WO2007025154A1 - Chemosensitivity tester - Google Patents
Chemosensitivity tester Download PDFInfo
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- WO2007025154A1 WO2007025154A1 PCT/US2006/033263 US2006033263W WO2007025154A1 WO 2007025154 A1 WO2007025154 A1 WO 2007025154A1 US 2006033263 W US2006033263 W US 2006033263W WO 2007025154 A1 WO2007025154 A1 WO 2007025154A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/14—Incubators; Climatic chambers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
Definitions
- the invention relates to a chemosensitivity tester used with biological artificial tissue slices, including those derived from any major organ, organ system, cloned tissue using somatic cell transfer, or any other stem cell based regimen, including cord blood, or any manner of neoplasms.
- the chemosensitivity tester may be used to evaluate, detect, and test drug candidates, drugs, and drug metabolites as a method of providing personalized medical treatments.
- the chemosensitivity tester can be used study diseases, such as carcinogenesis, in tissue that has been selected based upon phenotypic analysis, or any other proteomics, genomics, or metabonomic analysis methods, including nano-system biological approaches.
- liver damage is of great concern.
- other organs and systems, and how they react to foreign substances is extremely important.
- Conventional in vivo and in vitro tests utilizing small animals and cell culture techniques are therefore widely used to assess liver function in drug development.
- these conventional tests have particular disadvantages, such as individual variation, high costs to use large animals, and loss of naturally existing characteristics of liver in situ. The same is true for other organs.
- Bioartificial organ devices are currently in development. It is believed that organ function can only be replaced with the biological substrate, that is, for example, liver slices or a whole liver specimen, which requires the availability of liver tissue from xenogenic or human sources.
- the mechanical component of these hybrid devices serves both to remove toxins and to create a barrier between the patient's serum and the biologic component of the liver support device.
- the biologic component of these hybrid liver support devices may consist of liver slices, granulated liver, or hepatocytes from low-grade tumor cells or porcine hepatocytes. These biologic components are housed within chambers often referred to as bioreactors.
- bioreactors are housed within chambers often referred to as bioreactors.
- problems remain with respect to maintaining the functionality of the individual cell lines used in these devices. Most devices use immortalized cell lines. It has been found that over time these cells lose specific functions.
- Circe Biomedical® (Lexington, MA), Vitagen® (La JoIIa, CA), Excorp Medical (Oakdale, MN), and Algenix (Shoreview, MN).
- the Circe Biomedical device integrates viable liver cells with biocompatible membranes into an extracorporeal, bioartificial liver assist system.
- Vitagen's ELAD ® Extracorporeal Liver Assist Device
- Artificial Liver is a two-chambered hollow-fiber cartridge containing a cultured human liver cell line (C3A). The cartridge contains a semipermeable membrane with a characterized molecular weight cutoff.
- This membrane allows for physical compartmentalization of the cultured human cell line and the patient's ultrafiltrate.
- Algenix provides a system in which an external liver support system uses porcine liver cells. Individual porcine hepatocytes pass through a membrane to process the human blood cells.
- Excorp Medical's device contains a hollow fiber membrane (with 10OkDa cutoff) bioreactor that separates the patient's blood from approximately 100 grams of primary porcine hepatocytes that have been harvested from purpose-raised, pathogen-free pigs. Blood passes though a cylinder filled with hollow polymer fibers and a suspension containing billions of pig liver cells. The fibers act as a barrier to prevent proteins and cell byproducts of the pig cells from directly contacting the patient's blood but allow the necessary contact between the cells so that the toxins in the blood can be removed.
- a bioartificial organ for example a liver with improved efficiency, viability, and functionality for use in drug development would be beneficial.
- This longstanding need is addressed by the instant teachings, which provide for drug testing with bio-artificial tissue slices.
- Disclosed is a novel method for testing the chemosensitivity of a substance in a bioartificial organ system or cell culture by treating the bioartificial organ system or cell culture with at least one compound and observing the effect on the bioartificial organ system or cell culture.
- a business method for using the apparatus and methods of the present disclosure to provide for tissue and organ specific screening for patients in complement with cutting edge genomic, proteomic, and metabonomic analysis.
- Disclosed herein is a method for providing simulated in vivo conditions comprising providing a bioreactor for substantially duplicating in vivo tissue function in vitro, providing for the bioreactor to hold at least one aliquot of a tissue sample, and allowing at least one aliquot to generate useful data.
