WO2017161196A1 - Biological tissue processing apparatus and uses thereof - Google Patents

Abstract

An improved process of cleaning biological tissues containing materials (liquids, blood, proteins, and organic materials) is provided. An improved sterilization process for biological tissues that provides removal of both aerobic (Bacillus) and anaerobic (Clostridium) contaminants or microorganisms is also provided, as well as biological tissues that have a sterility assurance level of 10^-6 for Clostridium.

Description

Biological Tissue Processing Apparatus and Uses Thereof

Background

Prior methods for cleaning and sterilizing biological tissues, such as animal (including human) tissues have been described in the art. However, technical barriers to providing satisfactory "sterility assurance levels" for more resistant contaminating bio organisms apart from Bacillus, especially for aerobic organisms (such as Clostridium) to an acceptable sterility level (such as to at least 10^"6, while at the same time assuring that the biological tissue does not become degraded and/or otherwise changed in a manner that makes the tissue unsuitable for implantation, remain.

US Patent 7,108,832 relates to a sterilization method using carbon dioxide in a "supercritical state" (pressure, temperature) in a reaction chamber for reducing Bacillus and Bacillus spores, describing sterility level as 10^"6 for B. stear other mophilus, B. pumilus and B. subtilus bacteria and spores, in the presence of a chemical sterilization additive (about 0.001% to about 2.0% based on the total volume of the sterilant fluid in the reaction chamber). The sterilization additive was trifluoroacetic acid, acetic acid, peracetic acid, or Sporeclenz™, which is a mixture of peracetic acid, acetic acid and hydrogen peroxide. Mechanical agitation (vibration of the vessel) as part of the sterilization process was described.

"Lyophobic precipitation" technology (the removal of lipids) has been described for the removal of solvent from drug and other particles using an ultrasonic device in the presence of near- supercritical or supercritical temperature and pressure conditions with an anti-solvent gas, such as carbon dioxide, and is described in US Patent 7,744,923. However, the adaptation of sonication with supercritical carbon dioxide for cleaning and processing of biological tissues has not previously been described. In addition, because of the differences between various types of tissues and the biological consequences of temperature, chemical processing materials, processing times, and the combination of these parameters used in conjunction with carbon dioxide induced to a liquid in "supercritical" state, the art remained void of information that would be required to provide consistent and reproducible cleaning and sterilization protocols that could be easily adopted for biological of tissues. Unlike drug particles, biological tissues possess interstitial spaces, where anaerobic infectious microorganisms, such as Clostridium, often reside.

The process described in the current industry of tissue processing has failed to recognize anaerobic microorganisms as a serious contaminant that leads to graft failure and death in patients. FDA standards for processes in tissue sterilization relate to sterility for various Bacillus species as the standard against which sterility assurance level (SAL) is defined. However, growing concern concerning anaerobic microbial contamination, especially from Clostridium, continues to mount as the occurrence of failed graft, infection, and even death, is documented in patients. Thus, the medical arts in the area of tissue processing, remain in serious need for processes that provide tissues free of both aerobic and anaerobic microbial contamination, especially for anaerobic microorganisms such as Clostridium and spores thereof

The present invention provides a solution to these and other medical and health needs in the art.

Disclosure of the Invention

The present invention, in a general and overall sense, provides improved methods for processing biological tissues for use in human implant applications that are sterilized for anaerobic contaminating microorganisms, such as Clostridium, to a sterility assurance level of 10^"6. For this purpose, a challenge device that provides for Clostridium sporogens as a reference microorganism for sterility assurance is used. In some embodiments, the methods include the use of a sterilant additive that comprises hydrogen peroxide, such as SIGMA product 77240 (39 % peracetic acid, 45% acetic acid, 6% H₂0₂₎.

The invention also provides for superior cleaning processes for human biological tissues, the cleaning process providing for superior removal of lipids and other organic and inorganic materials in a tissue sample.

The invention also provides for an improved device for sterilizing human biological tissues.

In some embodiments, the device comprises: a chamber having inlets and outlets, said chamber being suitable for withstanding pressures and temperatures for generating a supercritical liquid form of C0₂ in the presence of an oxidizing reagent within the chamber, said chamber comprising; a first chamber inlet suitable for providing gaseous C0₂ into the chamber; a second chamber inlet suitable for providing a liquid into the chamber; a chamber outlet suitable for decompressing or evacuating fluids from the chamber; a specimen holding component suitable for retaining a solid tissue specimen in a fixed position within said chamber; a component suitable to receive a volume of water; a mechanism suitable for agitating the chamber at a desired speed and intensity level; and a regulatory device suitable for creating a desired temperature and pressure within said chamber for a selected period of time, a valve in communication with said first chamber inlet suitable for attaching to a source of gaseous C0₂; and a computerized regulatory component suitable for input of instructional information to regulate the processing of material in the chamber and the flow of gaseous C0₂ into the chamber.

Brief Description of the Drawings

Figure 1A— Supercritical Sonication. 101 - Pneumatic Ram; 102 - Chamber sealing lid; 103

- Recirculation line for access port; 104 - Electrical automation controls; 105 - Recirculation line for SCC0₂ fluid; 106 - Circulation Pump; 107 - Stainless Steel Reactor; 108 - Inlet; 109 - Outlet; 110— HMI; 111 - Temperature controlled heating strip.

Figure IB— Supercritical Sonication. 101 - Pneumatic Ram; 102— Chamber sealing lid; 103 - Recirculation line for access port; 104 - Electrical automation controls; 105 - Recirculation line for SCC0₂ fluid; 106 - Circulation Pump; 107 - Stainless Steel Reactor; 108 - Inlet; 109 - Outlet; 110 - HMI; 111— Temperature controlled heating strip; 113 - Chamber mount; 114 - Chamber mount.

Figure 1C - 20 Liter vessel, 316 Stainless, 2000 psi, MA amp about 300 O.D. 115 - Outlet for re-circulation line for access port; 116 - Sonication probe; 117 - inlet for re-circulation line for access port; 118— locking flange; 119 - Chamber; 120 - ASME Certified Drawing; 121 - formed flange; 122 - O-Ring groove for proper sealing; 124 - 9/16 MP Port; 102 - chamber sealing lid - comes straight out horizontally; 126 - 63; 127 - Chamber length.

