducted using higher concentrations of antimicrobial agents, the second group of dissected tissues is decontaminated effectively, though cell viability may be somewhat reduced. Nevertheless, such lower viability cryopreserved tissues can be used as effective allografts in specific instances where human graft material is required. In the preferred embodiment described below, the second antimicrobial regimen is comprised of at least one antimicrobial agent selected from each of the following families: celphalosporins, glycopeptides, amninoglycosides, linocosamides, and rifamycins. This second antimicrobial regimen is preferably conducted at 37°C and minimally reduces the cell viability of the tissues in the second group. According to a second aspect of this invention, a method for cryopreserving tissue is provided comprising the following steps: a. obtaining dissected tissues selected from the group consisting of heart valves, veins and musculoskeletal connective tissue; b. subjecting the tissue to a first antimicrobial regimen effective to reduce contamination of the dissected tissues while maintaining cell viability of the dissected tissues at a first level; c. subjecting at least a portion of the dissected tissues to a second antimicrobial regimen comprising at least one antimicrobial agent not included in the first antimicrobial regimen and effective to render at least some of the dissected tissues substantially free of contamination and to reduce cell viability of the dissected tissues minimally; and d. cryopreserving the dissected tissues after step c. Preferably, the second antimicrobial regimen is conducted at an elevated temperature of about 37°C using antimicrobial agents specifically chosen for effectiveness against commonly encountered bacteria such as staphylococci, streptococci, and propionibacteria. Because the tissue is subjected to two consecutive antimicrobial regimens, and because the first antimicrobial regimen maintains cell viability at the first level, this method is well suited for use in the method described initially above. The first antimicrobial regimen can be selected to be appropriate for high viability cryopreserved tissue that is initially less contaminated. This first antimicrobial regimen can be conducted while cultures are incubated that will allow the dissected tissues to be divided into first and second groups that vary in the degree of contamination, as described above. According to a third aspect of this invention, a method for cryopreserving tissue is provided comprising the following steps: a. obtaining dissected tissues selected from the group consisting of heart valves, veins and musculoskeletal connective tissue; b. subjecting the tissue to an antimicrobial regimen effective to render the tissues substantially free of contamination and to minimally reduce cell viability, said antimicrobial regimen comprising the step of exposing the dissected tissues to an antimicrobial solution comprising inhibitory concentrations of the following antimicrobial agents: sodium cefotaxime, vancomycin hydrochloride, netilmicin sulfate, lincomycin hydrochloride, rifampin; and c. cryopreserving the dissected tissues after step b. The antimicrobial regimen is preferably conducted at an elevated temperature of about 37°C for a period of at least four hours. These conditions have been found to decontaminate tissues such as heart valves effectively, even when other antimicrobial regimens that preserve a high level of cell viability have proven ineffective. This antimicrobial regimen may be conducted with other antimicrobial agents which are members of the same families. Such alternative antimicrobial regimens utilize at least one antimicrobial agent from each of the following families: cephalosporins, glycopeptides, aminoglycosides, lincosamides and rifamycins. The invention itself, together with further objects and attendant advantages, will best be understood by reference to the following detailed description, taken in conjunction with the accompanying drawings. Brief Description of the Drawings . Figure 1 is a flow diagram of a cryopreservation method that incorporates a first presently preferred embodiment of this invention. Figures 2a and 2b combine to form a flow diagram of the culture reading step 22 of Figure 1. Figure 3 is a flow diagram of a cryopreservation method that incorporates a second presently preferred embodiment of this invention. Detailed Description of the Presently Preferred Embodiments The following detailed description will first provide an overview of the first and second presently preferred embodiments of this invention, and will then provide further information regarding individual steps of these preferred methods. Overview Turning now to Figure 1, which illustrates a first presently preferred embodiment of this invention, the first step 10 is to dissect the tissues as appropriate for allograft transplants. A variety of tissues may be used, including heart valves, veins, and musculoskeletal connective tissue. As used herein, the term "musculoskeletal connective tissue" includes tissue such as tendons, ligaments and menisci, and