Cell separation, dissociation, and / or deaggregation using shock waves or mechanical shock.

Abstract

To provide an optimized and scalable method and device of obtaining a desired cellular fraction in a short period of time directly at the point-of-care, and without the need for multiple user intervention.SOLUTION: Methods provided by the present disclosure utilize extracorporeal shockwaves, mechanical impacts and / or principles of lithotripsy to break up a tissue sample into smaller fragments, e.g., clusters of cells and / or single cells, after which a desired cellular fraction can be isolated from the sample. Devices provided by the present disclosure deploy focused and / or directed shockwaves, and / or focused and directed mechanical impacts, to break apart a tissue sample. The devices maintain the sample in a sterile, closed environment during exposure to the shockwaves or mechanical impacts. Therefore, the shockwaves and / or mechanical impacts are generated outside of a closed device and are transmitted through one or more walls of the device into its interior, where the sample is located.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cellular bioprocessing is a form of biopharmaceutical manufacturing, and its goal is to produce therapeutic cells. To establish a reproducible and robust manufacturing process for the isolation and / or production of cells. And so it is. [Background technology]

[0002] Current cell manufacturing processes are highly user-dependent, and human intervention is required at numerous points. Intervention is needed. Due to this dependency, the current process is cumbersome and extremely It is diverse, expensive, and yields low results.

[0003] For example, many current manufacturing processes are carried out as follows: Tissue sample (fat) A sample (such as aspirate, whole blood, bone marrow, or umbilical cord blood) is obtained from the patient. The pull is transferred to the laboratory for bioprocessing. Cell separation takes place at the laboratory. The technique is used to obtain the desired cell fraction. The cell fraction is then isolated, and the disease Transferred to be returned to point-of-care equipment for human use, or cell swelling Either it is further processed by introducing it into a bioreactor. It is. Several known methods of cell separation are used to obtain the desired cell fraction. Ultrasonic cavitation requires the pull to come into contact with a foreign object (e.g., beads). This includes mechanical separation via a rectifier, enzymatic digestion, or the use of other mechanical means. This results in an increased regulatory compliance burden. The reason is that the enzyme needs to be in direct contact with the sample to produce the desired result. This is because it must be so. Also, enzymes can potentially impair the integrity of harvested cells. It has properties, and in some cases, it can change its characteristics. Sonication that can be given high power The use of the sonicator chip is similarly imperfect. The sonicator chip has a fixed frequency and is fine. It must be introduced into a cell or tissue suspension, otherwise it will negatively affect sterility. It has potential. Additionally, the use of such high-energy sound waves requires a large amount of energy. —is introduced into the sample, and it manifests as heat. This can potentially damage cells. This has properties that reduce the yield of usable cells.

[0004] Once obtained, the swollen cell product is then purified and concentrated in a biorea. It is moved from the center. The test sample is washed and concentrated for quality inspection. If removed from the cell product and the test results indicate an acceptable product, then ultimately, artificial The cell products created are prepared for long-term storage or for cryopreservation. Either it is administered to the patient from whom the sample was derived, or some of the aforementioned It is one of the combinations.