Figure ID - Top view of reaction vessel and cabinet. 128 - width of the chamber; 101— pneumatic ram; 102 - chamber sealing lid; 131— length of cabinet around chamber; 119 - chamber; 133 - length of chamber from right edge of cabinet; 134 - length of the cabinet.

Figure IE - Reaction vessel cabinet build out with dimensions. 128 - width; 134 - length; 135 - height.

Figure 2A - tissue before cleaning processing.

Figure 2B— tissue after C0₂ cleaning for 60 minutes in the presence of water.

Figure 3 A - tissue before cleaning processing.

Figure 3B - tissue after C0₂ cleaning for 90 minutes processing in the presence of water. Detailed Description of the Preferred Embodiments

Although the instant disclosure is described in detail below, it is to be understood that disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the instant disclosure, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they might be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments, which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

The practice of the instant disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA techniques which are explained in the literature in the field (e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations, such as "comprises" and "comprising," will be understood to imply the inclusion of a stated member, integer, or step or group of members, integers, or steps, but not the exclusion of any other member, integer, or step or group of members, integers, or steps. The term "comprise" also encompasses the terms "essentially consisting of and "consisting of unless the context clearly dictates otherwise. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents, unless the context clearly dictates otherwise.

Definitions The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

The following abbreviations are used throughout the application:

BTI— Bone Tendon Interface.

BI - Biological indicator.

SAL - Sterility Assurance Level.

PCD - Process Challenge Device.

HMI - Human Machine Interface

One embodiment of the invention comprises a method for sterilizing a human tissue to a sterility assurance level of at least 10^"6 for an anaerobic microorganism comprising:

(a) cleaning the human tissue by methods other than sonication, for a period of time sufficient to remove debris,

(b) incubating the cleaned human tissue in a volume of saline for a period of time suitable for providing germination of anaerobic microorganisms in the tissue at ambient temperature, to provide a pre-incubated human tissue;

(c) sterilizing the pre-incubated human tissue in the presence of a supercritical C0₂ sterilant fluid, water and a sterilant additive comprising a mixture of peracetic acid, hydrogen peroxide and acetic acid, for a period of from about 2 hours to about 12 hours, in a closed chamber, to provide a sterilized tissue; and

(d) rinsing the sterilized tissue to remove residual sterilant fluid

wherein the sterilized tissue has a sterility assurance level of at least 10^"6 for an anaerobic microorganism.

Another embodiment of the present invention comprises A method for sterilizing a human tissue to a sterility assurance level of at least 10^"6 for anaerobic bacteria comprising: (a) cleaning a harvested human tissue by methods other than mechanical means in the presence of supercritical C(¾ from about 30 minutes to about 360 minutes at ambient temperature;

(b) sterilizing the cleaned tissue in the presence of a supercritical C0₂ sterilant fluid, water and a sterilant additive comprising a mixture of peracetic acid, hydrogen peroxide and acetic acid, for a period of from about 2 hours to about 12 hours, in a closed chamber, while sonicating, to provide a sterilized tissue; and

(c) rinsing the sterilized tissue to remove residual sterilant fluid

wherein the sterilized tissue has a sterility assurance level of at least 10^"6 for an anaerobic microorganism.

A further embodiment of the invention comprises a method for sterilizing a human tissue to a sterility assurance level of at least 10^"6 for anaerobic microorganisms comprising:

(a) cleaning a harvested human tissue by methods other than mechanical means in the presence of supercritical C0₂ from about 30 minutes to about 360 minutes at ambient temperature;

(b) sterilizing the cleaned tissue in the presence of a supercritical C0₂ sterilant fluid, water and a sterilant additive comprising a mixture of peracetic acid, hydrogen peroxide and acetic acid, for a period of from 2 hours to about 12 hours, in a closed chamber, while sonicating, to provide a sterilized tissue; and

(c) rinsing the sterilized tissue to remove residual sterilant fluid ; and

(d) assessing the sterility of the sterilized human tissue obtained in step (c) for Clostridium sporogenes with a Clostridium challenge device, said challenge device comprising a test specimen of the tissue inoculated with Clostridium sporogenes, wherein said sterilized tissue has a sterility assurance level of at least 10^"6 for Clostridium microorganisms.

Another aspect of this invention embodies a device comprising:

a chamber having inlets and outlets, said chamber being suitable for withstanding pressures and temperatures for generating a supercritical liquid form of C(¾ in the presence of an oxidizing reagent within the chamber, said chamber comprising;

(a) a first chamber inlet suitable for providing gaseous C0₂ into the chamber;

a second chamber inlet suitable for providing a liquid into the chamber; a chamber outlet suitable for decompressing or evacuating fluids from the chamber; a specimen holding component suitable for retaining a solid tissue specimen in a fixed position within said chamber;

a component suitable to receive a volume of water;

a mechanism suitable for agitating the chamber at a desired speed and intensity level; and

a regulatory device suitable for creating a desired temperature and pressure within said chamber for a selected period of time,

(b) a valve in communication with said first chamber inlet suitable for attaching to a source of gaseous C0₂; and

(c) a computerized regulatory component suitable for input of instructional information to regulate the processing of material in the chamber and the flow of gaseous C0₂ into the chamber.

The device of this invention is shown in Fig. 1A - 1C and the dimensions of the chamber/stainless steel reactor is shown in Fig. ID. It comprises a chamber 119 within a 20 liter stainless steel reactor 107 that is capable of withstanding pressures of up to at least 2000 psi. This reactor is suitable for withstanding high temperatures and pressures involved in generating supercritical C0₂ (SCC0₂). The processing steps which may include one or more of cleaning, preincubation and sterilization steps, take place in this chamber where the sample may be subject to SCC0₂ and the oxidizing agent. The chamber is sealed at one end by a locking flange 118, whereas the other end is open for inserting and removing samples. During the processing steps, this open end is sealed with the chamber sealing lid 102 capable of horizontal movement in and out of a formed flange 121, and is held in place by a pneumatic ram 101. The chamber sealing lid is equipped with an O-ring for proper sealing of the lid with the formed flange. This chamber is surrounded by a heating strip 111 that controls the temperature inside the chamber. The entire chamber is mounted on chamber mounts 113 and 114.