excludes tissue such as bone. In the following example, the tissues will be assumed to be human heart valves. Prior art dissection techniques are preferred, such as those described in U.S. Patent 4,890,457, which is hereby incorporated by reference. Prior to dissecting the tissues, the donor of the tissues is screened to eliminate those donors who are septic at the time of procurement. After the heart valves have been dissected, they are then accumulated in a tissue storage solution such as sterile DMEM Solution B in step 12 and subjected to a first antimicrobial regimen in step 14. This first antimicrobial regimen may be similar to that described in the above-identified U.S. Patent 4,890,457. Generally speaking, the heart valves are immersed for 24 hours at a temperature of 2-8°C (preferably 4°C) in an antimicrobial solution which includes the following antimicrobial agents at the indicated concentrations (micrograms per milliliter) : cefoxitin 240; polymyxin B 100; vancomycin 50; lincomycin 120. Alternatively, the heart valves are immersed for 24 hours in imipenem (12 μg/ l) and vancomycin (50 μg/ml) at 30- 40°C (preferably 37°C) . The first antimicrobial regimen is designed to preserve a high level of cell viability in the cryopreserved heart valves, at least about 400 radioactive disintegrations per minute. Viability was determined by the procedure described below, which is excerpted from Appendix A of the corresponding U.S. Application Serial No. 07/800,916. Tissue viability was assessed by measurement of 3H-glycine incorporation into proteins. Protein synthesis was measured in either complete individual valve leaflets or bisected valve leaflets by scintillation counting. The tissues were placed in snap cap tubes (Falcon) containing 12 Cl of tritiated-glycine in 750 ul of DMEM supplemented with 15 ug/ml ascorbic acid. Radiolabeled glycine incorporation was determined after a 48 hour incubation at 37°C in an atmosphere consisting of 5% carbon dioxide in air. The tissue was then washed four times with phosphate-buffered saline solution, dehydrated with ethanol and ether, air dried and weighed. The tissue samples were then rehydrated with 200 1 of water and solubilized by addition of 1 M NaOH, incubation at 60°C for 1 hour and two 20-second sonications. The ho ogenates were centrifuged at 12,00 x g and 100 1 aliquots of the supernatants were placed on glass fiber filter discs (Whatman No. 1822024; Whatman Chemical Separation Inc., Clifton, N.J.). The filter discs were dried, and the proteins precipitated by addition of ice-cold 10% trichloroacetic acid for 30 minutes. This was followed by five ethanol rinses, two ether rinses, and drying. The discs were then placed in 1 ml of Protosol (Dupont) , to solubilize the proteins, followed by addition of 10 ml of scintillation fluid (Ecoscint A; National Diagnostics, Inc., Somerville, N.J.) and tritium incorporation was measured by scintillation counting (Beckman Instruments, Inc. , Palo Alto, CA) . All results are expressed in disintegrations per minute (DPM) per mg of dry tissue. A microbial analysis is performed while the dissected heart valves are accumulated and subjected to the first antimicrobial regimen. This microbial analysis comprises the steps 16, 18, 20, 22, 24 of preparing appropriate cultures, incubating the cultures, and then reading the cultures after an incubation time of 24 hours. Separate cultures are prepared for each dissected heart valve, and the results of the microbial analysis are used in step 26 to classify individual dissected heart valves in either a first group or a second group. The first group includes heart valves that are most likely to be contaminated with microbes against which the first antimicrobial regimen is effective. Thus, the first group includes those which are likely to be rendered free from microbial contamination by the first antimicrobial regimen. Heart valves in the first group are immediately packaged, cryopreserved and stored in steps 28 and 30. The packaging and cryopreservation techniques may be similar to those described in the above-identified U.S. Patent 4,890,457. Heart valves which are classified in the second group are heart valves which are not likely to be rendered free from microbial contamination by the first antimicrobial regimen, but which may be rendered free from microbial contamination by a second antimicrobial regimen. This second group of heart valves is subjected to the second antimicrobial regimen in step 32, which uses additional antimicrobial agents. The incubation temperature for the second antimicrobial regimen is preferably 30-40°C and the incubation time is preferably 4-26 hours. Preferably, step 32 includes the step of immersing the heart valves of the second group in an antimicrobial solution at 37°C for 24 hours, wherein the antimicrobial agents of Table I are present in the solution at no less than the concentrations set out in Table I. TABLE I Final Concentration Antimicrobial Agent (micrograms/milliliter) sodium cefotaxime 120 vancomycin hydrochloride 100 netilmicin sulfate 50 lincomycin hydrochloride 120 rifampin 60 This second antimicrobial