[0005] As can be recognized, some of these steps involve moving the sample from one container to another. It requires transfer to a container and requires significant user intervention. This refers to the mislabeling or mishandling of one or more samples during processing. This not only increases the risk of errors, but in some cases, it can also compromise the sterility and identity of the sample. This adversely affects the purity or efficacy. Additionally, processing the sample in this manner... The time required to do so means that the desired cell fraction is not ready for delivery to the patient, even over a period of several hours (not to mention days), which can have an adverse impact on the patient's health. The potential for compromised sterility, and the unreasonably long period of time required to process the cell sample, are unacceptable for the patient, especially for a patient with a compromised immune system or a patient who requires immediate treatment, and are also unacceptable for the provider who needs to provide timely and high-quality treatment to many patients. (even if this is not the case), Regarding the potential for compromised sterility, and the unreasonably long period of time required to process the cell sample, are unacceptable for the patient, especially for a patient with a compromised immune system or a patient who requires immediate treatment, and are also unacceptable for the provider who needs to provide timely and high-quality treatment to many patients. Regarding the potential for compromised sterility, and the unreasonably long period of time required to process the cell sample, are unacceptable for the patient, especially for a patient with a compromised immune system or a patient who requires immediate treatment, and are also unacceptable for the provider who needs to provide timely and high-quality treatment to many patients. Regarding the potential for compromised sterility, and the unreasonably long period of time required to process the cell sample, are unacceptable for the patient, especially for a patient with a compromised immune system or a patient who requires immediate treatment, and are also unacceptable for the provider who needs to provide timely and high-quality treatment to many patients. Regarding the potential for compromised sterility, and the unreasonably long period of time required to process the cell sample, are unacceptable for the patient, especially for a patient with a compromised immune system or a patient who requires immediate treatment, and are also unacceptable for the provider who needs to provide timely and high-quality treatment to many patients. Regarding the potential for compromised sterility, and the unreasonably long period of time required to process the cell sample, are unacceptable for the patient, especially for a patient with a compromised immune system or a patient who requires immediate treatment, and are also unacceptable for the provider who needs to provide timely and high-quality treatment to many patients. SUMMARY OF THE INVENTION

[0006] Therefore, there is a need for an optimized and scalable method to directly obtain the desired cell fraction in a short period of time without the need for multiple user interventions at the point of care. There is also a need for a device that can implement such a method. Such a method and device are provided by the present disclosure. The disclosed method and device eliminate the need to transfer the cell sample to a laboratory for processing, and also provide the desired cell sample in a very short period of time without contacting the cell sample with foreign matter during separation and with minimal user contact, resulting in a processed sample that enables the optimization of expensive technical resources. Therefore, there is a need for an optimized and scalable method to directly obtain the desired cell fraction in a short period of time without the need for multiple user interventions at the point of care. There is also a need for a device that can implement such a method. Such a method and device are provided by the present disclosure. The disclosed method and device eliminate the need to transfer the cell sample to a laboratory for processing, and also provide the desired cell sample in a very short period of time without contacting the cell sample with foreign matter during separation and with minimal user contact, resulting in a processed sample that enables the optimization of expensive technical resources. Therefore, there is a need for an optimized and scalable method to directly obtain the desired cell fraction in a short period of time without the need for multiple user interventions at the point of care. There is also a need for a device that can implement such a method. Such a method and device are provided by the present disclosure. The disclosed method and device eliminate the need to transfer the cell sample to a laboratory for processing, and also provide the desired cell sample in a very short period of time without contacting the cell sample with foreign matter during separation and with minimal user contact, resulting in a processed sample that enables the optimization of expensive technical resources. Therefore, there is a need for an optimized and scalable method to directly obtain the desired cell fraction in a short period of time without the need for multiple user interventions at the point of care. There is also a need for a device that can implement such a method. Such a method and device are provided by the present disclosure. The disclosed method and device eliminate the need to transfer the cell sample to a laboratory for processing, and also provide the desired cell sample in a very short period of time without contacting the cell sample with foreign matter during separation and with minimal user contact, resulting in a processed sample that enables the optimization of expensive technical resources. Therefore, there is a need for an optimized and scalable method to directly obtain the desired cell fraction in a short period of time without the need for multiple user interventions at the point of care. There is also a need for a device that can implement such a method. Such a method and device are provided by the present disclosure. The disclosed method and device eliminate the need to transfer the cell sample to a laboratory for processing, and also provide the desired cell sample in a very short period of time without contacting the cell sample with foreign matter during separation and with minimal user contact, resulting in a processed sample that enables the optimization of expensive technical resources. Therefore, there is a need for an optimized and scalable method to directly obtain the desired cell fraction in a short period of time without the need for multiple user interventions at the point of care. There is also a need for a device that can implement such a method. Such a method and device are provided by the present disclosure. The disclosed method and device eliminate the need to transfer the cell sample to a laboratory for processing, and also provide the desired cell sample in a very short period of time without contacting the cell sample with foreign matter during separation and with minimal user contact, resulting in a processed sample that enables the optimization of expensive technical resources. Therefore, there is a need for an optimized and scalable method to directly obtain the desired cell fraction in a short period of time without the need for multiple user interventions at the point of care. There is also a need for a device that can implement such a method. Such a method and device are provided by the present disclosure. The disclosed method and device eliminate the need to transfer the cell sample to a laboratory for processing, and also provide the desired cell sample in a very short period of time without contacting the cell sample with foreign matter during separation and with minimal user contact, resulting in a processed sample that enables the optimization of expensive technical resources. Therefore, there is a need for an optimized and scalable method to directly obtain the desired cell fraction in a short period of time without the need for multiple user interventions at the point of care. There is also a need for a device that can implement such a method. Such a method and device are provided by the present disclosure. The disclosed method and device eliminate the need to transfer the cell sample to a laboratory for processing, and also provide the desired cell sample in a very short period of time without contacting the cell sample with foreign matter during separation and with minimal user contact, resulting in a processed sample that enables the optimization of expensive technical resources.