The chamber has a first chamber inlet 108 for providing gaseous C0₂ into the chamber and an outlet 109 to vent C0₂ from the chamber after the process steps have concluded. The first chamber inlet also has a valve suitable for attaching a source of gaseous C0₂. A computerized regulatory component 110 (HMI) capable of receiving instructional information, and controls capable of directing the inflow and outflow of C0₂, and controlling the temperature (via the heating strip) and pressure within the reactor/chamber for a prolonged period of time are also provided (e.g. electrical automation control 104). A second inlet for providing liquid into the chamber may also be present. The chamber also has a recirculating line 105 which is connected to chamber via an outlet for re-circulation 115 and an inlet for re-circulation 117. This recirculation line is also connected to a circulation pump 106 which compresses the gaseous C0₂ to a supercritical state and maintains the pressure required for the C0₂ to be in supercritical state for the duration of the process.

During one or more of the process steps, the chamber hold the tissue samples in a specimen holding component which is capable of holding a solid tissue sample in a fixed position within the chamber. The chamber also includes a component suitable for holding a certain volume of water. A mechanism suitable for agitating the chamber at desired frequency and intensity level is also present. A sonication probe 116 is also provided to sonicate the samples at frequencies ranging from 20 - 200 kHz.

An oxidizing agent may be used during the cleaning, pre-incubation and sterilization steps. The preferred oxidizing agent for the cleaning and pre-incubation steps is hydrogen peroxide (H₂0₂), whereas the preferred oxidizing agent during the sterilization step is a mixture of peracetic acid, acetic acid and hydrogen peroxide. Such a mixture is available from Sigma Aldrich® under product number 77240 ("Sigma 77240") and comprises 39% peracetic acid, 45% acetic acid and 6% hydrogen peroxide.

The component suitable for holding water is generally a porous substance which may be, but is not limited to an absorbent material, such as a sterile pad of surgical grade gauze or wound dressing material, or other absorbent material

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

In order that the disclosure described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner. Example 1 Tissue Cleaning Procedure

Once a tissue has been harvested from, for example, a human or animal body, it must first be cleaned to remove blood, lipids, fats and other materials. The cleaning procedure must be as complete as possible to enhance the overall suitability of the sample for the sterilization process, and ultimately for use in surgical implantation applications.

The present example presents a comparison of the level of cleaning achieved using a mechanical agitation of the tissue with supercritical carbon dioxide compared to methods other than mechanical means with supercritical carbon dioxide. Such methods may include but are not limited to sonication. While both procedures were useful, the process employing other mechanical means such as sonication resulted in additional removal of fats/lipids, about 10 - 17% by weight more removal of lipids as compared to the use of supercritical C0₂ with mechanical manipulation alone, for a similar amount of processing time.

Care should be taken to avoid the exposure of the tissue specimen to reagents and experimental conditions that could degrade and/or denature the tissue. In addition, the type of tissue being cleaned will dictate the harshness of reagents and mechanical/sonication levels that may be used in the cleaning process so as to avoid damage to the tissue.

For soft tissue, such as amniotic membrane, a cleaning step with a saline solution and gentle rinsing is used. Care should be taken to avoid triton-containing fluids on soft tissue. Soft tissues, especially amniotic membrane tissues, will result in a graft failure when harsher cleaning agents are used.

The tissue type cleaned in the present example was Achilles tendon. The cleaning process was evaluated for a process using supercritical C0₂ in combination with sonication of the tissue specimen in a chamber. A total of 3 runs of 30 minutes, 60 minutes or 90 minutes each, were performed. The weight of each tissue was measured in grams before and after the supercritical C0₂ + sonication cleaning process. Results are stated below in Table 1. The tissue samples examined in Table 1 were processed through a "first cleaning" protocol of normal saline soak, sonication with Triton X-100 / for 10-15 minutes (per-cycle), centrifuge (Program 2: 10 minutes, 4500rpms); normal saline soak (rehydrate until delip (delipidization) cycle); supercritical C0₂ and mechanical agitation for 90 minutes, before undergoing a second cleaning process of supercritical C0₂ and sonication. The results of second cleaning process are shown in Table 1 and show a net reduction in percent change in weight correlating to greater delipidization. Table 1 - Cleaning Process - Results

Run #1 : 30 minute cycle with sonication

Run #2: 60 minute SCC0₂ "run" cycle with sonication

Run #3: 90 minute SCC0₂ "Delip run" cycle with sonication

The standard "run" period for cleaning tissue using SCC0₂ mechanical agitation in saline is 90 minutes with mechanical agitation. This present data shows evidence that a reduced time period of a 60 minute run cycle with SCC0₂ sonication will remove more bioburden from the tissues than a 90 minute run cycle with mechanical agitation. A modified Bone Tendon Bone Rinse protocol was developed based on these results.

Results: Standard Cleaning Protocol provides for the following steps:

Step l : Normal saline soak.

Step 2: Sonicate with Triton X-100 / Centrifuge: Sonicate with Triton X-100 for 10- 15 minutes (per cycle), centrifuge (Program 2: 10 minutes, 4500rpms; normal saline soak (rehydrate until delip). Step 3: Delipidization ("delip") step: (Program 1 : 90 minutes, 95°F, 1436 psi). SCC0₂, mechanical agitation.

Step 4: Process Stop.

Step 5: Process Start.

Step 6: Normal saline soak (soak until thawed, if necessary) (if applicable).

Step 7: Sonicate with Triton X-100 / Centrifuge: Sonicate with Triton X-100 for 10- 15 minutes (per cycle), Centrifuge (Program 2: 10 minutes, 4500rpms); and normal saline rinse.

Step 8: Sonicate with hydrogen peroxide / normal saline soak: Sonicate in ¾0₂ 5- 10 minutes (each cycle), no more than 20 minutes total. Centrifuge (Program 2: 10 minutes, 4500rpms), and normal saline soak.

Step 9: Delipidization ("delip") step: (Programl : 90 minutes, 95°F, 1436 psi). SCC0₂, mechanical agitation.

Step 10: Sonicate / Normal Saline Rinse / Centrifuge: Sonicate for 5 minutes, Centrifuge (Program 2: 10 minutes, 4500rpms).