- a method for substantially simulating in vivo conditions comprising, obtaining a bioreactor for substantially duplicating in vivo tissue function in vitro, obtaining a tissue sample, and using at least one aliquot of the tissue sample to generate useful data.
- tissue sample dividing the tissue sample into tissue slices, including the tissue slices as a part of a bioartificial tissue system, and allowing the bioartificial tissue system to be used by treating the tissue slices with at least one drug regimen to generate useful data.
- a similar method comprising obtaining a bioartificial tissue system containing tissue slices, using the bioartificial tissue system by treating the tissue slices with at least one drug regimen to generate useful data, and comparing the data to determine mitotic activity, toxicity of a compound, or histopathology, choosing a drug regimen based on the comparisons of data.
- a business method for chemosensitivity testing which comprises providing a bioreactor-based system for housing tissue slices, populating the bio- reaction based system with tissue, testing a regimen on the tissue, and collecting results.
- Fig. 1 is a schematic diagram of a system of an embodiment of a bioartificial organ system
- Fig. 2 is a perspective view of an embodiment of a complete bioartificial organ system
- Fig. 3 is a perspective view of an embodiment of a bioreactor installed in a bioartificial organ system
- Fig. 4 is a perspective view of an embodiment of a bioreactor unit
- Fig. 5 is an perspective view of an embodiment of a bioreactor unit showing the placement of a tissue slice apparatus
- Fig. 6 is a exploded view of an embodiment of an tissue slice apparatus with containing an tissue slice
- Fig. 7A is a side sectional view of an tissue slice arrangement of an embodiment of a bioartificial organ system
- Fig. 7B is a perspective view of the tissue slice arrangement of Fig. 7A;
- Fig. 8 is a graphical representation of in vitro lidocaine clearance with continuous and intermittent perfusion using a bioartificial organ system
- Fig. 9 is a graphical representation of in vitro lidocaine clearance with a
- Fig. 10 is a graphical representation of in vitro DMX concentration with a 6-hour and a 24-hour run using the bioartificial organ system.
- Fig. 11 is a graphical representation of in vitro ammonia clearance with a 6-hour and a 24-hour run using the bioartificial organ system
- Fig. 12 is a schematic showing the process undertaken by the tissue slice apparatus of the present disclosure.
- Fig. 13 is a flowchart of an embodiment demonstrating a method of using a bioartificial organ system to obtain useful results, according to embodiments of the present disclosure.
- the term "regimen” shall be understood to mean one or more drugs, compounds, therapeutic agents, nucleic acids, peptides, metabolites, viruses, bacteria, or other agents that may be applied to a cell or tissue.
- the present inventor has discovered an improved modular system for circulating plasma about slices of organs from animals to create a system for chemosensitivity testing, inter alia.
- Another object of the present disclosure is to provide an effective method using a platform for testing of the efficacy of compounds prior to actual administration of the compounds to live patients.
- the compound's toxic and pharmacologic effects are realized through in vivo and in vitro animal testing.
- the present disclosure allows for the use of tissues, both human and animal, to be cultured and for the testing of the compound on the tissue.
- the FDA will ask, at a minimum, the new drug applicant to: (1) develop a pharmacologic profile of the drug; (2) determine the acute toxicity of the drug in at least two species of animals; and (3) conduct short-term toxicity studies ranging from 2 weeks to 3 months, depending on the proposed duration and use of the substance in the proposed clinical studies. The process is complicated and costly, with hundreds and sometimes thousands of compounds being tested.
- a further object of the present disclosure is to provide a method upon which a drug regimen, for example, a chemotherapeutic regimen, can be personalized for individual users.
- a drug regimen for example, a chemotherapeutic regimen
- not all regimens work the same way in all patients.
- a drug cocktail that is effective in one patient will be ineffective in a different patient.
- the present disclosure provides a method to test the efficacy of a drug regimen prior to administration to patients, thereby administering only the most effective regimen to each patient.
- a further object of the present disclosure is to provide a research platform and method for the study of disease and the ways in which compounds affect a tissue.
- tissue sample, tissue slice, or tissue aliquot refers to a bioartificial organ system or cell cultures of tissue cells that are not part of a bioartificial organ system.
- a chemosensitivity testing method that uses a bioartificial organ system for evaluation, detection, and testing of drug candidates, drugs and drug metabolites as incorporated by reference.
- the system has an tissue slice culture apparatus.
- Other similar systems that allow for testing of a bioartificial organ are expressly contemplated.
- the methods of the present disclosure may be used with conventional tissue samples.