[0007] The devices and methods disclosed herein have several features, and one of them does not alone cause its desirable attributes. The following claims The devices and methods disclosed herein have several features, and one of them does not alone cause its desirable attributes. The following claims Without limiting the scope, specific features of the disclosed devices and methods are discussed concisely. This will be done. After considering this discussion, and in particular, "in order to carry out the invention" After reading the chapter titled "Forms of," a person skilled in the art will be able to describe the characteristics of the device and method. However, it is important to understand whether it offers several advantages over conventional systems and methods. This will happen.

[0008] In one aspect, the present disclosure relates to the bioprocessing of cell samples, for example, self-stem Suitable for bioprocessing of lipid tissue samples processed for cell therapy. It provides a completely closed system. The system is not open to the atmosphere, and therefore, Throughout the bioprocessing process, sterile sample processing and sample Enables transport.

[0009] In the first aspect, a method is provided for isolating a cell fraction from a tissue sample, the method being applied to the target The steps include obtaining a tissue sample from a shock wave, a force from a mechanical shock, or both. The process involves a step of contacting the tissue sample and a step of isolating the cell fraction from the tissue sample. The source of force from shock waves and / or mechanical shocks, including the suction cup, is the tissue sample and Avoid physical contact.

[0010] In some embodiments, the tissues are adipose tissue, brain tissue, pharyngeal tissue, laryngeal tissue, and cardiac tissue. Arterial tissue, muscle tissue, liver tissue, gallbladder tissue, kidney tissue, small intestine tissue, large intestine tissue, lymph nodes Tissues, lung tissue, spleen tissue, bone marrow tissue, stomach tissue, venous tissue, pancreatic tissue, bladder tissue, bone, teeth, Dentin tissue, gingival tissue, skin tissue, pineal gland tissue, pituitary tissue, thyroid tissue, adrenal gland tissue, pancreas The tissue is selected from organ tissue, ovarian tissue, and testicular tissue.

[0011] In some embodiments, the tissue is adipose tissue.

[0012] In some embodiments, a tissue sample is brought into contact with a shock wave, and the source of the shock wave is It is a shock wave applicator powered by a shock wave generator.

[0013] In some embodiments, the tissue sample is subjected to force from mechanical impact, and the machine The source of force from the impact is an impact arm powered by a motor. .

[0014] In some embodiments, the tissue sample is subjected to forces from shock waves and / or mechanical shocks. It is washed once or multiple times before contact.

[0015] In some embodiments, the shock wave is used to break down a tissue sample into multiple small cell groups, multiple individual It breaks down into individual cells, or both.

[0016] In some embodiments, the force from mechanical impact causes the tissue sample to break down into multiple small cells. It breaks down into groups, multiple individual cells, or both.

[0017] In some embodiments, the cell fraction consists of multiple small cell groups, multiple individual cells, and This includes both, and the isolation of the cell fraction is performed by centrifugation.