regimen is selected to decontaminate may of the heart valves not decontaminated by the first antimicrobial regimen, while minimally or only slightly reducing cell viability of valves in the second group. After the second antimicrobial regimen has been completed the heart valves of the second group are packaged, cryopreserved and stored in steps 34 and 36. Once again these packaging and cryopreserving steps may be similar to those described above in U.S. Patent 4,890,457. Preferably, microbiological tissue samples are obtained prior to packaging the first and second groups and after the antimicrobial regimens, and these additional samples are subjected to microbial analysis in step 38 to confirm that the cryopreserved heart valves in both the first and second groups have been effectively decontaminated. The method described above provides a number of important advantages. First, since all of the dissected tissues are subjected to the first antimicrobial regimen (step 14) , this method does not overly delay the cryopreservation of heart valves in the first group. The initial microbial analysis, including incubation of the samples removed initially from the dissected tissues, takes place prior to or at the same time as the first antimicrobial regimen. In this way, the microbial analysis is available for classifying the dissected tissues into the first and second groups without overly delaying the processing of the first group. This ensures that the cell viability of the cryopreserved heart valves in the first group is not compromised. Second, the heart valves in the second group are subjected to both the first and second antimicrobial regimens, and the prospect of effectively decontaminating the valves in the second group is increased. The second antimicrobial regimen utilizes antimicrobial agents specifically selected for effectiveness against the most frequently encountered contaminating bacteria when the process of U.S. Patent 4,890,457 is used, and it is conducted at an elevated temperature at which such antimicrobial agents are maximally effective. In this way, the yield of cryopreserved heart valves in the second group is optimized. The result is that additional minimally reduced viability cryopreserved heart valves are made available (the second group) without substantially reducing the yield of the preferred, high cell viability cryopreserved heart valves of the first group. The process of U.S. Patent 4,890,457 has resulted in a yield of high viability cryopreserved valves of about 75%. That is, about 25% of the dissected valves are not suitable for transplantation, principally due to contamination. The process described above has resulted in a yield of high viability cryopreserved valves of about 70% and a yield of minimally reduced viability cryopreserved valves of almost 30%. Turning now to Figure 3 which illustrates a second presently preferred embodiment of this invention, the first step 100 is to dissect the tissues as appropriate for allograft transplants. This is done as described for step 10 above. Then in step 120 the dissected tissues are accumulated as described in step 12 above, and all the accumulated tissues are subjected to one antimicrobial regimen 150. Preferably, step 150 includes the step of immersing the heart valves in an antimicrobial solution at 37°C for 24 hours, wherein the antimicrobial agents of Table I above are present in the solution at no less than the concentrations set out in Table I. Then the tissues are packaged and cryopreserved in steps 280 and 300. Once again, the packaging and cryopreservation techniques may be similar to those described in the above-identified U.S. Patent 4,890,457. Microbial Analysis (Steps 16, 18, 20. 22, 24) For each dissected heart valve, the samples of step 16 may include (1) a piece of external heart muscle tissue, (2) a piece of the mitral or tricuspid valve, (3) a piece of muscle tissue proximal to the aortic or pulmonary valve, and (4) several milliliters of solution in which the heart was received. In step 18, the microbiological samples are grouped into batches according to time of arrival, refrigerated for later plating if necessary, homogenized when appropriate, and then plated on the following media: blood agar plate (5-10% C0 2 ) 0.025 ml; chocolate agar plate (5-10% C0 2 ) 0.025 ml; blood agar plate (anaerobic) 0.025 ml; enriched thioglycollate broth 1.0 ml. Both the blood agar and the chocolate agar plates in C0 2 are incubated at 35°C for 4 days and observed daily. The anaerobic blood agar plate is incubated in an anaerobic jar at 35°C for 6 days and observed on the 2nd, 4th and 6th days. The thioglycollate broth is incubated at 350C for 14 days and observed daily for the first 7 days and periodically thereafter. The culture reading step 22 is flow charted in Figures 2a and 2b. After the cultures have been incubated for 24 hours in step 20, the cultures are observed for growth at decision block 60. This examination consists of visual examination of the plates and broth and microscopic examination of the broth by gram stain at lOOOx magnification. If no growth is observed, the corresponding