- the present disclosure provides a method for chemosensitivity testing that provides a way to personalize chemotherapeutic regimens in individual patients, to predict toxicity of a compound in normal tissues, and to study disease.
- a sample of tissue is extracted.
- the sample is sliced into a plurality of tissue slices.
- Various chemotherapeutic regimens are applied to each tissue slice.
- the results are compared to determine the efficacy of each regimen.
- personalized chemotherapeutic regimens may be designed based on the results from the tests.
- Other applications for a chemosensitivity tester are to study and evaluate toxicity, and study and evaluate histopathology. Fig.
- FIG. 1 is a schematic representation of drug testing system 10 in accordance with the present disclosure.
- culture medium 13 is introduced into the bioreactor 15.
- tissue slice apparatus 20 which comprises at least one tissue slice 23 arranged between two wire meshes 21 (see Figs. 7A and 7B) and placed vertically parallel within bioreactor 15.
- the culture medium level begins to rise until it comes into contact with the tissue slices, which allows tissue slices 23 to contact a myriad of compounds that are introduced via the culture medium.
- Oxygenated gas is introduced by gas valve 151 in the top of the chamber.
- gas valve is shown in the top of the chamber, it is also contemplated herein that the gas valve could be on the side or bottom of the chamber, provided with an appropriate seal to prevent leakage of liquid medium.
- the gas is preferably a mixture of 95% O 2 by volume and 5% CO 2 by volume, and is supplied at a pressure ranging from 1 to 10 ATM to the chamber through the gas valve and discharged therefrom, while controlling the pressure by a pressure controller (not shown).
- a solenoid valve (also not shown) may be coupled with the pressure controller to maintain a pre-set gas pressure.
- Gas sterilizing device 18 for example, a syringe filter having a pore size of about 0.22 ⁇ m, is preferably installed in gas valve 151 to filter out microbes, thereby sterilizing the supply gas to the chamber.
- Gas check valve 11 with gas sterilizing device 18 is positioned on the medium reservoir and serves to equalize the pressure between the reservoir and atmosphere. Stabilization of the tissue slices is an important feature of the invention.
- the tissue slices are cultured under the supplies of the culture medium and an oxygenated gas.
- the liquid culture medium, or the plasma is supplied through the reservoir into the chamber and the oxygenated gas is supplied through the top of the chamber.
- Each is supplied at regular intervals so that each of the tissue slices is exposed alternately to the medium and to the gas at an exposure-time ratio ranging from about 1 :1 to about 1:4.
- a ratio of about 1 :2.5 to about 1 :3.5 has been found to be effective, and a ratio of about 1 :1 or 1 :3 has also been found to be effective, although changing these parameters are certainly within the normal skill level of an artisan.
- Pump 19 controls the flow of the culture medium.
- the rate in which a tissue slice is alternately exposed to gas and culture medium corresponds roughly to the rate of metabolism.
- Waymouth MB 752/1 culture medium is preferred over plasma.
- the particular choice of the type of culture medium or plasma will be known to a person of ordinary skill and may vary from cell type to cell type.
- the gas mixture described above is 95% O 2 and 5% CO 2 . Since this mixture may produce free oxygen radicals, which are often toxic to tissue culture cells.
- glutathione and vitamin E as oxygen free radical scavengers and anti-oxidants, are added and supplemented with 10% inactivated fetal bovine serum and L-glutamine.
- Bioartificial organ system 10 comprises one or more bioreactors 15 disposed in incubator 32 that regulates temperature and humidity within the chamber.
- Incubator 32 allows users to regulate the conditions of a bioartificial organ, ensuring that the bioartificial organ is exposed to optimal conditions for viability over time.
- the choice of a suitable incubator system for tissue culture is well known in the art and requires no further recitation.
- Disposed in an incubation chamber are one or more bioreactors 15.
- Each bioreactor 15 holds one or more tissue slices or samples.
- Rotator 30 turns bioreactor 15 for wet and dry phases respectively. The wet phase corresponds to the time that the tissue slice is substantially exposed to culture medium.
- control module 34 provides an interface for controlling the parameters of bioartificial organ system 10 operation. For example, using control modules, the time period that tissue slices are exposed to gas and culture medium may be regulated, which roughly corresponds to metabolism rates. Similarly the gas to culture medium ratios may be regulated using control module 34, as well as other necessary operating parameters.
- gas and culture medium supplies are provided as would be understood by a person of ordinary skill in the art.