[0018] In some embodiments, centrifugation is performed for 3 minutes to 30 minutes, from 500g to 2,000g. It is done at this speed.

[0019] In some embodiments, centrifugation is performed at 1,200g for 10 minutes.

[0020] In some embodiments, the isolated cell fraction is resuspended after centrifugation.

[0021] In some embodiments, the cell fraction is isolated within 30 minutes.

[0022] In a second aspect, a method is provided for isolating stem cells from adipose tissue, wherein the method involves extracting the adipose tissue from the subject. The steps involve obtaining a fat tissue sample and placing the tissue sample in a container or cartridge. The first step involves exposing the tissue sample to mechanical shock to release the stem cells. The steps are: separating the stem cell fraction from the adipose tissue, and centrifuging the stem cell fraction. This includes ensuring that the source of force from mechanical impact does not physically contact the adipose tissue.

[0023] In some embodiments, the adipose tissue is once before being subjected to forces from mechanical impact. It is washed multiple times.

[0024] In some embodiments, the source of the mechanical impact force is powered by a motor. The impact arm is a motor, and the motor includes a gearing, and the gearing is a motor The speed of the impact arm can be reduced when it is operating at full speed.

[0025] In some embodiments, forces from mechanical impact pass through the walls of the container or cartridge. It is then delivered to adipose tissue.

[0026] In some embodiments, the gearing is 1 / 10 of the speed in the impact arm This enables deceleration.

[0027] In some embodiments, the motor includes a gear diameter of 0.75 inches and 3,000 At a motor speed of rpm, the impact arm has a speed of 117.8 inches per second. They are doing it.

[0028] In some embodiments, the separation of the stem cell fraction is achieved by separating the adipose tissue from the aqueous layer. This makes it possible for the aqueous layer to contain stem cells.

[0029] In some embodiments, stem cells are isolated from adipose tissue within 30 minutes.

[0030] In some embodiments, centrifugation is performed for 3 to 10 minutes, from 500 g to 1,600 g. It will be held at [location].

[0031] In a third aspect, a method is provided for isolating stem cells from adipose tissue, wherein the method involves extracting the adipose tissue from the subject. The steps involve obtaining a fat tissue sample and placing the tissue sample in a container or cartridge. The steps include exposing tissue samples to shock waves to release stem cells, and adipose tissue The process includes the steps of separating the stem cell fraction and centrifuging the stem cell fraction, The source of the shock waves does not physically contact the adipose tissue.

[0032] In some embodiments, the adipose tissue is washed once or multiple times before being exposed to shock waves. To be purified.

[0033] In some embodiments, the shock wave source is powered by a shock wave generator. It is a shock wave applicator.

[0034] In some embodiments, shock waves penetrate the walls of a container or cartridge through the fatty tissue. It will be delivered to [destination].

[0035] In some embodiments, adipose tissue has a power of 0.5 bar to 5.0 bar. They are exposed to a shock wave.

[0036] In some embodiments, the container is a vinyl bag having walls 0.25 mm thick. The adipose tissue is then exposed to shock waves with a power of 2.0 bar to 2.5 bar.

[0037] In some embodiments, the adipose tissue is subjected to shock waves at any one point in time. The total area is 1 cm². 2 From 100cm 2 It is within the range.

[0038] In some embodiments, the container is a 19-ounce vinyl container with walls 0.25 mm thick. This is a bag containing a total volume of adipose tissue sample, 30cc, at any one point in time. The total area of ​​adipose tissue exposed to the shock wave is 5 cm². 2 That is the case.

[0039] In some embodiments, the adipose tissue is in the range of 5,000 to 100,000. You will be exposed to several shock waves.

[0040] In some embodiments, the separation of the stem cell fraction is achieved by separating the adipose tissue from the aqueous layer. This makes it possible for the aqueous layer to contain stem cells.

[0041] In some embodiments, centrifugation is performed for 3 to 10 minutes, from 500 g to 1,600 g. It will be held at [location].