tissue is classified at block 62 as in Group 1, to be cryopreserved as a high viability valve. On the other hand, if growth is observed in block 60, the procedure branches depending upon whether or not any of the plates are visually positive for growth (block 64) . If visual growth is not observed on the plates, then the corresponding tissue is classified in block 70 as in Group 2, to be treated with the second antimicrobial regimen described above. Returning to block 64, if any of the C0 2 plates are visually positive for growth, the broth is then examined in block 72 to determine whether it is positive for growth. If so, all of the plate growth is then visually assessed in block 76 to determine whether or not it is all classified visually as staph. If not, the corresponding tissue is classified as in Group 2. If so, the broth is then observed in block 80 to determine whether or not all of the broth growth is gram positive and cocci (not in chains) . If not, the corresponding tissue is classified in block 70 as in Group 2. If so, the plate growth is then analyzed in block 82. If it is all gram positive cocci, all slide catalase positive, and all negative for Staph aureus, then the corresponding tissue is classified in block 62 as in Group 1. Otherwise, the corresponding tissue is classified in block 70 as in Group 2 . Returning again to block 72, if the broth is negative for growth, then the plate growth is visually assessed in terms of its morphology in block 74. If its morphology indicates something other than staph in any part, the corresponding tissue is classified in block 70 as in Group 2. Otherwise, the plate growth is analyzed in block 82 as before. The tests and other procedures described above are individually well known to those skilled in the art. For example, a suitable test for identification of Staph aureus may be obtained from Wellcome Diagnostics under the tradename STAPHAUREX. In this way, the cultures are read at 24 hours and the corresponding tissues are classified into either Group 1 or Group 2. As explained above, the tissues in these two groups are processed differently in order to decontaminate both groups properly prior to cryopreservation. The Second Antimicrobial Regimen (Step 32 and Step 150) The antimicrobial solution for the second antimicrobial regimen of the first presently preferred embodiment and for the only antimicrobial regimen of the second presently preferred embodiment is preferably prepared as follows. First, three stock solutions are prepared. The first is a cefotaxime stock solution made up of 2.0 grams of sterile sodium cefotaxime and 3 ml of sterile water. Sodium cefotaxime may be obtained under the tradename CLAFORAN (Hoechst-Roussel) . Cefotaxime and Cefoxitin (a cephomycin) belong to the cephalosporin family. (Cefoxitin is used for tissue decontamination in U.S. Patent 4,890,457) . Cefotaxime has a broader spectrum of activity than cefoxitin, is especially effective against the staphylococci and streptococci, and is reported to have synergy with an aminoglycoside against Pseudomonas species. The second stock solution is preferably made of 500 milligrams of sterile vancomycin hydrochloride and 10 ml of sterile water. Vancomycin hydrochloride may be obtained under the tradename VANCOCIN HCI (Lilly) . Vancomycin, which is also used for decontamination in U.S. Patent 4,890,457 described above, is in the glycopeptide family and is one of the antimicrobial agents with the broadest spectrum of activity against gram positive organisms, including staphylococci and streptococci, and specifically Streptococcus faecalis. It has been reported to have synergy with the aminoglycosides and/or rifampin when treating patients for viridans streptococci, which is of particular interest in this application. The third stock solution contains rifampin and can be formed for example of 600 milligrams of sterile rifampin and 10 ml of sterile water. Rifampin may be obtained under the tradename RIFADIN (Merrell Dow) . Rifampin is in the rifamycin family and is very active against propionibacteria, staphylococci and streptococci including Enterococcus faecalis. It has been reported to have synergistic activity with vancomycin and in the treatment of Staphylococcus epidermidis and diphtheriods. Once these three stock solutions have been prepared, a 10X intermediate solution can be prepared from 250 ml of sterile DMEM solution B (Gibco Labs CAT #380-2320, page 15, Catalog and Reference Guide, 1987 ed) , 1.0 ml of the cefotaxime stock solution, 5.0 ml of the vancomycin stock solution, 2.5 ml of the rifampin stock solution, 1.25 ml of a commercially available netilmicin sulfate solution (100 mg/ml) and 1.0 ml of a commercially available lincomycin hydrochloride solution (300 mg/ml) . This intermediate solution may be stored at -80°C. Netilmicin sulfate, which can be obtained under the tradename NETROMYCIN (Settering) , is in the aminoglycoside family and is effective against gram negative aerobic bacteria and against all gram positive bacteria (streptococci, staphylococci and propionibacteria fall into this group) . Netilmicin has exceptional activity