Step 11 : Sonicate with isopropyl alcohol: 70% IP A; Sonicate: 5 minutes.

Step 12: Sonicate / Normal Saline Rinse: Sonicate: 5 minutes; if alcohol smell or lipids appear, repeat the first rinse; Centrifuge (Program 2: 10 minutes, 4500rpms).

Step 13: Dry to packaging.

The percent (%) change in weight presented above is correlated to a removal of lipids/fats from the tissue specimen. An increase in the amount of lipid removed of from 7 to 10% was achieved using a process that employed both supercritical carbon dioxide and sonication of the sample for 60 minutes.

The level of sonication that may be used with a sample will vary, depending on the type of tissue being processed. For example, lower sonication speeds should be used with delicate tissues, such as amniotic membrane. Tissues with greater structural integrity would be processed for cleaning using a higher sonication setting with supercritical carbon dioxide.

First delipidization ("delip") process:

Cleaning - cleaning a price of tissue requires that fats/lips, as well as other impurities, like blood, and other organic materials, be removed from the tissue sample.

With initial supercritical C0₂ plus mechanical manipulation, the tissue sample had a reduced weight of about 0 - 3 % by weight, indicating that 0 - 3 % of the weight of the sample constituted lipids and other organic material that should be removed from the sample prior to the sterilization process. However, as the data here demonstrates, even further removal of impurities from the tissue sample may be achieved when the tissue sample is processed with supercritical C0₂ while being sonicated.

Development of Modified Bond Tendon Bone Rinse Protocol - the modified and improved tissue cleaning process developed includes the following steps (1-8):

Step 1 : Normal saline soak.

Step 2: Sonicate with Triton X-100 / Centrifuge: Sonicate with Triton X-100 for 10- 15 minutes (per cycle). Centrifuge (Program 2: 10 minutes, 4500rpms; normal saline soak (rehydrate until delip).

Step 3: Delipidization ("delip") step: (Program 1 : 60 minutes, 95°F, 1436 psi). SCC0₂, Sonication.

Step 4: Sonicate with Triton X-100 / Centrifuge: Sonicate with Triton X-100 for 10- 15 minutes (per cycle). Centrifuge (Program 2: 10 minutes, 4500rpms), normal saline rinse.

Step 5: Sonicate with hydrogen peroxide / normal saline soak: Sonicate in H₂0₂, 5-

10 minutes (each cycle), no more than 20 minutes total. Centrifuge (Program 2: 10 minutes, 4500rpms), normal saline soak.

Step 6: Sonicate with isopropyl alcohol (70% IP A): Sonicate for 5 minutes.

Step 7: Sonicate / Normal saline rinse: Sonicate for 5 minutes; if alcohol smell or lipids appear; repeat first rinse. Centrifuge (Program 2 for 10 minutes, 4500rpms).

Step 8: Dry to packaging.

Example 2— CQ2 Sterilization Process with Human Bone-Tendon-Bone

(BTB), Process without Added Water

The present example is provided to demonstrate the effect that the presence or absence of water in the supercritical C0₂ processing chamber has on sterilization capability against Clostridium sporogenes in a tissue packaged in Tyvek.

Bone samples were processed in a method employing sonication and supercritical carbon dioxide machine for 2 hours, 4 hours and 6 hours, using 0.05% SIGMA 77240 as a sterilant additive (SIGMA 77240: 39% peracetic acid, 45% acetic acid, 6% H₂0₂), at a temperature of 95°- 104°F, at 1200 PSI (1280 or 1300 PSI) in an about 20 liter treatment chamber. The time of the run was varied depending on the type of tissue being processed.

The strength of the sonication setting (setting number) will vary depending on the type of tissue being processed. Relatively lower sonication levels are recommended for more delicate tissues, such as amniotic membrane.

All the samples mentioned below were BTI (bone tendon interface). These runs failed without the addition of moisture. A failed run means that the challenge devise with the Clostridium, did not achieve a sterility level (SAL). All tissues were inoculated with Clostridium sporogenes, 1.0 x 10⁶.

Samples:

I Two (2) hours with no water in SCC0₂ and sonication

GEN150015-025 - No Clostridium Growth Observed

GEN150031-022 - Clostridium Growth Observed

II Four (4) hours with no water in SCC0₂ sonication

SDS142235-017 - Clostridium Growth Observed

SDS 142306-013 - No Clostridium Growth Observed

III Six (6) hours with no water in SCC0₂ sonication

SDS 142306-014 - No Clostridium Growth Observed

TDS 143456-023 - No Clostridium Growth Observed.

Results: Partial kill of the Clostridium sporogenes was observed in the C0₂ sterilization run that did not rely on water. These results demonstrate that processing with supercritical C0₂ and sonication will not be effective to sterilize tissue of Clostridium in the absence of water packaged in a Tyvek container.

Example 3 - Location Study

Samples. These samples will resemble an actual load with tissue and PCD's for mapping purposes and a possible worst case load parameter. Location Study (Left to Right in chamber), uniformity to sterilization of tissues throughout the 20 liter treatment chamber.

LETSD05629-015 (PCD)

AOC201008810-001

LETSD 12065-011

SNTE07021-037

SNTS 10204-012

AOC201014766-022

LETSD05629-017 (PCD)

LETSD 11455-011

AOC200918163-008

LETSD11455-010

AOC201007467-013

AOC200917050-006

TDS144619-015 (PCD)

Conditions:

Pressure 1250 psi

Target 36°C

temperature

Target pressure 1250 psi

Processing time 120 min

Water volume 5 mL

Sigma 77240 (39 2.5 mL

% peracetic acid,

45% acetic acid,

6% H₂0₂)

Notes:

Lenox nozzle used to deliver water and SIGMA 77240

Indirect water spray

Indirect SIGMA 77240 spray 15 min. flush conducted

Results: The processed samples were sent to a reference lab to determine microbial growth/presence of Clostridium sporogenes. (Specimen type BI, test code BTI- 14 day). No Clostridium sporogenes growth was detected. LETSD05629-015, (PCD) LETSD05629-017 (PCD), TDS144619-015 (PCD).

LETSD05629-015 - No Clostridium Growth Observed.

LETSD05629-017 - No Clostridium Growth Observed

TDS 144619-015 - No Clostridium Growth Observed.