- Fig. 3 there is shown a close-up view of a bioreactor 15 installed in incubator 32. For ease of ingress and egress, bioreactor 15 and rotator 30 may be affixed to a platform that slides into and out of incubator 32.
- each bioreactor 15 When installed in incubator 32, each bioreactor 15 is connected to gas and culture medium supplies, 36 and 38 respectively.
- gas valves 151 (one for each tissue slice apparatus chamber 155) connects to gas supply 36.
- manifold system 17 is disposed between gas valves 151 and gas supply 36.
- Gas filter 18, as previously described, is installed between gas supply 36 and gas valves 151.
- culture medium valves 153 are connected to culture medium supply 38.
- Culture medium filter 18, as previously described, is disposed between culture medium supply 38 and culture medium valves 153.
- FIG. 4 there is shown an embodiment of bioreactor 15.
- the embodiment shown in Fig. 4 comprises a plurality of tissue slice apparatus chambers 155 (see Fig. 5).
- Each tissue slice apparatus chamber 155 is formed by a sealed cavity when bioreactor base 157 and bioreactor cover 158 are interconnected.
- Gas valves 151 may are typically connected to gas supply 36 and are in gas communication with tissue slice apparatus chamber 155 for the purpose of providing tissue slices contained in tissue slice apparatus chamber 155 with a supply of a desired gas mixture.
- Culture medium valves 153 are in fluid communication with tissue slice apparatus chamber 155 and serve the same function for culture medium as gas valve 151 does for gas.
- bioreactor cover sealers 159 seal bioreactor cover 158 and bioreactor base 157.
- Bioreactor cover sealer 159 may be an O-ring or other similar device that prevent fluid leakage when bioreactor base 157 and bioreactor cover 158 are in an interconnected configuration, and when inverted the actual choice of device serving as bioreactor cover sealer 159 will be understood and appreciated by a person of ordinary skill in the art.
- each tissue slice apparatus chamber 155 accommodates at least one tissue slice apparatus 20.
- tissue slice apparatus 20 When uninterconnected, tissue slice apparatus 20 may be inserted into a cavity forming a part of tissue slice apparatus chamber 155 in bioreactor base 157.
- a single tissue slice apparatus 20 is inserted into each cavity; a plurality of tissue slice apparatuses 20, however, may be employed in a single bioreactor 15 by providing a plurality of tissue slice apparatus chambers 155 in a bioreactor 15.
- the present disclosure contemplates configurations of bioreactor 15 that comprise various numbers of tissue slice apparatus chambers 155, and various numbers of tissue slice apparatuses 20 per tissue slice apparatus chamber 155.
- bioreactor cover 158 is interconnected with bioreactor base 157. Once interconnected, bioreactor 15 is sealed with bioreactor cover sealer 159. Bioreactor 15 may then be placed into incubator 52 and connected with gas supply 36 and culture medium supply 38 and experiments accordingly conducted.
- tissue slice apparatus 20 may comprise a plurality of meshes 21.
- Meshes 21 may be made of stainless steel or other materials that would be known by a person of ordinary skill in the art, as described previously.
- tissue slices 23 are placed between adjacent meshes 21 and meshes 21 are clasped together with tissue slice apparatus clips 24. It will be understood by artisans that size and thickness of tissues slices may need be optimized for each protocol and may vary from experiment to experiment and tissue to tissue.
- tissue slice apparatus 23 two meshes 21 form tissue slice apparatus 23.
- the tissue slices 23 are disposed in the lower 40% of tissue slice apparatus 20 according to the exemplary embodiment. This configuration of tissue slices 23 in tissue slice apparatus 20 ensures that the tissue slice is fully exposed to both the dry and wet cycles.
- Figs. 7A and 7B show similar embodiments of tissue slice apparatus 20.
- Two stainless steel meshes 21 the size of which can be chosen based on the dimensions of the chamber. These two meshes are preferably arranged in parallel. In an embodiment, the meshes have about a 0.26 mm pore size. Also, in an embodiment, the meshes are pressed to ensure consistent flatness. Between meshes 21 is a plurality of tissue slices 23, such as liver slices arranged in an orderly fashion. The two meshes are positioned on each side of the tissue slices with enough room so as to not crush the tissue slices, but also to hold them sufficiently so that they do not get washed away by the culture medium.
- tissue slices 23 used in the present disclosure may be obtained from a suitable animal, for example, a rabbit, pig, dog, rodent, or human, depending on the intended use of the apparatus.
- Tissue slices 23 may be of any size or shape suitable for maintaining the viability and essential functions thereof.