against staphylococci and streptococci, which are of particular interest in this application. It has been reported to have synergistic activity in combination with vancomycin against Staphylococcus aureus, the viridans streptococci, and Streptococcus Group D, non-Enterococcus (also of specific interest in this application) . Depending upon the actual isolate, it may also be synergistic with cefotaxime against Enterococcus species, which are also of specific interest here. Lincomycin hydrochloride can be obtained under the tradename LINCOCIN (Upjohn) . Lincomycin, which is in the lincosamide family and is used in the process of U.S. Patent 4,890,457 to decontaminate heart valves, has effective antibacterial activity against the staphylococci and streptococci and many anaerobic bacteria. In order to make the final working solution 34 ml of the intermediate solution described above is combined with 253 ml of sterile DMEM solution B. The resulting antimicrobial solution contains no less than the desired concentrations of the stated antimicrobial agents, as defined in Table I above. This solution has been proven effective in eliminating contamination by a range of microbes, including those of the viridans streptocoσcal group, propionibacteria, and coagulase-negative staphylococσus. The concentrations of these antimicrobial agents have been chosen to be at least 4 to 8 times the minimum inhibitory concentrations of the targeted microbes. Four to eight times the minimum inhibitory concentration of an organism is also generally the antimicrobial agent serum level which physicians attempt to achieve when treating systemic bacterial infections. Polyenes such as amphotericin B have been excluded to enhance cell viability. Packaging (Steps 28. 34 and 280. The packaging steps 28, 34 and 280 utilize a three bag system. Two of these bags made from a 4 mil polyimide/FEP film (#400 FN 022 - Kapton/Dupont) made up of a lamination of Teflon and Kapton, and the third bag is a peel pouch made of a 2.5 mil polyethylene/polyester laminate. The Kapton pouches are 4" x 6" and 6" x 9" with a 3/8" seal on three sides and the peel pouch is 5" x 8" with a 3/8" seal on three sides. A heart valve being prepared for freezing is placed in a sterile DMEM solution containing 10% DMSO with a total volume (including tissue) of 100 ml. The tissue and liquid are transferred to the smaller Kapton bag and that bag is sealed with an impulse heat sealer. The bag now containing the heart valve is then placed into the peel pouch which is similarly sealed. Finally, this peel pouch and its contents are placed in the larger of the Kapton bags and sealed. The valve is now ready for cryopreservation. All packaging is sterile prior to use and the packaging process is carried out in a clean room. Further details regarding the bags may be found in U.S. patent application Serial No. 395,279, filed August 17, 1989, which is hereby incorporated by reference. Cryopreservation .Steps 30. 36 and 300. Table II shows the presently preferred protocol for the cryopreservation steps 30, 36 and 300. The packaged heart valves are placed in a cooling chamber (CryoMed Model 1010A) and the temperature of the cooling chamber is controlled according .to chamber temperature (indicated by the letter 'C 1 in column 3 of Table II), sample temperature (indicated by the letter 'S 1 in column 3 of Table II) , and time. Sample temperature is obtained with an instrumented sample substitute that has thermal properties that match those of an actual sample. As shown in Table II, the chamber is held in Step 1 at 4°C for 8-10 minutes to allow equilibration, and chamber temperature is then reduced at 20°C/minute in Step 2 until the chamber reaches a temperature of -25°C. Chamber temperature is then lowered at 5°C/min until it reaches -30°C (Step 3) . Next, chamber temperature is lowered at 1.5°C/min until the sample temperature reaches -3°C (Step 4). The remaining Steps 5-12 are followed until the sample temperature falls to -80°C. The protocol of Table II has been designed to produce a substantially constant sample cooling rate of l°C/minute from +4°C to -80°c. TABLE II Cryopreservation Cooling Protocol Holding Temperature (°C_ * Time (Min 4 (C) 8-10 -25 (C) -30 (C) -3 (S) -160 (C) -110 (C) -110 (C) 8 -70 (C) -70 (C) 10 -40 (C) -40 (C) 3 -80 (S) * C = Chamber Temperature S = Sample Temperature Additional Microbial Analysis (Step 38) Second and third sets of samples are taken for microbial analysis as follows. Following the exposure to antimicrobial agents in the first antimicrobial regimen (Step 14) , and following exposure to antimicrobial agents in the second antimicrobial regimen (Step 32) , the tissues are rinsed in antimicrobial agent free medium to reduce their antimicrobial agent residual levels and packaged. The second sets of samples which are taken at this time are plated in duplicate as described below and are composed of (1) a piece of tissue proximal to the aortic or pulmonary valve, (2) a piece of the mitral or tricuspid valve, and (3) several milliliters of solution used to wash the antimicrobial agents from the tissue after treatment. These