Example 4 - Sterilization under SCCQ2 without SIGMA 77240,

120 minute sterilization period

All samples were inoculated with Clostridium sporogenes at a concentration of 1.3 x 10⁶ spores as the challenge organism prior to processing through the sterilization conditions identified in Run 1 and Run 2.

After sterilization according to the parameters listed below the sample set, the sterilized samples were sent to a referenced lab to determine the presence of growth of the challenge organism, Clostridium sporogenes.

Run 1 - The present Run 1 provides data related to the sterilization of human bone tendon tissue to determine sterilization effectiveness of the present system under the below defined conditions at a 120 minute sterilization time period under supercritical conditions and while undergoing sonication agitation. No SIGMA 77240 was used.

Samples:

PCD Samples

CTD 15008654-011

LETSD05653-030

Conditions:

Pressure 1250 psi

Target 36°C

temperature

Target pressure 1250 psi Processing time 120 min

Water volume 4 mL

SIGMA 77240, O mL

volume (39 %

peracetic acid,

45% acetic acid,

6% H₂0₂)

Notes

Lenox nozzle used to deliver water

Indirect water spray

No SIGMA 77240 used

No flush conducted

Sample location (front to back)

Results: The processed samples were sent to a reference lab to determine microbial growth/presence of Clostridium sporogenes. (Specimen type BI, test code BTI- 14 day). No Clostridium sporogenes growth was detected in one sample (LETS5653-030). Clostridium was present in (CTD15008654-011) sample.

LETS5653-030 - No Clostridium Growth Observed

CTD15008654-011 - Clostridium Growth Observed

Run 2: Here, SIGMA 77240 was used.

13 samples (3 PCD + 10 filler) were part of the present Run 2.

PCD Samples:

CTD150865-010

GEN150153-011

LETSD05653-028

Conditions:

Pressure 1250 psi

Target 36° C

temperature Target pressure 1250 psi

Processing time 120 min

Water volume 5 mL

SIGMA 77240, 5 mL

volume (39 %

peracetic acid,

45% acetic acid,

6% H₂0₂)

Notes:

Lenox nozzle used to deliver water and SIGMA 77240

Indirect water spray

Indirect SIGMA 77240 spray

15 min. flush conducted

Sample location (left to right)

LETSD05653-028 (PCD)

AOC201008810-001

LETSD12065-011

SNTB07021-037

SNTS10204-012

AOT201014766-022

GEN150153-011 (PCD)

LETSDE11455-011

AOC200918163-008

AOC201007467-0133

AOC200917050-006

LETSD11455-010

CTD150865-010 (PCD)

Results: The processed samples were sent to a reference lab to determine microbial growth/presence of Clostridium sporogenes. (Specimen type Bl, test code BTI- 14 day). CTD 150865-010 No Clostridium Growth Observed

GEN150153-011 No Clostridium Growth Observed

LETSDO5653-028 No Clostridium Growth Observed

The above results demonstrate that uniform sterilization of tissue sample was achieved throughout the chamber under these conditions.

Example 5 - Supercritical Machine

Size: 20 liter, square design or round if possible. The basic structure for the machine/device to be used in the processing of the present invention is provided at Figure 1. The basic functional parameters for which the machine will be constructed for use include the following:

Temperature: minimum = 32°C, maximum 104°F (Fahrenheit).

Pressure: 0 to 2000 psi with pulsation of 1000 psi and 2000 psi, optimal operating pressure at about 1400 psi.

Flow: continuous and uniform circulation of gas flow within the chamber.

Depressurization: slow rate OR fast exhaust regulated.

Sonication: 20 - 200 kHz.

Vacuum: 760 Torr to 70 mTorr.

A sketch of the prototype is provided at Figure 1.

Example 6 - Clostridium Challenge Device

The present example described the preparation of a Clostridium challenge device. This device may be used to assess the sterility level of a processed biological tissue for contamination of

Clostridium to a sterility assurance level of 10^"6.

The present example will describe a Clostridium challenge device that would be used to assess SAL for Clostridium in a Bone-Tendon-Bone (BTB) tissue.

The present example described the preparation of a Clostridium challenge device. This device may be used to assess the sterility level of a processed biological tissue for contamination of

Clostridium to a sterility assurance level of 10^"6.

The present example will describe a Clostridium challenge device that would be used to assess SAL for Clostridium in a Bone-Tendon-Bone (BTB) tissue. Inoculate a representative tissue sample with Clostridium 1.0 x 10⁶ and strategically place it within the sterilization vessel to verify total sterility assurance.

Step 1 Obtain a representative sample that has been cleaned in the same manner as the tissue to be sterilized.

Step 2 Lyophilize the sample to preserve the inoculum.

Step 3 Aseptically inoculate the sample with Clostridium 1.0 x 10⁶.

Step 4 Provide 10 PCD's for testing and 1 for representative control. PCD expiration dates will have to be established.

Step 5 Strategically place 10 PCD's into the vessel with a predetermined number of tissue samples for sterilization and initiate the sterilization cycle.

Example 7 - Pre-incubation Step - Anaerobic Microorganisms

The present example described the use of a pre-incubation step in the processing of tissues. The tissue should be first cleaned, and then subject to the present pre-incubation step prior to being processed through the sterilization step. In this manner, anaerobic microorganisms may be coaxed out of interstitial spaces in tissues, and eradicated. This step will eliminate the possible infection of an implant recipient with the processed tissue with Clostridium and other anaerobic microorganisms. Without the pre-incubation step, tissue is at risk of harboring undetected Clostridium and other anaerobic infectious agents, while still demonstrating a 10^"6 sterility level for conventional, aerobic contaminating microorganisms such as Bacillus.

The present investigators have found that through an independent D-value study, that the most resistant microorganism for human tissue is Clostridium spores. This discovery was quite unexpected from the conventional teachings in the art, which instead considered Bacillus to be the most appropriate challenge microorganism for assessing tissue sterility.

Step 1 : Clean the tissue in accordance with established protocol for Soft Tissue Rinse,

Cortical Bone Rinse or Bone Tendon Bone Rinse.

Step 2: Prepare PCD's for sterility testing.

Step 3: Strategically place 10 PCD's into the vessel with a predetermined number of tissue samples.