- thickness of tissue slices 23 shown to be effective have a thickness ranging from about 10 ⁇ m to about 2,000 ⁇ m.
- a thickness is from about 100 ⁇ m to about 500 ⁇ m has been determined to effective in particular experiements.
- the present disclosure is ideally suited to methods of testing the toxicity and efficiency of a drug.
- the testing is accomplished by exposing tissue slices to a drug or drug candidate and observing the ability of the tissues, such as liver, to metabolize a compound, which compound or its metabolites can be detected.
- a compound which compound or its metabolites can be detected.
- ammonia and lidocaine are common compounds that can be metabolized by healthy liver.
- the following examples show this testing, as applied to liver-slices. Those skilled in the art will recognize the utility of the present disclosure as applied to other organs.
- At least one compound is applied to at least one tissue aliquot in the bioartificial organ system. After a predetermined time elapses, data is gathered. In each experiment, the conditions may be duplicated.
- the data are compared. Comparison of the data provides for various utilities of the present disclosure, including, for example, detection of mitotic activity, cyto-toxicity parameters, and histopathology. Once derived from the data, these results are useful in formulation of the most effective chemotherapeutic regimen for a patient, for general prediction of the toxicity of a given compound or compounds, or for study of carcinogenesis, for example. Naturally, other inferences may be derived from the data, and variations in the design of the experiments using the tissue slices or aliquots allow for variations in the information derived.
- Figs. 8-11 demonstrate the utility of the principles and apparatuses disclosed herein using liver slices as the tissue sample.
- the data presented in Figs. 8 and 9 demonstrates the ability of the bioartificial organ system to clear lidocaine over time.
- Fig. 10 shows the increase in concentration being managed over time, which substantially simulates in vivo physiology of samples.
- Fig. 11 demonstrates the ability of the teachings of the present disclosure to detoxify ammonia.
- the data presented in Figs. 8-11 are not intended to be limiting or to demonstrate the actual results the teachings of the present disclosure will achieve, but merely to demonstrate the achieved utility of the teachings of the present disclosure. It is intended that various configurations will accomplish similar results from configuration to configuration that are not exactly duplicative of the data presented herein.
- bioreactor 15 is loaded with tissue slices 23 and sealed.
- Bioreactor 15 may be identical to embodiments disclosed herein or other apparatuses with similar functionality.
- Bioreactor is connected to at least one culture medium reservoir 12 which contains a supply of culture medium.
- bioreactor comprises a plurality of tissue slice apparatus chambers 155
- different culture medium reservoirs may be used to supply culture medium depending on the specific goals of the experiment sought.
- bioreactor 15 comprises 6 tissue slice apparatus chambers 155 (see, e.g., Fig. 5).
- the first chamber may be used as a control wherein culture medium is supplied with no additives.
- the other 5 chambers may be supplied with culture medium containing a compound to be tested on the tissue slices 23 such as lidocaine or ammonia in various concentrations.
- tissue slice apparatus chambers 155 may be supplied with the exact same compounds in order to have multiple sets of results or to prevent fouling of a particular tissue slice apparatus chamber 155 from preventing retrieval of results.
- the exact experimental design and protocol are configurable in many variations as would be known and understood by a person of ordinary skill in the art.
- filter 18 may be disposed between culture medium reservoir 12 and bioreactor 15.
- Bioreactor 15 is also connected to a gas supply 40.
- Gas supply may supply gasses in various combinations and concentrations according to experimental designs and protocols.
- a single gas supply 40 may be connected to all tissue slice apparatus chambers 155. Nevertheless, a plurality of gas supplies 40 may be used if desired and called for by experimental design or protocol.
- filter 18 may be disposed between gas supply 40 and bioreactor 15. After culture medium and gas is supplied to each tissue slice apparatus chamber 155 bioreactor 15 is incubated for a given period of time. During incubation, tissue slices are alternately exposed to culture medium and gas. This may be accomplished in multiple ways.
- culture medium and gas may be injected and recovered alternately so that either gas or culture medium is in tissue slice apparatus chamber 155 at any one given time.
- bioreactor 15 may be rotated so that tissue samples are alternately exposed to culture medium and gas, which are both held in tissue slice apparatus chamber 155. This may be accomplished by ensuring that tissue samples 23 occupy only a certain volume of tissue slice apparatus chamber 155 so that it is fully submerged in culture medium in one configuration, but upon rotation is fully exposed to gas.