second samples are parallel processed tissue samples which are representative of the aortic and/or pulmonary valves, which cannot be tested directly without destroying the valves themselves. The third sets of samples are composed of several milliliters of the packaging solution alone. The second sets of samples from Group 2 (step 32) are treated to remove remaining antimicrobial agents and the second sets of samples from both Group 1 and Group 2 are then plated on the following media: blood agar plate (C0 2 ) 0.025 ml; chocolate agar plate (C0 2 ) 0.025 ml; blood agar plate (anaerobic) 0.025 ml; enriched thioglycollate broth 1.0 ml; sabourauds agar w/Gentamicin 0.025 ml; sabourauds heart infusion agar with blood 0.025 ml. Antibiotics may be removed with the device sold under the tradename ARD (Becton Dickinson) . The third sets of samples are plated on the following media: blood agar plate (C0 2 ) 0.025 ml; chocolate agar plate (C0 2 ) 0.025 ml; blood agar plate (anaerobic) 0.025 ml; enriched thioglycollate broth 1.0 ml. The bacterial agar plates and broths are incubated as described above in conjunction with Step 20. The sabourauds plates are incubated at 30°C, and the plates are observed 2 times the first 2 weeks and weekly thereafter at weeks 3 and 4. Gram stains are prepared of all positive cultures and accepted state-of-the-art microbiological biochemical tests are utilized to establish genus and species (where appropriate) . Gram stains are prepared from any negative thioglycollate broths to confirm "negative" visual observations. Tissues whose cultures demonstrate microorganisms on them as determined by the incoming microbiological samples are classified into 2 groups: those uncontaminated or contaminated with only "permitted" organisms (Table III) , and those contaminated with one or more "prohibited" organisms (Table IV) . Those tissues with "permitted" organisms are considered for release if the cultures of the second and third sets of samples (after antimicrobial treatment) are determined to be negative. Those tissues from Group 1 determined to be contaminated with "prohibited" organisms on the incoming microbiological samples are withheld from implantation (rejected) . Those tissues from Group 2 initially contaminated with "prohibited" organisms are considered for release if the second and third sets of samples (after antimicrobial treatment) are determined to be negative. Finally, all the tissues are examined histologically, and the pathologist always observes for bacteria and fungi on the preparations. TABLE III Permitted Organisms (Prior to Antimicrobial Treatment) Agrobacter radiobacter Bacillus Corynebacterium sp., except A5, D2, JK Diphtheroids Eikenella Haemophilus sp. , except aphrophilus and influenzae Kingella Lactobacillus sp. (other than Lacto. brevis and Lacto. casei; must be referred for speciation) Micrococcus sp. Moraxella sp. , including Moraxella (formerly Branhamella) catarrhalis Propionibacterium sp. Staphylococcus, coagulase negative TABLE IV Prohibited Organisms Acinetobacter sp. Actinobacilus actinomycetemcomitans Actinomyces sp. Aeromonas hydrophila Bacteroides Capnocytophaga sp. Campylobacter sp. Cardiobaσterium hominis CDC Group IV E Citrobacter diversus Clostridium sp. Corynebacterium Group A5 Corynebacterium Group D2 Corynebacterium Group JK Enterobacter sp. Enterococcus group Escherichia sp. Fungi Haemophilus aphrophilus Haemophilus influenzae Klebsiella sp. Lactobacillus brevis Lactobacillus casei Neisseria gonorrhoeae Neisseria meningitis Proteus sp. Pseudomonas sp. Serratia sp. Staphylococcus aureus Streptococcus anginosus (Group F) Streptococcus agalactiae (Group B) Streptococcus Group D, non-Enterococcus Streptococcus pneumoniae Streptococcus pyogenes (Group A, Beta hemolytic) Viridans streptococcus group Conclusion From the foregoing description it should be apparent that an improved cryopreservation method has been described which provides important advantages in terms of both improved decontamination steps and improved yields. Though this process has been described specifically with reference to the cryopreservation of heart valves, it is believed to be readily adapted for use with other tissues such as veins and musculoskeletal connective tissue such as tendons, ligaments and menisci. Of course, a wide range of changes and modifications can be made to the preferred embodiment described above. For example, the packaging and cryopreservation steps can be altered as appropriate for the individual application. Typically, a variation in packaging will often result in a variation in the specific protocol used for cryopreservation. Additionally, the specific antimicrobial agents used and the concentrations and the temperatures at which they are used as well as other variables such as the cryoprotectant may all be adapted as appropriate for the particular application. It is therefore intended that the foregoing detailed description be regarded as illustrative rather than limiting, and that it be understood that it is the following claims, including all equivalents, which are intended to define the scope of this invention.