Step 4: The length of time required to perform preconditioning is based on the density of the tissue being sterilized. Amniotic Tissue - 1 hour.

Soft Tissue - 1 hour.

Bone Tendon Bone - 2 hour.

Cortical Bone - 2 hours.

Step 5: Prepare the vessel with 25 mL of saline and a predetermined amount of SIGMA 77240 (e.g. 5.0 ml SIGMA 77240).

Step 6: Close the vessel and initiate the warming of the vessel with mechanical agitation. The pre-incubation step for various tissue samples comprises the following steps:

Bone Tendon Bone Rinse

Step 1 : Normal saline soak.

Step 2: Sonicate with Triton X-100 / Centrifuge: Sonicate with Triton X-100 for 10-15 minutes (per cycle), centrifuge (Program 2: 10 minutes, 4500rpms; normal saline soak (rehydrate until delip).

Step 3: Delipidization ("delip") step: (Program 1 : 90 minutes, 95°F, 1436 psi). SCC0₂, mechanical agitation.

Step 4: Process Stop.

Step 5: Process Start.

Step 6: Normal saline soak (soak until thawed, if necessary) (if applicable).

Step 7: Sonicate with Triton X-100 / Centrifuge: Sonicate with Triton X-100 for 10-15 minutes (per cycle), Centrifuge (Program 2: 10 minutes, 4500rpms); and normal saline rinse.

Step 8: Sonicate with hydrogen peroxide / normal saline soak: Sonicate in H₂0₂ 5-10 minutes (each cycle), no more than 20 minutes total. Centrifuge (Program 2: 10 minutes, 4500rpms), and normal saline soak.

Step 9: Delipidization ("delip") step: (Programl : 90 minutes, 95°F, 1436 psi). SCC0₂, mechanical agitation.

Step 10: Sonicate / Normal Saline Rinse / Centrifuge: Sonicate for 5 minutes, Centrifuge (Program 2: 10 minutes, 4500rpms).

Step 11 : Sonicate with isopropyl alcohol: 70% IP A; Sonicate: 5 minutes.

Step 12: Sonicate / Normal Saline Rinse: Sonicate: 5 minutes; if alcohol smell or lipids appear, repeat the first rinse; Centrifuge (Program 2: 10 minutes, 4500rpms).

Step 13: Dry to packaging. Soft Tissue Rinse

Step 1 : Normal Saline Rinse / Soak

Step 2: Delipidization ("delip") step: (Program 1 : 90 minutes, 95°F, 1436 psi). SCC0₂, mechanical agitation.

Step 3: Process Stop.

Step 4: Process Start.

Step 5: Normal saline soak (soak until thawed, if necessary) (if applicable).

Step 6: Normal Saline Rinse (if lipids appear repeat the rinse)

Step 7: Rest to Packaging

Cortical Bone Rinse

Step 1 : Normal saline soak / Centrifuge: Sonicate w/saline for 30-60 minutes (per cycle) Centrifuge (Program 2: 10 minutes, 4500rpms; runs 3 & 4 may not be required.)

Step 2: Sonicate with Triton X-100 / Centrifuge: Sonicate with Triton X-100 for 15-10 minutes (per cycle), centrifuge (Program 2: 10 minutes, 4500rpms.)

Step 3: Delipidization ("delip") step: (Program 1 : 90 minutes, 95°F, 1436 psi). SCC0₂, mechanical agitation.

Step 4: Process Stop.

Step 5: Process Start.

Step 6: Normal saline soak (soak until thawed, if necessary) (if applicable).

Step 7: Sonicate with Triton X-100 / Centrifuge: Sonicate with Triton X-100 for 15-20 minutes (per cycle), Centrifuge (Program 2: 10 minutes, 4500rpms).

Step 8: Sonicate with Hydrogen Peroxide / Normal Saline Soak: Sonicate in H₂0₂ 5-10 minutes (each cycle), no more than 20 minutes total. Centrifuge (Program 2: 10 minutes, 4500rpms), and normal saline soak 5-10 minutes.

Step 9: Delipidization ("delip") step: (Programl : 90 minutes, 95°F, 1436 psi). SCC0₂, mechanical agitation.

Step 10: Sonicate / Normal Saline Rinse / Centrifuge: Sonicate for 5 minutes, Centrifuge (Program 2: 10 minutes, 4500rpms).

Step 11 : Sonicate with isopropyl alcohol: 70% IPA; Sonicate: 5 minutes.

Step 12: Sonicate / Normal Saline Rinse: Sonicate: 5 minutes; if alcohol smell or lipids appear, repeat the first rinse; Centrifuge (Program 2: 10 minutes, 4500rpms).

Step 13: Dry to packaging. Amniotic Membrane

Step 1 : Place the epithelial side of the membrane onto the dissection tray, or down.

Step 2: Use your hand or a gentle blotting with a lab wipe in combination with saline to clean the membrane and remove as much blood and chorion as possible. Do not rub excessively, as it will cause damage to the tissue layers. It may be required to turn the membrane over and rinse with saline to remove blood deposits on the epithelial layer to ensure the tissue is clean.

Step 3; Gently flip the membrane over so that the chorion layer is facing down on the dissection tray.

Step 4; With the chorion layer side down on the dissection tray lay a piece of nitrocellulose paper over the membrane, so that is can attach. When the nitrocellulose has attached to the membrane, creating surface tension.

Step 5; Flip the nitrocellulose paper over exposing the membrane size and place in Tyvek packaging.

Example 8 - Sample Variations of Spray Directly onto the Sample

versus Indirectly into the Chamber

This sampling is intended to determine if the PCD's need to be sprayed with water and SIGMA 77240 in the SSC0₂ sonication chamber to achieve no growth or if the addition of the water and SIGMA 77240 into the vessel while in SCC0₂ sonication will provide the same results of no growth.

