KR20080056163A - Novel enhanced processes for drug testing and screening using human tissue - Google Patents

Abstract

A novel method for testing human tissue in a testing system is more effective than conventional cell culture systems and functions by treating the human tissue slice system's samples with at least one compound and observing the effect on the human tissue slices resident therein, or cells, tissue samples or other derivatives from the testing process.

Description

NOVEL ENHANCED PROCESSES FOR DRUG TESTING AND SCREENING USING HUMAN TISSUE

The present invention is directed to biological, including umbilical cord blood, all forms of neoplasm, specifically those derived from all major organs, organ systems, tissues cloned using somatic metastasis, or all stem cell based therapies, including human tissue. A test system using tissue flakes. The present tester can be used to evaluate, detect, and test drug candidates, drugs, drug metabolites, as a method of providing personalized medical treatment. Ultimately, such testers may be used to study diseases such as carcinogenesis in selected tissues based on phenotypic assays or any other proteomic, genomic or metabolic assays, including nano-system biological approaches.

Current therapies have identified two major deficiencies with regard to pre-sale and post-sale trials. As an example, the complete defeat of VIOXX® has clearly shown that if some of the candidate populations have potential for side effects, efforts are needed to select candidate substances for treating specific disease states. Using currently available genomic and proteomic assays, high-risk patients and patient populations can select tissue before administering these potentially hazardous, pathologically toxic compounds.

Likewise, increasing costs have influenced this calculation, highlighting and highlighting the need pointed out herein. This was made too clear by the record number studies identified here.

According to a study by the Tufts Center for Drug Development Research, the average cost of drug development in 2001 exceeded $ 800 million. According to the study, about $ 16 million was spent for each pharmaceutical company for preclinical studies. Therefore, the reduction of test time and cost in drug development is a decisive factor for the survival of most pharmaceutical companies. In addition, there is usually more than one competitor on the same New Testament battlefield, requiring all competitive advantages. Most of the cost of drug development comes from the FDA certification process. However, most of these costs are difficult to cover in the same way as preclinical costs. In order to address soaring preclinical costs, in vivo and ex vivo testing and selection of effective, costly and timely potential new drug candidates has become a significant area of concern in the art.

In the development of new drugs, toxicity has always been an important consideration. Since the liver metabolizes most drugs, liver damage is of great concern. Similarly, it is important whether other organs and organ systems and how they react to adventitious materials. Conventional in vivo and ex vivo assays using small animal and cell culture techniques are therefore used to evaluate drug function development time. However, these conventional test methods have certain disadvantages such as individual variation, high cost when using large animals, and loss of the original characteristics of the liver itself. The same is true for other institutions. As our knowledge of how these different organs and mammals respond to various prodrugs, drugs, compounds and systems has increased, the relative importance of the present invention has become more and more important.

To overcome this disadvantage, cell culture systems were used. However, such cell to cell linkage interactions cannot be maintained for a desired length of time. Once cell-to-cell connectivity is lost, the test will soon fail because it can no longer control organ, organ system, or organism-level responses.

Artificial organ devices are currently under development. It is known that the function of a living organ can only be replaced by a liver lamella or an entire liver sample that requires the effectiveness of a biological substrate, eg, a heterologous or human-derived liver tissue. Recently, efforts have been made to combine mechanical and biological support systems within a hybrid liver support device. The mechanical components of such hybrid devices are used to remove toxins and form a barrier between the serum of the patient and the biological components of the liver support device. The biological components of such hybrid liver support devices may consist of liver cells derived from liver flakes, granulated liver, or benign tumor cells or liver cells of porcine pigs. Such biological components are contained within compartments often referred to as bioreactors. However, there remains a problem regarding maintaining the functionality of the individual cell lines used in such devices. Most devices use an infinite cell line. Over time, they found that these cells lost certain functions.

Several types of intercellular devices under development, such as Circe Biomedical®, Lexington, Mass., Vitagen®, La Jolla, Calif., Excorp Medical, Oakdale, MN), and Algenix (Algenix, Shoreview, MN). The Kirke Biomedical Device integrates viability liver cells with biocompatible membranes into in vitro and artificial liver support systems. Vitagen ELAD® (In Vitro Liver Assist Device) Artificial liver is a hollow-fiber cartridge with two compartments containing cultured human liver cell lines (C3A). This cartridge contains a semipermeable membrane that blocks certain molecular weights. This membrane allows for physical isolation of cultured human cell lines and patients' ultrafiltrations. Algenix provides a system in which the external liver support system uses porcine liver cells. Individual porcine liver cells pass through the membrane to process human blood cells. Exco Medical's device includes a hollow fiber membrane (10 kDa cutoff) bioreactor that separates the patient's blood from about 100 g of primary pig liver cells collected from intentionally bred, pathogen-free pigs. Blood passes through a cylinder filled with a suspension containing hollow polymer fibers and hundreds of millions of pig liver cells. The fiber acts as a barrier to allow the toxins in the blood to be removed by allowing essential contact between the cells while blocking the protein and cell by-products of the pig cells from directly contacting the blood of the patient.