Claims: WE CLAIM: 1. A method for cryopreserving tissues comprising the following steps: (a) obtaining a plurality of dissected tissues selected from the group consisting of heart valves, veins and musculoskeletal connective tissue; (b) dividing the dissected tissues into at least two groups including a first group characterized by a lower level of microbial contamination and a second group characterized by a higher level of microbial contamination; (c) subjecting at least the first group of dissected tissues to a first antimicrobial regimen effective to render at least some of the first group of dissected tissues substantially free of contamination and to maintain cell viability of the first group of dissected tissues at a selected level; (d) cryopreserving the first group of dissected tissues after completion of step (c) ; (e) subjecting the second group of dissected tissues to at least a second antimicrobial regimen effective to render at least some of the second group of dissected tissues substantially free of contamination and to reduce cell viability only minimally below the selected level; and (f) cryopreserving the second group of dissected tissues after step (e) . 2. The method of Claim 1 wherein the dissected tissues in both the first and second groups are subjected to the first antimicrobial regimen. 3. The method of Claim 1 wherein step (c) is completed before step (b) . 4. The method of Claim l wherein step (b) comprises the following steps: (bl) preparing cultures of material associated with the dissected tissues; (b2) obtaining readings of the cultures prepared in step (bl) after an incubation period; (b3) dividing the dissected tissues into said at least two groups based on the readings obtained in step (b2) . 5. The method of Claim 4 wherein the incubation period of step (b2) occurs while at least the first group of dissected tissues is being subjected to the first antimicrobial regimen in step (c) . 6. The method of Claim 1 wherein step (e) comprises the following step: (el) exposing the second group of dissected tissues to an antimicrobial solution at a temperature of about 37° C for at least about 4 hours. 7. The method of Claim 6 wherein the antimicrobial solution comprises at least one antimicrobial agent selected from each of the following families: cephalosporins, glycopeptides, aminoglyσosides, lincosamides, and rifamycins; and wherein the antimicrobial agents when used in the second antimicrobial regimen are effective to substantially eliminate infection by at least the following microbes: streptococci, propionibacteria, and staphylococci. 8. The method of Claim 6 wherein the antimicrobial solution comprises inhibitory concentrations of the following antimicrobial agents: sodium cefotaxime, vancomycin hydrochloride, netilmicin sulfate, lincomycin hydrochloride, and rifampin. 9. The method of Claim 8 wherein step (el) comprises the step of exposing the second group of dissected tissues to the antimicrobial solution at a temperature of about 37° C for about 24 hours. 10. The method of Claim 9 wherein the antimicrobial agents are present in the antimicrobial solution in at least the following concentrations in units of micrograms per milliliter: sodium cefotaxime 120; vancomycin hydrochloride 100; netilmicin sulfate 50; lincomycin hydrochloride 120; rifampin 60. 11. The method of Claim 1 wherein the dissected tissues are human heart valves. 12. A method for cryopreserving tissues comprising the following steps: (a) obtaining a plurality of dissected tissues selected from the group consisting of heart valves, veins, and musculoskeletal connective tissue; then (b) subjecting the dissected tissues to a first antimicrobial regimen effective to reduce contamination of the dissected tissues while maintaining cell viability of the dissected tissues at a first level; then (c) subjecting at least a portion of the dissected tissues to a second antimicrobial regimen comprising at least one antimicrobial agent not included in the first antimicrobial regimen and effective to render at least some of the dissected tissues substantially free of contamination and to reduce cell viability of the dissected tissues only minimally below the first level; and (d) cryopreserving the dissected tissues after step (c) . 