1. Samples - 120 min/ Direct Spray 4 ml H₂0/Indirect Spray 2.5 ml SIGMA 77240 PAA/ 1.25 ml per sample

PCD Samples:

NMDS141415-008

NMDS141415-009

Conditions:

Target Temperature 36° C

Target pressure 1250 psi

Process time 120 min

Water volume 4 mL per sample

SIGMA 77240, volume (39 % 2.5 mL / batch (1.25 peracetic acid, 45% acetic acid, 6% mL/sample)

¾0₂ )

Notes:

50 mm Lenox nozzle deliver water and SIGMA 77240

Direct water spay

Indirect spray of SIGMA 77240 (39% peracetic acid, 45% acetic acid, 6% H₂0₂)

15 min system flush was conducted with sonication

Results: Processed samples were sent to a reference lab to determine microbial growth/presence of Clostridium sporogenes. (Specimen type BI, test code BTI-14 day). No Clostridium sporogenes growth was detected.

2. Samples - 120 min/Indirect 4 ml H20/ Indirect 2.5 ml SIGMA 77240/ 4 ml water

PCD Samples:

NMDS140899-007

NMDS140899-008

Conditions:

Target Temperature 36° C

Target pressure 1250 psi

Process time 120 min

Water volume 4 mL per sample

SIGMA 77240, volume (39 5.0 mL / batch (2.5

% peracetic acid, 45% acetic mL/sample)

acid, 6% ¾<¾)

Notes:

50 mm Lenox nozzle deliver water and SIGMA 77240.

Direct water spray.

Indirect SIGMA 77240 spray (39% peracetic acid, 45% acetic acid, 6% H₂0₂)

15 min system flush was conducted with sonication. Results: Processed samples were sent to a reference lab to determine microbial growth/presence of Clostridium sporogenes. (Specimen type BI, test code BTI- 14 day). No Clostridium sporogenes growth was detected.

NMDS1413070-009 - No Clostridium Growth Observed

TDS 150532-008 - No Clostridium Growth Observed

3. Samples - 120 min/Indirect 4 ml H20/Indirect 2.5 ml PAA/1.25 ml SIGMA 77250/

4 ml water per sample

PCD Samples:

TDS144288-010

TDS144288-011

Conditions:

Target Temperature 36° C

Target pressure 1250 psi

Process time 120 min

Water volume 4 mL per sample

SIGMA 77240, volume (39 % 2.5 mL / batch (1.25

peracetic acid, 45% acetic mL/sample)

acid, 6% H₂0₂)

Notes:

50 mm Lenox nozzle deliver water and SIGMA 77240

Direct water spray

Indirect SIGMA 77240 spray (39% peracetic acid, 45% acetic acid, 6% H202)

15 min system flush was conducted with sonication

Results: Process samples were sent to a reference lab to determine microbial growth/presence of Clostridium sporogenes. (Specimen type BI, test code BTI- 14 day). No Clostridium sporogenes growth was detected.

NMDS 140899-007 - No Clostridium Growth Observed

NMDS140899-008 - No Clostridium Growth Observed

4. Samples: 120 minutes/direct 4 ml H20/Indirect 2.5 ml PAA/1.25 ml per sample. PCD Samples:

NMDS1413070-009 TDS150532-008

Conditions:

Target Temperature 36° C

Target pressure 1250 psi

Process time 120 min

Water volume 4 mL per sample

SIGMA 77240, volume 2.5 mL / batch (1.25

(39 % peracetic acid, mL/sample)

45% acetic acid, 6%

¾0₂)

Notes:

50 mm Lenox nozzle deliver water and SIGMA 77240

Direct water spray

Indirect SIGMA 77240 spray (39% peracetic acid, 45% acetic acid, 6% H202)

15 min system flush was conducted with sonication

Results: Process samples were sent to a reference lab to determine microbial growth/presence of Clostridium sporogenes. (Specimen type Bl, test code BTI- 14 days). No Clostridium sporogenes growth was detected.

NMDS1413070-009 - No Clostridium Growth Observed

TDS 150532-008 - No Clostridium Growth Observed

Example 9 -

The present example demonstrates the sterilization of Achilles tendon tissues using a sterilant additive (SIGMA 77240) under supercritical carbon dioxide conditions, where the samples are subject to sonication during the sterilization period of 120 minutes, 180 minutes and 240 minutes. The challenge organism used was Clostridium sporogenes. In all incubation periods, a total kill of Clostridium sporogenes was observed.

1. Samples: - 120 minute incubation

PCD Samples:

TDS 144288-010

TDS144288-011 Conditions:

Pressure 1250 psi

Target Temperature 36'

Process time 120 min

Water volume 4 mL per sample

SIGMA 77240, volume 2.5 mL batch (1.25

(39 % peracetic acid, mL/sample)

45% acetic acid, 6%

H₂0₂)

Notes:

Nozzles installed to deliver water and SIGMA 77240 directly to the pouches

No problems with system

System flush was carried out and 15 min sonication followed.

The sample pouches had moisture visible on interior

The paper clips used to hold sample in place left rust spots on pouches

Wire ties will be used on subsequent runs

Results: Processed samples were sent to a reference laboratory to determine microbial growth/presence of Clostridium sporogenes. (Specimen type BI, test code BTI- 14 day). No Clostridium sporogenes growth was detected.

TD SI 44288-010 - No Clostridium Growth Observed

TDS144288-011 - No Clostridium Growth Observed

2. Samples: Process time 180 minute incubation

PCD Samples:

TDS144288-012

TDS144288-013

Conditions:

Target Temperature 36° C

Target pressure 1250 psi

Process time 180 min

Water volume 4 mL per sample

SIGMA 77240, volume 2.5 mL per sample (39 % peracetic acid,

45% acetic acid, 6%

¾0₂)

Notes:

Nozzles (chamber inlet) to deliver water and SIGMA 77240directly to the pouches containing tissue specimen.

HPLC Pump

Inlet to the HPLC pump to deliver water and SIGMA 77240 to sample. SIGMA 77240, volume (39% peracetic acid, 45% acetic acid, 6% H202)

SIGMA 77240 was not delivered to sample TDS144288-012.

Spraying water and SIGMA 77240 directly on sample might be unnecessary if sample TDS144288-012 is SAL 10^"6 for Clostridium.

System flush was carried out and 15 min sonication followed.

The sample pouches had moisture visible on interior

Wire ties used to hold samples

Results: Processed samples were sent to a reference lab to determine microbial growth/presence of Clostridium sporogenes. (Specimen type BI, test core BTI- 14 day). No Clostridium sporogenes growth was detected.