Various features of these devices show advances in existing technology, but they still have certain drawbacks. The effectiveness of all these devices using individual liver cells is limited due to the lack of cell-to-cell interactions that characterize liver conditions in vivo. Thus, livers having improved efficacy, bioactivity, and functionality when used in developing artificial organs, such as new drugs, are advantageous. This long aspiration has been solved by the present invention in which drug testing is performed on artificial tissue flakes.

As the technology of artificial organ systems has evolved, so has compound compounds. The present application discloses a method of utilizing recent advances in an artificial organ system, and particularly a method applied to advanced procedures of human tissue.

Summary of the Invention

Treating the artificial organ system or cell culture with at least one compound, and observing the effect on the artificial organ system or cell culture, and the human tissue within the artificial organ system, cell culture, and human tissue itself. A new method of testing is disclosed. In addition, those of ordinary skill in the art will be able to supplement commercial genomics, proteomics, and metabolic assays to provide commercial methods of the present invention that provide tissue and organ specific selection for patients using instruments and methods. It can be easily understood that it is disclosed as.

This specification provides a bioreactor that substantially replicates tissue functions in vivo in vitro, immobilizing at least one aliquot of a tissue sample in the bioreactor, and data available as at least one aliquot. It provides a method for providing simulated in vivo conditions comprising the step of generating.

Likewise, obtaining a bioreactor that substantially replicates tissue function in vivo in vitro, obtaining a tissue sample, and generating usable data using at least one aliquot of the tissue sample A method of substantially simulating in vivo conditions is disclosed.

In addition, treating the tissue flakes with at least one drug regimen using a step of providing a human tissue sample, including the tissue flakes as part of the artificial tissue system, dividing the tissue sample into the tissue flakes, and using the artificial tissue system. A method of substantially simulating in vivo conditions is then disclosed that includes generating usable data.

The present disclosure provides a method for obtaining an artificial biological tissue system containing human tissue flakes, generating data available by treating the tissue flakes with at least one drug therapy using the artificial tissue system, and mitotic activity, A similar method is disclosed that includes comparing data to determine toxicity, or histopathology, and selecting drug therapy based on the comparison of the data.

Finally, providing a bioreactor-based system for storing human tissue flakes, anchoring human tissue to the bio-reaction based system, testing the therapy on human tissue, and collecting the results Disclosed is a commercial method for testing human tissue comprising.

BRIEF DESCRIPTION OF THE DRAWINGS The above-mentioned features and objects of the present invention will become more apparent with reference to the following detailed description given in conjunction with the accompanying drawings, wherein like reference numerals denote like elements, wherein:

1 is a schematic diagram of a system in an embodiment of an artificial living organ system;

2 is a perspective view of an embodiment of a human tissue and artificial organ system in accordance with the present invention;

3 is a perspective view of an embodiment of a bioreactor installed in human tissue and artificial organ system according to the present invention;

4 is a perspective view of an embodiment of the system of FIGS. 1-3;

5 is a perspective view of an embodiment of the present system showing the location of a human tissue lamella device;

6 is an exploded view of an embodiment of a tissue flake device containing human tissue flakes;

FIG. 7A is a side cross-sectional view of a tissue flake alignment state in an embodiment of human tissue and artificial organ system;

FIG. 7B is a perspective view of the human tissue lamella alignment state of FIG. 7A; FIG.

8 is a graph of in vitro lidocaine removal by continuous and intermittent perfusion using an artificial living organ system;

FIG. 9 is a graph of in vitro lidocaine removal according to 6 and 24 hour drive using an artificial organ system;

FIG. 10 is a graph of in vitro DMX concentrations at 6 and 24 hour drive using an artificial organ system;

FIG. 11 is a graph of ex vivo ammonia removal over 6 and 24 hour runs using an artificial living organ system; FIG.

12 is a schematic diagram of a method performed by a human tissue lamella device of the present invention;

13 is a flowchart of an embodiment showing how to use human tissue and an artificial organ system to obtain useful results, in accordance with an embodiment of the present invention.