- Fig. 6 demonstrates an embodiment reflecting this idea, wherein tissue sample 23 occupies 40% of tissue slice apparatus 20. Other variations on this idea will be understood by a person of ordinary skill in the art.
- drain pump 60 may remove an aliquot of culture medium. Once removed, culture medium may have any gas extracted at the same time captured in bubble trap 70.
- the culture medium aliquot is held and tested in a processed culture medium reservoir 50. Once tested, it may be returned to bioreactor 15 or discarded. Filters 18 disposed within the system maintain sterility.
- FIG. 13 illustrates a method of testing chemosensitivity of various compounds and substances.
- a tissue sample is extracted during surgery.
- Tissue may be human or animal depending on the desired use of the tissue.
- tissue is removed from the patient for whom the regimen is to be created.
- cancerous tissue may be removed and exposed to a variety of cancer fighting drug cocktails to determine the best cocktail for the particular cancer tested.
- tissue from any suitable human or animal host may be used.
- animal tissue may be used initially. After a compound is deemed safe in animal studies, human tissue sample may then used to further test toxicity of the compound or substance.
- Tissue slices may be obtained incidental to other surgeries or in procedures designed specifically to obtain the tissue sample, for example a biopsy.
- tissue For personalized chemotherapeutic regimens, tissue must be obtained from the patient, necessitating taking a tissue sample directly from the patient either during a non-related surgery or during an operation specifically designed to obtain a tissue sample.
- Other chemosensitivity applications may use other sources of tissue samples depending on the particular protocol.
- tissue samples are used to obtain results when various compounds are tested on them. Such results, including personalized medicine related matters, are within the normal skill level of artisans and likewise may be found in at least one of U.S. Letters Patents Nos. 6,678,669; 6,905,816; 6,983,227 and 6,999,607 each of which are expressly incorporated herein by reference as if fully set forth herein.
- the tissue sample is sliced or otherwise divided into at least one aliquot of tissue.
- each slice or aliquot is then individually cultured in tissue slice apparatus chamber 155 and bioreactor 15 as previously described, such as using the method shown in Fig. 12.
- the use of multiple, duplicative tissue slices allows researchers and doctors to expose the same tissue slices to compounds at the organ level where the only variable at play is the regimen administered.
- the present disclosure provides a powerful tool for evaluation the efficacy of each given regimen compared to other viable regimens. Moreover, because the present system and methods are designed to allow testing at the organ, system, and in some cases, organism level, as described in the examples below. Thus, using the methods and apparatus of the present disclosure, researchers and doctors have a powerful tool to evaluate mitotic activity, cyto-toxicity parameters, histopathology, and many other related applications at the organ, system, and organism level.
- a system or organism can be recreated in vitro using the instant techniques, but provide results that are indicative of in vivo processes by using tissue slices from a variety of tissues in a system and connecting them in parallel. Connection occurs via transfer of culture medium from one tissue type to the next, which allows researchers to observe the stepwise effects of a regimen on various organ samples.
- tissue slices or different tissue types from tissue slices may be combined in single tissue slice apparatus 20 in various permutations. Experiments of this type would allow researchers to control for tissue type in an in vivo system in an in vitro environment for study and therapeutic applications. The many variations in the use of the apparatuses and methods of the present disclosure in the observation of the effects and efficacy of regimens will be understood by a person of ordinary skill in the art. Similarly, the various methods of controlling variables in a in vivo system will appeal to artisans as a powerful means of obtaining results that would otherwise be impossible short of human or animal experiments. For example, according to an embodiment, a plurality of animal liver slices positioned securely within bioreactor 15 so as to maximize the surface area of the liver slices exposed to a culture medium.
- the tissue slice apparatus chamber is preferably thermoregulated.
- the temperature is preferably kept at about 36.5 degrees C.
- rodent tissue slices it is kept between about 36 to 38 degrees C.
- pig tissue slices are very sensitive to temperature fluctuation and it must be maintained at 38 degrees C, the normal body temperature of pigs.
- a doctor may use the teachings of the present disclosure to determine the optimum concentrations of a chemotherapy drug cocktail for a cancer patient.
- the doctor takes a biopsy of a cancerous tissue from the patient and divides the tissue sample into aliquots.
- the doctor divides aliquots into two groups. The doctor uses the first group to determine the most effective drug cocktail for the patient in question. The doctor then uses the second group of aliquots to determine the optimal concentration of the drug cocktail.
- the doctor uses the group to determine the most effective drug cocktail.
- Tissue aliquots are cultured as described previously.
- Various drug cocktails are administered to the tissue aliquot.