13. The method of Claim 12 wherein the first antimicrobial regimen comprises exposing the dissected tissues to a first antimicrobial solution, and wherein the second antimicrobial regimen comprises exposing the dissected tissues to a second antimicrobial solution at a temperature between 30° and 40°C. 14. The method of Claim 13 wherein the second antimicrobial regimen is conducted at a temperature of about 37°C. 15. The method of Claim 13 wherein the second antimicrobial solution comprises at least one antimicrobial agent selected from each of the following families: cephalosporins, glycopeptides, aminoglycosides, lincosamides, and rifamycins; and wherein the antimicrobial agents when used in the second antimicrobial regimen are effective to substantially eliminate infection by at least the following microbes: streptococci, propionibacteria, and staphylococci. 16. The method of Claim 13 wherein the second antimicrobial solution comprises inhibitory concentrations of the following antimicrobial agents: sodium cefotaxime, vancomycin hydrochloride, netilmicin sulfate, lincomycin hydrochloride, and rifampin. 17. The method of Claim 16 wherein the antimicrobial agents of the second antimicrobial solution are present in the second antimicrobial solution in at least the following concentrations in units of micrograms per milliliter: sodium cefotaxime 120; vancomycin hydrochloride lOO; netilmicin sulfate 50; lincomycin hydrochloride 120; rifampin 60; 18. The method of Claim 12 wherein the dissected tissues are human heart valves. 19. The method of Claim 12 wherein the second antimicrobial regimen comprises exposing the dissected tissues to the second antimicrobial solution for at least 4 hours. 20. A method for cryopreserving tissues comprising the following steps: (a) obtaining a plurality of dissected tissues selected from the group consisting of heart valves, veins and musculoskeletal connective tissue; then (b) subjecting the dissected tissues to at least one antimicrobial regimen effective to render at least some of the dissected tissues substantially free of contamination and to reduce cell viability of the dissected tissues minimally, wherein the at least one antimicrobial regimen comprises the step of exposing the dissected tissues to an antimicrobial solution comprising inhibitory concentrations of the following antimicrobial agents: sodium cefotaxime, vancomycin hydrochloride, netilmicin sulfate, lincomycin hydrochloride, rifampin; and (c) cryopreserving the dissected tissues after step (b) . 21. The method of Claim 20 wherein the antimicrobial agents of the antimicrobial solution are present in the antimicrobial solution in at least the following concentrations in units of micrograms per milliliter: sodium cefotaxime 120; vancomycin hydrochloride 100; netilmicin sulfate 50; lincomycin hydrochloride 120; rifampin 60. 22. The method of Claim 20 wherein the dissected tissues are human heart valves. 23. The method of Claim 20 wherein the antimicrobial regimen comprises exposing the dissected tissues to the antimicrobial solution for at least 4 hours. 24. The method of Claim 20 wherein the antimicrobial regimen comprises the step of maintaining the antimicrobial solution at a temperature between 30° and 40°C. 25. The method of Claim 24 wherein the temperature is about 37°C. 26. A method for cryopreserving tissues comprising the following steps: (a) obtaining a plurality of dissected tissues selected from the group consisting of heart valves, veins and musculoskeletal connective tissue; then (b) subjecting the dissected tissues to at least one antimicrobial regimen effective to render at least some of the dissected tissues substantially free of contamination and to reduce cell viability of the dissected tissues minimally, wherein the at least one antimicrobial regimen comprises the step of exposing the dissected tissues to an antimicrobial solution comprising inhibitory concentrations of at least one antimicrobial agent selected from each of the following families: cephalosporins, glycopeptides, aminoglycosides, lincosamides, and rifamycins; wherein the antimicrobial regimen is effective to substantially eliminate infection by at least the following microbes: streptococci, propionibacteria, and staphylococci; (c) cryopreserving the dissected tissues after step (b) . 27. The method of Claim 26 wherein the dissected tissues are human heart valves. 28. The method of Claim 26 wherein the antimicrobial regimen comprises exposing the dissected tissues to the antimicrobial solution for at least 4 hours. 29. The method of Claim 26 wherein the antimicrobial regimen comprises the step of maintaining the antimicrobial solution at a temperature between 30° and 40°C. 30. The method of Claim 29 wherein the temperature is about 37°c.

Inventors: ANDERSON STEVEN G, BROCKBAND KELVIN G M, COBLE CHERYL A, SCHELL GERALD H, THORNSBERRY CLYDE, ANDERSON, STEVEN, G., BROCKBAND, KELVIN, G., M., COBLE, CHERYL, A., SCHELL, GERALD, H., THORNSBERRY, CLYDE
Applicants: CRYOLIFE INC, CRYOLIFE, INC.
Application year: 1992
Main classification: F25C5
IPC codes: A01N1