As used herein, the term "therapy" is understood to mean one or more drugs, compounds, therapeutic agents, nucleic acids, peptides, metabolites, viruses, bacteria, or other agents that can be applied to cells or tissues. shall.

The present invention discloses, in particular, an advanced modular system for circulating plasma around slices of animal organs to form a system for testing human tissue.

It is another object of the present invention to provide an effective method of using a platform for testing compound efficacy prior to the actual administration of the compound to survive the patient. Toxicity and pharmaceutical effects of the compounds are realized through in vivo and ex vivo animal testing. However, the present invention allows the use of tissues of both humans and animals to be cultured, and the compounds are tested for that tissue. For new drug compounds, FDA will, to the applicant of the new drug, at least: (1) develop the pharmaceutical properties of the drug; (2) measure acute toxicity of the drug in at least two animals; And (3) to conduct short-term toxicity studies in the range of two weeks to three months, depending on the duration and use of the substance proposed in the proposed clinical study. This procedure is complex and expensive because of the need to test hundreds and sometimes thousands of compounds.

Another object of the present invention is to provide a method by which drug therapy, for example chemotherapy, can be personalized for each individual user. In general, in chemotherapy, not all therapies work the same way on all patients. Drug cocktail therapy that works for one patient will not work for another. The present invention provides a method of testing the efficacy of drug therapy prior to administration to a patient so that only the most effective therapy is administered to each patient.

It is a further object of the present invention to provide a research platform and method for the study of the pathways in which diseases and compounds affect tissues, in particular using human tissues.

For the purposes of the present invention, the term tissue sample, tissue slice, or tissue aliquot refers to a cell culture of tissue cells that are not part of an artificial organ system or an artificial organ system, and in particular human tissue.

According to an exemplary embodiment of the present invention, a method for testing human tissue using an artificial organism organ system for evaluation, detection, and testing of drug candidates, drugs, and drug metabolites included as reference materials is provided. The system includes a human tissue flake culture device. Other similar systems are particularly contemplated that allow for testing of human tissues and artificial organs. In addition, in another embodiment, the method of the present invention may use a conventional tissue sample. However, human tissue is most effective in obtaining real time and practical results.

The present invention provides a human tissue testing method that provides a method for personalizing chemotherapy regimens for individual patients, predicting the toxicity of the compound in normal tissues, and studying the disease. According to an embodiment of the present invention, a tissue sample is taken during surgery, such as a biopsy. This sample is cut into a number of tissue slices. Various chemotherapy regimens are applied to each tissue slice. After this therapy is completed, the results are compared to determine the efficacy of each therapy. By comparing the various therapies, one can design a personalized chemotherapy regimen based on the test results. Other uses for pharmacosensitivity testers are for the study and evaluation of toxicity, and for the study and evaluation of histopathology.

1 is a schematic diagram of a drug test system 10 according to the present invention. Culture medium 13 is introduced into the bioreactor 15 from the reservoir 12. Within the bioreactor 15 there is at least one tissue flake device 20, which is disposed between two wire meshes 21 (see FIGS. 7A and 7B) and is vertically in the bioreactor 15. At least one tissue lamella 23 positioned in parallel. As the culture medium is introduced into the bioreactor, the culture medium level rises until it comes in contact with the tissue flakes, thereby bringing the tissue flakes 23 into contact with the myriad compounds supplied through the culture medium.

Oxygen gas is introduced by the gas valve 151 above the compartment. The gas valve is shown above the compartment, but in the present invention the gas valve can be designed to be present on the side or bottom of the compartment and is provided with suitable sealing means to block leakage of the liquid medium. The gas is preferably a mixture of 95% O ₂ and 5% CO ₂ by volume, supplied to the compartment via a gas valve in a pressure range of 1 to 10 ATM by adjusting the pressure by a pressure regulator (not shown), from Discharged. A solenoid valve (not shown) is combined with the pressure regulator to maintain a preset gas pressure. In order to filter out the microorganisms, it is preferable to install a gas sterilization apparatus 18, for example, an injection filter having a pore size of about 0.22 m in the gas valve 151 to sterilize the compartment feed gas. A gas check valve 11 with a gas sterilization device 18 is placed on the medium reservoir and used to equalize the pressure between the reservoir and the atmosphere.