- the percent of apoptosis of the cancerous tissue in the aliquots is measured by methods that would be common to a person of ordinary skill in the art.
- the doctor selects the drug cocktail inducing the greatest degree of apoptosis in cancerous tissue compared to the healthy tissue.
- the doctor uses the second group of tissue aliquots to determine the optimal concentration of the drug cocktail to use on the cancerous tissues.
- Tissue aliquots are cultured in the same manner as the first group of tissue aliquots.
- the doctor applies various concentrations of the drug cocktail to each tissue aliquot and selects the concentration of the drug cocktail that imparts the greatest degree of apoptosis in cancerous tissue as compared to healthy tissue.
- the patient will receive the treatment that imparts the greatest benefit while minimizing undesirable side effects. If the doctor wishes, the same result may be obtained in a single experiment where various concentrations of a plurality of drug cocktails is applied to a plurality of tissue aliquots.
- the teachings of the present disclosure may be used to predict the toxicity of compounds to healthy tissue. Such results would be useful in data generated and submitted to the FDA pursuant to approval of a new drug application or abbreviated new drug application.
- An animal or human tissue sample is obtained by taking a biopsy or as part of surgery. Usually two species of animals, one rodent and one non-rodent are used because a drug may affect one species differently than another. Other organs likewise provide key data and are useful within the scope of the present disclosure.
- the tissue is taken as part from a deceased organ donor, cloned, regenerated or otherwise supplied by techniques known to those skilled in the art ranging from cord blood to stem cells by somatic cell transfer, among other things.
- Tissue aliquots are derived from the tissue sample and cultured as previously described. Once the tissue is cultured, a compound is applied to each tissue aliquot to determine the efficacy of the compound to achieve a desired result as described previously. Data is gathered and interpreted as prescribed by the FDA or as according to parameters set by a person of ordinary skill in the art in the determination of a compound's efficacy in a tissue.
- disease studies may be performed by using various compounds in the study of a disease.
- inhibitors and stimulators of compounds in the tissues may be used to study their effects on chemical pathways in the tissue.
- compounds may be applied to the tissue in an effort to observe their effects on the tissue level as opposed to the cellular level or organism level.
- the present disclosure provides the methods to use a bioartificial organ system; experimental parameters would be readily apparent to a person of ordinary skill in the art without the need for undue experimentation.
- the testing may be performed by an independent third party in order to rule out any appearance of bias. Every effort is made to ensure that as few animals as possible are used as a source of tissue samples, and that they are treated humanely. Alternately, the present disclosure also contemplates the use of human tissues, samples of which may be obtained from organ donors. Since most drugs are metabolized in the liver, toxicity studies naturally focus on the effects on the liver.
- Tissue slices may be obtained from a variety of organs. These then are placed in parallel into multiple bioreactor tissue slice apparatus chambers. Culture medium is applied to a first chamber and permitted to come into contact with the tissue slice for a time period. Following the initial time period, the culture medium is removed from the first tissue slice apparatus chamber and moved into a second tissue slice apparatus chamber containing a tissue sample from a different organ or the same organ under different experimental conditions, such as increased metabolism, a different primary cell type, or a different concentration of cell type of interest. Prior to or concurrently with moving the culture medium to the second tissue slice apparatus chamber, samples of the culture medium may be obtained to interim testing, in embodiments. The culture medium moved into the second tissue slice apparatus chamber is then reacted for a time period. The procedure is repeated for each tissue slice apparatus chamber until the experiment is concluded.
- samples of tissue may therefore be taken from inside the mouth, esophagus, stomach, small intestine sections corresponding to the duodenum, jejunum, and ileum, and the large intestine.
- a sample of culture medium with a protein sample may be therefore reacted with each disparate tissue type to determine the effect of a particular organ on the proteins to be digested on a system level.