TDS144288-012 - No Clostridium Growth Observed

TDS144288-013 - No Clostridium Growth Observed

3. Samples: Process time 240 minute incubation

PCD Samples:

TDS144288-014

TDS144288-015

Conditions:

Pressure 1250 psi

Target Temperature 36°

Process time 440 min

Water volume 4 mL per sample

SIGMA 77240, volume 2.5 mL per sample (39 % peracetic acid,

45% acetic acid, 6%

H₂0₂

Notes:

Nozzles installed to deliver water and SIGMA 77240 directly to the pouches

Sonicator #1 and #3 was turned off after 120 min to help drop vessel temperature

System flush was carried out and 15 min sonication followed.

The sample pouches had moisture visible on interior

Wire ties used to hold samples

Results: Processed samples were sent to a reference lab to determine microbial growth/presence of Clostridium sporogenes. (Specimen type BI, test code BTI- 14 day). No Clostridium sporogenes growth was detected.

TDS 144288-014 - No Clostridium Growth Observed

TDS144288-015 -No Clostridium Growth Observed

Bibliography

The following references are specifically incorporated herein by reference:

1. US Patent 7,744,923

2. US Patent 7,108,832 3. Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds.,

Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

4. White et al. (2006), Journal of Biotechnology, 123(4): 504-515.

5. Christopher, Renee (2009), Journal of Orthopedics, 6(2): e9.

Claims

Claims What is claimed is:

1. A method for sterilizing a human tissue to a sterility assurance level of at least 10^{" 6} for an anaerobic microorganism comprising:

(a) cleaning the human tissue by agitating the tissue by other than sonication, for a period of time sufficient to remove debris, to provide a cleaned human tissue;

(b) incubating the cleaned human tissue for a period of time and under an appropriate temperature to permit germination of anaerobic microorganisms;

(c) sterilizing the incubated human tissue in the presence of supercritical C0₂ sterilant fluid, water and a sterilant additive comprising a mixture of peracetic acid, hydrogen peroxide and acetic acid, for a period of from about 2 hours to about 12 hours, in a closed chamber, to provide a sterilized tissue; and

(d) rinsing the sterilized tissue to remove residual sterilant fluid, wherein the sterilized tissue has a sterility assurance level of at least 10^"6 for an anaerobic microorganism.

2. The method of claim 1 wherein the tissue is agitated in the presence of supercritical C0₂ for about 30 minutes to about 360 minutes at ambient temperature.

3. The method of claim 1 wherein the tissue is agitated by sonication.

4. The method of claim 1 wherein the anaerobic microorganism is Clostridium sporogenes.

5. The method of claim 3 further comprising the step of assessing the presence of Clostridium sporogenes in the sterilized tissue.

6. The method of claim 1 wherein the sterilant additive comprises a mixture of peracetic acid, hydrogen peroxide and acetic acid, and the additive is provided at a concentration of about 0.05% by volume of the sterilant fluid.

7. The method of claim 1 wherein the human tissue is a human bone tissue or a human bone-tendon-bone tissue.

8. The method of claim 1 wherein the agitation is mechanical agitation.

9. The method of claim 1 wherein the tissue is sterilized for 6 hours in the presence of supercritical C02.

10. A method for sterilizing a human tissue to a sterility assurance level of at least 10^{" 6} for anaerobic bacteria comprising: (a) cleaning a harvested human tissue by methods other than mechanical means in the presence of supercritical C0₂ from about 30 minutes to about 360 minutes at ambient temperature;

(b) sterilizing the cleaned tissue in the presence of a supercritical C0₂ sterilant fluid, water and a sterilant additive comprising a mixture of peracetic acid, hydrogen peroxide and acetic acid, for a period of from about 2 hours to about 12 hours, in a closed chamber, while sonicating, to provide a sterilized tissue; and

(c) rinsing the sterilized tissue to remove residual sterilant fluid

wherein the sterilized tissue has a sterility assurance level of at least 10^"6 for an anaerobic microorganism.

11. A method for sterilizing a human tissue to a sterility assurance level of at least 10^"aerobic microorganisms comprising:

(a) cleaning a harvested human tissue by methods other than mechanical means in the presence of supercritical C0₂ from about 30 minutes to about 360 minutes at ambient temperature;

(b) sterilizing the cleaned tissue in the presence of a supercritical C0₂ sterilant fluid, water and a sterilant additive comprising a mixture of peracetic acid, hydrogen peroxide and acetic acid, for a period of from 2 hours to about 12 hours, in a closed chamber, while sonicating, to provide a sterilized tissue; and

(c) rinsing the sterilized tissue to remove residual sterilant fluid ; and

(d) assessing the sterility of the sterilized human tissue obtained in step (c) for Clostridium sporogenes with a Clostridium challenge device, said challenge device comprising a test specimen of the tissue inoculated with Clostridium sporogenes, wherein said sterilized tissue has a sterility assurance level of at least 10^"6 for Clostridium microorganisms.

12. The method of claim 11 wherein the human tissue is a bone tendon bone tissue.

13. The method of claim 11 wherein the anaerobic microorganism is Clostridium.

14. A device comprising:

(a) a chamber having inlets and outlets, said chamber being suitable for withstanding pressures and temperatures for generating a supercritical liquid form of C0₂ in the presence of an oxidizing reagent within the chamber, said chamber comprising;

a first chamber inlet suitable for providing gaseous C0₂ into the chamber; a second chamber inlet suitable for providing a liquid into the chamber; a chamber outlet suitable for decompressing or evacuating fluids from the chamber; a specimen holding component suitable for retaining a solid tissue specimen in a fixed position within said chamber; a component suitable to receive a volume of water; a mechanism suitable for agitating the chamber at a desired speed and intensity level; and a regulatory device suitable for creating a desired temperature and pressure within said chamber for a selected period of time,

(b) a valve in communication with said first chamber inlet suitable for attaching to a source of gaseous C0₂; and

(c) a computerized regulatory component suitable for input of instructional information to regulate the processing of material in the chamber and the flow of gaseous C0₂ into the chamber.

15. The device of claim 14 wherein the chamber has a volume of about 20 liters.

16. The device of claim 14 wherein the chamber is non-cylindrical.

17. The device of claim 14 wherein the agitating step comprises sonication.