Stabilization of tissue flakes, including human tissue, is an important feature of the present invention. Tissue flakes are cultured under a supply of culture medium and oxygenate gas. Liquid culture medium, or plasma, is supplied into the compartment through the reservoir and oxygenated gas is supplied through the top of the compartment. Each is supplied at regular intervals so that each human tissue slice is alternately exposed to the medium and gas at an exposure-time ratio ranging from about 1: 1 to about 1: 4. Changes in these variables are clearly within the general scope of the art, but it has been found that ratios of about 1: 2.5 to about 1: 3.5 are effective, and ratios of about 1: 1 or 1: 3 are effective. The pump 19 regulates the flow of the culture medium. The rate at which tissue slices are alternately exposed to gas and culture medium corresponds approximately to the rate of metabolism.

Waymouth MB 752/1 culture medium is preferred over plasma in the present invention. The particular choice of type of culture medium or plasma is known to those skilled in the art and may vary by cell type. To prevent central necrosis, the gas mixtures described above are 95% O ₂ and 5% CO ₂ . Because this mixture can produce free oxygen radicals, it is often toxic to tissue culture cells. For example, liver samples are added with high concentrations of glutathione and vitamin E as oxygen free radical scavengers and antioxidants and supplemented with 10% inactive fetal bovine serum and L-glutamine.

Referring to Figure 2, it shows an embodiment of the human tissue and artificial organ system 10. The artificial organism organ system 10 includes one or more bioreactors 15 disposed within an incubator 32 that regulates temperature and humidity in the compartment. The incubator 32 allows the user to adjust the conditions of the artificial living organs such that the biological living organs are exposed to optimal conditions for bioactivity over time. The selection of a suitable incubator system for tissue culture is well known in the art and no further explanation is necessary.

One or more bioreactors 15 are disposed in the culture compartment. Each bioreactor 15 holds one or more human tissue slices or samples. The rotor 30 rotates the bioreactor 15 with a wetting machine and a dryer, respectively. The wetting period corresponds to the time at which the tissue slices have been exposed to the culture medium. Likewise, the dryer corresponds to the time when the tissue flakes were substantially exposed to the gas. The regulation module 34 provides an interface for adjusting the parameters of the ABI system 10 operation. For example, a control module can be used to control the time that human tissue flakes are exposed to gas and culture medium, which generally corresponds to the rate of metabolism. Similarly, the ratio of gas to culture medium can also be adjusted using the control module 34, and other necessary operating parameters can also be adjusted. In the incubator 32, gas and culture medium supply are provided in a manner that would be understood by one skilled in the art.

3, an enlarged view of the bioreactor 15 installed in the incubator 32 is shown. In order to facilitate supply and discharge, the bioreactor 15 and the rotor 30 may be fixed to a platform that slides in and out of the incubator 32. When installed in incubator 32, each bioreactor 15 is connected to a gas and culture medium feeders 36 and 38, respectively. As shown in the embodiment of FIG. 3, a gas valve 151 (one for each tissue lamella device compartment 155) is connected to a gas supply 36. As shown in FIG. 3, a delivery system 17 is disposed between the gas valve 151 and the gas supplier 36. As described above, the gas filter 18 is installed between the gas supply 36 and the gas valve 151. Similarly, culture medium valve 153 is connected to culture medium supply 38. As described above, the culture medium filter 18 is disposed between the culture medium supply 38 and the culture medium valve 153.

4, the embodiment of the bioreactor 15 is illustrated. While many arrangements are possible, it will be appreciated by those skilled in the art, and the embodiment shown in FIG. 4 includes a number of human tissue lamella device compartments 155 (see FIG. 5). Each tissue lamella device compartment 155 is formed by a sealed cavity when the bioreactor body 157 and the bioreactor cover 158 are connected to each other. Gas valve 151 is generally connected to gas supply 36 and allows gas exchange of tissue lamella device compartment 155 for the purpose of supplying a desired gaseous mixture to tissue flakes contained in tissue lamella device compartment 155. do. The culture medium valve 153 is in fluid communication with the tissue lamella device compartment 155 and acts the same on the culture medium as the gas valve 151 does for gas. Looking at the embodiment, the bioreactor cover seal 159 seals the bioreactor cover 158 and the bioreactor body 157. If the bioreactor body 157 and the bioreactor cover 158 are structured to engage each other, the bioreactor cover seal 159 may be an O-ring or other similar device that blocks fluid leakage and may be replaced. In this case, the actual selection of the device to act as the bioreactor lid seal 159 is understood and apparent to those skilled in the art.