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Abstract
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2006282873A AU2006282873A1 (en) | 2005-08-24 | 2006-08-24 | Chemosensitivity tester |
CA002620837A CA2620837A1 (en) | 2005-08-24 | 2006-08-24 | Chemosensitivity tester |
EP06802350A EP1931988A4 (en) | 2005-08-24 | 2006-08-24 | Chemosensitivity tester |
JP2008528204A JP2009505662A (en) | 2005-08-24 | 2006-08-24 | Chemotherapy sensitivity tester |
IL189672A IL189672A0 (en) | 2005-08-24 | 2008-02-21 | Chemosensitivity tester |
Applications Claiming Priority (12)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/210,511 US7829325B2 (en) | 2003-12-16 | 2005-08-24 | Drug testing with bio-artificial organ slices including for example those derived from liver |
US11/210,511 | 2005-08-24 | ||
US71296405P | 2005-08-30 | 2005-08-30 | |
US60/712,964 | 2005-08-30 | ||
US78202906P | 2006-03-13 | 2006-03-13 | |
US60/782,029 | 2006-03-13 | ||
US78530806P | 2006-03-23 | 2006-03-23 | |
US60/785,308 | 2006-03-23 | ||
US79196606P | 2006-04-14 | 2006-04-14 | |
US60/791,966 | 2006-04-14 | ||
US11/466,730 | 2006-08-23 | ||
US11/466,730 US20070048732A1 (en) | 2005-08-30 | 2006-08-23 | Chemosensitivity tester |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2007025154A1 true WO2007025154A1 (en) | 2007-03-01 |
Family
ID=37771946
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2006/033263 WO2007025154A1 (en) | 2005-08-24 | 2006-08-24 | Chemosensitivity tester |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1931988A4 (en) |
AU (1) | AU2006282873A1 (en) |
CA (1) | CA2620837A1 (en) |
IL (1) | IL189672A0 (en) |
WO (1) | WO2007025154A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113416629A (en) * | 2021-06-03 | 2021-09-21 | 中南大学湘雅三医院 | Tumor chemotherapy drug sensitivity detection kit and detection method |
Citations (4)
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---|---|---|---|---|
JPH08163996A (en) * | 1994-12-13 | 1996-06-25 | Sumitomo Bakelite Co Ltd | Testing method of toxicity of animal tissue |
US5976870A (en) * | 1994-11-09 | 1999-11-02 | Park; Sung-Su | Artificial liver composed of a liver-slice culture apparatus |
US20040265997A1 (en) * | 2003-06-27 | 2004-12-30 | Park Sung-Soo | Bio-artificial liver system |
WO2005061694A1 (en) * | 2003-12-16 | 2005-07-07 | Hepahope, Inc. | Drug testing system with bio-artificial liver |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4308351A (en) * | 1980-04-18 | 1981-12-29 | Joseph Leighton | System for growing tissue cultures |
US6372495B1 (en) * | 1995-10-06 | 2002-04-16 | Seed Capital Investments-2 (Sci-2) B.V. | Bio-artificial organ containing a matrix having hollow fibers for supplying gaseous oxygen |
US5827729A (en) * | 1996-04-23 | 1998-10-27 | Advanced Tissue Sciences | Diffusion gradient bioreactor and extracorporeal liver device using a three-dimensional liver tissue |
-
2006
- 2006-08-24 EP EP06802350A patent/EP1931988A4/en not_active Withdrawn
- 2006-08-24 AU AU2006282873A patent/AU2006282873A1/en not_active Abandoned
- 2006-08-24 WO PCT/US2006/033263 patent/WO2007025154A1/en active Application Filing
- 2006-08-24 CA CA002620837A patent/CA2620837A1/en not_active Abandoned
-
2008
- 2008-02-21 IL IL189672A patent/IL189672A0/en unknown
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5976870A (en) * | 1994-11-09 | 1999-11-02 | Park; Sung-Su | Artificial liver composed of a liver-slice culture apparatus |
JPH08163996A (en) * | 1994-12-13 | 1996-06-25 | Sumitomo Bakelite Co Ltd | Testing method of toxicity of animal tissue |
US20040265997A1 (en) * | 2003-06-27 | 2004-12-30 | Park Sung-Soo | Bio-artificial liver system |
WO2005061694A1 (en) * | 2003-12-16 | 2005-07-07 | Hepahope, Inc. | Drug testing system with bio-artificial liver |
Non-Patent Citations (1)
Title |
---|
See also references of EP1931988A4 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113416629A (en) * | 2021-06-03 | 2021-09-21 | 中南大学湘雅三医院 | Tumor chemotherapy drug sensitivity detection kit and detection method |
CN113416629B (en) * | 2021-06-03 | 2023-09-22 | 中南大学湘雅三医院 | Tumor chemotherapeutic drug sensitivity detection kit and detection method |
Also Published As
Publication number | Publication date |
---|---|
EP1931988A1 (en) | 2008-06-18 |
EP1931988A4 (en) | 2009-01-28 |
CA2620837A1 (en) | 2007-03-01 |
AU2006282873A1 (en) | 2007-03-01 |
IL189672A0 (en) | 2008-06-05 |
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