According to the embodiment of the invention shown in FIG. 5, each human tissue lamella device compartment 155 houses at least one tissue lamella device 20. When not connected to one another, the tissue lamella device 20 is inserted into a cavity that forms part of a tissue lamella device compartment 155 within the bioreactor body 157. According to an exemplary embodiment, one human tissue lamella device 20 is inserted into each cavity; Multiple human tissue lamella devices 20 are, however, used in one bioreactor 15 by providing multiple tissue lamella device compartments 155 within the bioreactor 15. Nevertheless, the present invention contemplates the structure of the bioreactor 15 including a plurality of human tissue lamella device compartments 155 per human tissue lamella device compartment 155, and a plurality of tissue lamella device 20. After each tissue lamella device 20 is inserted into the human tissue lamella device compartment 155, the bioreactor cover 158 is mutually coupled with the bioreactor body 157. Once interconnected, the bioreactor 15 is sealed with the bioreactor lid seal 159. The bioreactor 15 is located in the incubator 52 and connected to the gas supply 36 and the culture medium supply 38, and the experiment is performed.

As shown in the embodiment of FIG. 6 and described above, the human tissue lamella device 20 may include a plurality of network structures 21. The network structure 21 is made of stainless steel or other materials known to those skilled in the art as described above. One or more tissue flakes 23 are positioned between adjacent network structures 21, which are fastened to each other by tissue flake device clips 24. It will be appreciated by those skilled in the art that the size and thickness of human tissue flakes can be optimized for each procedure, which can vary from experiment to experiment and from tissue to tissue.

According to the experimental embodiment shown in FIG. 6, two network structures 21 form a human tissue lamella device 23. According to an experimental embodiment, the tissue flakes 23 are disposed at 40% or less of the human tissue flake device 20. Placement of tissue flakes 23 in tissue flake device 20 allows tissue flakes to be fully exposed to both drying and wetting cycles.

7A and 7B show a similar embodiment of the human tissue lamella device 20. The size of the two stainless steel mesh structures 21 is selected based on the compartment size. These two network structures are preferably arranged in parallel. In one embodiment, this network structure has a pore size of about 0.26 mm. Also, in one embodiment, the network structure is crimped to ensure constant planarity. A plurality of tissue flakes 23, such as regularly arranged liver flakes, are located between the network structures 21. The two network structures have spaces on each side of the human tissue flakes such that the human tissue flakes are not compressed, but are sufficiently positioned to secure them so that they are not removed by the culture medium. While FIGS. 7A and 7B show only a relatively few tissue slices, located between the network structures, it can be appreciated that the efficacy of the instrument may vary depending on the number of tissue slices and the size of tissue slices used. Additionally, although two network structures 21 are shown, it should be considered that multiple network structures 21 may be used. If a single network structure 21 is used, it is formed, at least in part, to surround the human tissue flakes 23 to form a space and retain them there. For example, this network structure may be formed into a U-shape which is a suitable form.

Human tissue flakes 23 used in the present invention are obtained from a suitable source, which depends on the intended use of the device. Obviously human tissue is considered, but can be used interchangeably with animal tissue. However, one skilled in the art will recognize the inherent benefits of human tissue. Tissue flakes 23 may be of any size and shape suitable for maintaining bioactivity and their essential functions. In the present invention, the thickness of the tissue flakes 23 which is effective is in the range of about 10 μm to about 2,000 μm. In certain experiments it was determined that a thickness range of about 100 μm to about 500 μm was effective.

The present invention is ideally suited for methods of testing the toxicity and efficacy of drugs. This test is performed by exposing tissue flakes to a drug or drug candidate and observing the ability of the tissue, such as the liver, to metabolize the compound, where the compound or its metabolites can be detected. For example, ammonia and lidocaine are common compounds that can be metabolized by healthy livers. The following examples show the experiments applied to the liver-flakes. Those skilled in the art will appreciate that the utility of the present invention may be applied to other organizations.

To test chemotherapy regimens on tissue slices or aliquots, at least one compound is applied to at least one tissue aliquot in an artificial organ system. After a predetermined time elapsed, data was collected. In each experiment, the conditions were replicated.

Upon completion of the test for each tissue slice or aliquot, the data is compared. Comparison of the data provides various usefulities of the present invention, including, for example, detection of mitotic activity, cell-toxic variables, and histopathology. Derived from the data, for general prediction of the toxicity of a given compound or compounds, or for carcinogenicity studies, for example, these results are useful in formulating the most effective chemotherapy regimens for patients. Naturally, other conclusions can be derived from the data, and changing the experimental design using tissue slices or aliquots changes the derived information.

8-11 illustrate the utility of the present principles and devices using liver flakes as tissue samples. The data shown in FIGS. 8 and 9 show the lidocaine removal capacity of other proliferative organ systems over time. Similarly, FIG. 10 shows an increase in other concentrations over time, which substantially mimics the physiological response of the sample in vivo. Finally, Figure 11 shows the ammonia detoxifying ability of the present invention. The data shown in FIGS. 8-11 are not intended to limit or verify actual results. The subject matter of the present invention will be achieved only by demonstrating the achieved utility of the subject matter of the present invention. Various batch structures are intended to achieve similar results for each batch structure where the data presented herein is not fully redundant.

12, an embodiment of a method of using a device disclosed herein is shown. According to the exemplary embodiment of the present invention, the bioreactor 15 carries a human tissue flake 23 and is sealed. Bioreactor 15 may be a device having the same or similar function as the embodiment described herein. The bioreactor is connected with at least one culture medium reservoir 12 containing a source of culture medium. Looking at the embodiment, the bioreactor includes a plurality of tissue flake device compartments 155, and other culture medium storage containers may be used for the culture medium supply depending on the specific results of the experiments to be sought. For example, in accordance with an embodiment of the present invention, bioreactor 15 includes six tissue lamella device compartments 155 (eg, see FIG. 5). The first compartment was used as a control and no additive was supplied to this culture medium. The remaining five compartments are fed to the tissue slices 23 with culture medium containing the compounds to be tested, such as lidocaine or ammonia at various concentrations. Similarly, all or part of the tissue lamella device compartment 155 is supplied with exactly the same compound, either to obtain multiple result sets or to block the regeneration of results to contaminate the specific tissue lamella device compartment 155. This is to block. Accurate experimental designs and procedures, however, can be constructed in a variety of variations known or understood by those skilled in the art. Looking at the embodiment, the filter 18 may be disposed between the culture medium reservoir 12 and the bioreactor 15.

Bioreactor 15 is also connected to gas supplier 40. The gas supply can supply gas in various combinations and concentrations according to experimental design and procedures. In general, a single gas supplier 40 is connected to all tissue lamella device compartments 155. Nevertheless, multiple gas supplies 40 may be used if desired or required by experimental design or procedures. Looking at the embodiment, the filter 18 may be disposed between the gas supply 40 and the bioreactor 15.

Culture medium and gas are supplied to each tissue lamella device compartment 155 and bioreactor 15 and then incubated for a given time. During incubation, tissue slices are alternately exposed to culture medium and gas. This is done by various methods. For example, the culture medium and gas are alternately injected and recovered such that the gas or culture medium is present within tissue lamella device compartment 155 for a given time. Alternatively, the bioreactor 15 may be rotated to alternately expose the tissue samples to culture medium and gas immobilized within the tissue lamella device compartment 155. This can be accomplished by allowing the tissue sample 23 to occupy only a certain volume of the tissue lamella device compartment 155, thereby allowing it to be completely submerged in the culture medium under one batch structure and to be sufficiently exposed to gas upon rotation. 6 shows an embodiment incorporating this idea, wherein the tissue sample 23 accounts for 40% of the tissue lamella device 20. Other variations of this idea may be understood by those skilled in the art.

Samples of the culture medium may be removed for testing at various time points during the experiment. According to the embodiment shown in FIG. 12, the drain pump 60 can remove a part of the culture medium. Once removed, the culture medium may simultaneously hold all extract gas trapped in the defoamer 70. A portion of this culture medium is then fixed and tested in the treated culture medium reservoir 50. If tested, they are returned to the bioreactor 15 or discarded. The filter 18 placed in this system maintains sterilization.

The embodiment shown in FIG. 13 shows a human tissue test method using various compounds and materials. In an exemplary method, the tissue sample is extracted during the procedure. The organization is preferably human. For example, to devise a personalized chemotherapy regimen, tissue is removed from the patient in need of the therapy. For example, cancerous tissue is removed and exposed to various anticancer drug cocktails to determine the optimal cocktail regimen for the particular cancer tested. Similarly, in toxicity test applications, tissues in suitable human or animal hosts may be used. Looking at the embodiment, animal tissue may be used first. After the compound is considered safe for animal testing, the human tissue sample can be further used for toxicity testing of the compound or substance.

Tissue flakes may be obtained incidentally during other procedures, or in particular through procedures designed to obtain tissue samples, such as biopsies. For personalized chemotherapeutic therapies, tissue should be obtained from patients who need to extract tissue samples directly from the patient during irrelevant surgery or especially procedures designed to obtain tissue samples. Other drug-sensitive applications may use other tissue sample sources that depend on specific procedures.

Looking at the embodiment, tissue samples of suitable size were used to obtain results when various compounds were used. Such results, including problems associated with personalized therapeutic agents, are within the scope of those skilled in the art, and likewise these include at least one US patent document 6,678,669; 6,905,816; 6,905,816; 6,983,227 and 6,999,607, each of which is expressly incorporated herein by reference as fully described herein.

When removed from the patient, the tissue sample is sliced or otherwise divided into at least one aliquot of tissue. In one embodiment, each flake or aliquot is then incubated in the tissue lamella device compartment 155 and bioreactor 15, respectively, for example using the embodiment of FIG. Researchers and clinicians use the same number of replicated tissue slices to expose the same tissue slices to the compound at the level of human organs under conditions in which the only variable that works is the administered therapy.

Then analyze the results. Since the only variable is the administered regimen, the present invention provides a powerful means of assessing the efficacy of each given therapy over other available therapies. In addition, this is because the systems and methods of the present invention are designed to enable testing at the organ, system, and, in some cases, at the organism level, which will be described in the Examples to be described below. Thus, when using the methods and devices of the present invention, researchers and clinicians have a powerful means of assessing mitotic activity, cell-toxic parameters, histopathology, and many other related applications at the organ, system, and organism levels. do.

According to an embodiment of the present invention, a system or organism can be regenerated in vitro using the present techniques and provides results in vivo showing the use of tissues from the tissues in the system and similarly connected various tissues. Linkage occurs through the transfer of culture medium from one tissue type to another, allowing researchers to observe the step effect of therapy on various organ samples.

According to a similar embodiment, tissue flakes or other tissue types taken from tissue flakes may be combined into a single tissue flake device 20 in various permutations. This type of experiment allows researchers to control tissue types in in vivo systems in in vitro environments for research and therapeutic uses. Those skilled in the art will understand many variations using the devices and methods of the present invention to observe the efficacy and efficacy of the therapy. Similarly, various methods of regulating variables in in vivo systems will serve as a powerful means for those skilled in the art to achieve results that would be nearly impossible without human or animal experimentation.

For example, according to an embodiment of the present invention, a plurality of human liver flakes are stably located in the bioreactor 15 to maximize the surface area of the liver flakes exposed to the culture medium. Means are provided for selectively providing and removing the culture medium into the tissue lamella device compartment to raise the level so that the culture medium in the compartment contacts the tissue lamella. The culture medium is loaded into the compartment so that the liver flakes are completely submerged. The same procedure can be used to reverse the culture medium in contact with the tissue flakes. There is a means of supplying gas over the compartment to alternately expose the tissue slices to gas and culture medium. This has been described above. In addition, the reservoir is provided to contain culture medium entering and exiting the compartment. This compartment is preferably temperature controlled. For human tissue flakes, the temperature is preferably maintained at about 36.5 ° C. For rodent tissue flakes, it is maintained between about 36 and 38 ° C. However, since pig tissue slices are very sensitive to temperature changes, they should be maintained at 38 ° C, the normal body temperature of pigs.

Example 1

In one embodiment, the clinician can use the subject matter of the present invention to determine the optimal concentration of chemotherapeutic drug cocktail for cancer patients. The clinician takes a biopsy of the cancerous tissue from the patient and divides the tissue sample into aliquots. The clinician divides the aliquot into two groups. The clinician uses the first group to determine the most effective drug cocktail for the patient in question. The clinician then uses a second group of aliquots to determine the optimal concentration of drug cocktail.

Aliquots of the first group are used by clinicians to determine the most effective drug cocktail. Tissue aliquots are cultured as described above. Various drug cocktails are administered to tissue aliquots. The percentage of apoptosis of cancerous tissue in the aliquots is determined by methods generally known to those skilled in the art. The clinician then selects a drug cocktail that includes the highest apoptosis rate of cancerous tissue as compared to healthy tissue.

The clinician then uses a second group of tissue aliquots to determine the optimal concentration of drug cocktail used for the cancerous tissue. Tissue aliquots are cultured in the same manner as the tissue aliquots of the first group. The clinician applies varying concentrations of the drug cocktail to each tissue aliquot and selects the concentration of drug cocktail that indicates the highest apoptosis rate of cancerous tissue as compared to healthy tissue.

Depending on the results of the optimization of the chemotherapy regimen that the patient will receive for cancer treatment, the patient will receive the treatment with the greatest benefit while minimizing unnecessary side effects. If the clinician desires, the same result will be obtained in a single experiment applying multiple drug cocktails of various concentrations to multiple tissue aliquots.

Example 2

In other embodiments of the invention, the subject matter of the present invention may be used to predict the toxicity of a compound to healthy tissue. These results are useful for data generated and submitted to FDA for approval of a new drug application or short drug application. Animal or human tissue samples are taken biopsies or obtained as part of surgery. In general, two species of animals, one rodent and one non-rodent, are used because drugs can act on one species differently than on other species. Other agencies likewise provide key data, which are useful within the scope of the present invention. In another embodiment of the invention, the tissue may take part from other organs that are provided by techniques known to those of skill in the art, ranging from deceased organ donors, clones, regenerative or umbilical cord blood to stem cells by somatic metastasis. Can be.

Tissue aliquots are derived from tissue samples and cultured as described above. When the tissue is cultured, the compound is applied to each tissue aliquot to determine the efficacy of the compound to achieve the desired result as described above. Data is collected and interpreted according to the FDA's regulations regarding the determination of the efficacy of compounds in tissues or according to variables set by those skilled in the art.

Example 3

Similarly, disease research can be performed using various compounds used to study disease. For example, inhibitors and stimulants of compounds in tissues can be used to study the effects on chemical pathways in tissues. In addition, compounds may be applied to tissues to observe their effect at the tissue level compared to the cellular or organismal level. The present invention provides a method of using an artificial living organ system; Experimental variables are apparent to those skilled in the art without requiring undue experimentation.

Testing was conducted by an independent third party to exclude the appearance of any prejudice. Only as few animals as possible were used as a source of tissue samples and every effort was made to treat them humanely. In addition, the present invention is designed for use in human tissue, such a sample obtained from an organ donor. Since most drugs are metabolized in the liver, toxicological studies are naturally focused on their effects on the liver.

Example 4

The invention also provides a novel method of observing interactions between organ systems. Tissue flakes are obtained from various organs. They are then placed in parallel into the plurality of bioreactor tissue flake device compartments. The culture medium is provided in the first compartment to allow contact with the tissue flakes for a period of time. After the initial time has elapsed, the culture medium is removed from the first tissue lamella device compartment and subjected to other organs or samples of the same organ under different experimental conditions, such as increased metabolism, different primary cell types, or concentrations of other target cells. Move to a second tissue lamella device compartment that contains. Samples of the culture medium are obtained in an intermediate test in a running state before or simultaneously with the culture medium moving to the second tissue lamella device compartment. The culture medium transferred into the second tissue lamella device compartment then reacts for a period of time. Repeat this procedure in each tissue lamella compartment until the end of the experiment.

For example, researchers are interested in protein digestion. Thus, tissue samples are taken from the oral cavity, esophagus, stomach, duodenum, jejunum, and the small intestine portion corresponding to the ileum, and the large intestine. Samples of culture medium containing protein samples thus react with each heterologous tissue type to determine the effect of specific organs of the protein digested on system level.

While such devices and methods have been described in terms of the most practical and preferred embodiments currently contemplated, it should be understood that the invention is not limited to the disclosed embodiments. It is intended to cover various modifications and similar arrangements included within the spirit and scope of the present invention, which is to be accorded the widest interpretation and encompasses all such modifications and similar structures. The invention includes all embodiments of the following claims.

Claims (12)

Providing a bioreactor that mimics in vivo tissue function in vitro; Securing at least one aliquot of the tissue sample to the bioreactor; And Generating data usable with at least one aliquot In vitro use of human tissue for drug testing and screening comprising a. The method of claim 1, wherein the in vivo condition is achieved by maintaining cell to cell interaction in the tissue. The method of claim 1, wherein the human tissue based data is generated using a combination of tissue sample and bioreactor. The method of claim 1, wherein the bioreactor is used for at least one of determining the optimal chemotherapy regimen, predicting the toxicity of the regimen, or studying the disease. The method of claim 1, wherein the data represents at least one of a group of mitotic activity, toxicity studies, and histopathology. The method of claim 1, wherein the bioreactor substantially replicates tissue function at the organism level. The method of claim 6, wherein the bioreactor substantially replicates tissue function at the system level. 8. The method of claim 7, wherein the bioreactor substantially replicates tissue function at the organ level. The method of claim 1, wherein the available data is generated by applying at least one therapy. Securing an artificial biological tissue system containing human tissue flakes; Using an artificial tissue system by treating the tissue slice with at least one therapy to produce usable data; Comparing the data to determine mitotic activity, toxicity of the compound, or histopathology; And Selecting therapies based on the data comparison Test method of explanted human tissue comprising a. Providing a bioreactor-based system for containing human tissue flakes; Anchoring human tissue to the bio-reaction based system; Testing the therapy on tissue; And Steps to Collect Results Commercial method of testing at least one of the extracted, harvested and explanted human tissue comprising a. 12. The method of claim 11, further comprising using the result to determine at least one of mitotic activity, toxicity, or histopathology.

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