[0042] The features and advantages of this disclosure are described in the following detailed description and accompanying text describing exemplary embodiments. Further explanation can be given with reference to the attached drawings. [Brief explanation of the drawing]

[0043] [Figure 1]This is an overview of a method for isolating a cell fraction from a tissue sample according to embodiments provided in this disclosure. [Figure 2] This figure shows typical examples of extracorporeal shock wave devices suitable for use in the systems and methods provided in this disclosure. [Figure 3] This figure shows typical examples of mechanical impact devices suitable for use in the systems and methods provided in this disclosure. [Figure 4] This figure shows a first representative example of a rigid, self-contained cartridge suitable for use with external shock wave and / or mechanical shock devices provided by this disclosure. In the embodiments shown, some internal components are flexible containers. [Figure 5] This figure shows a typical example of a rigid, self-contained cartridge suitable for use with the external shock wave and / or mechanical shock devices provided in this disclosure. [Modes for carrying out the invention]

[0044] Before describing embodiments of this disclosure, such embodiments are provided merely as examples. This means that, and the various alternatives to the embodiments of the present disclosure described herein. It should be understood that alternative examples may be used when implementing this disclosure. Numerous modifications, variations, and substitutions can be conceived by those skilled in the art without departing from this disclosure. It will become attached.

[0045] Unless otherwise defined, all technical and scientific terms used herein refer to the same technical and scientific terms used herein. The terms have the same meaning as those generally understood by those skilled in the art to whom this disclosure belongs. Methods and materials similar to or equivalent to those described in the specification may be used in the implementation of this disclosure or It may be used in testing, but the appropriate methods and materials are described below. In such cases, the patent specification, including the definition, will prevail. In addition, the materials and methods The examples provided are for illustrative purposes only and are not intended to be limiting. Examples of shapes, variations, and substitutions are conceivable to those skilled in the art without departing from the present disclosure. Yes.

[0046] As used in the specification and claims, the singular "one (a)", "1 "An" and "the" clearly indicate that the context does not. If not, it includes multiple references. For example, the term "one cell" can refer to multiple cells (such as (Contains a mixture of the above.)

[0047] As used herein, “extracorporeal shock wave,” “multiple extracorporeal shock waves,” and / Alternatively, "ESW" refers to a rapid, high-amplitude pulse of mechanical energy, similar to a sound wave. These are generated outside the sample of interest and then transmitted to that sample. It is transmitted. In that respect, the source of the ESW does not make physical contact with the sample. The shock wave itself The device comes into contact with the sample, but the source does not come into contact with the sample. Therefore, it is an extracorporeal shock wave. It is generated by a source located some distance from the sample, and then the sample It is transmitted to the air, and / or the sample is positioned within it. This can be done through the walls of the container. ESW is, for example, Sun Concentrate ESW within the area of ​​interest, covering either the entire pull or only a portion of the sample. To that end, it can be focused and / or directed.

[0048] As used herein, “subject” or “patient” means a mammal, for example, a human. It represents the interval.

[0049] The methods provided in this disclosure are for external shock waves, mechanical shocks, vibrations and / or crushing. Using the principles of lithography, tissue samples can be broken down into smaller fragments, i.e., cell groups and / or The cells can be pulverized into single cells, and then the desired cell fraction can be isolated from the sample. In various embodiments, this method involves the step of isolating a sample from a subject, and The pull includes the cell type of interest, and the step and sample to generate ESW. Instead of bringing the sample into contact with the device used for this purpose, it is brought into contact with an external shock wave to reduce its size. The steps of breaking down the rhinoceros cell population and / or individual cells, and then the broken-down sample The procedure includes the step of isolating the cell type of interest from the sample.

[0050] The devices provided by this disclosure are focused and / or directed Shock waves, and / or focused and directed mechanical shocks. This is used to break down the sample of interest. In various embodiments, this disclosure The provided device is designed to protect against exposure to shock waves or mechanical shocks in a sterile, enclosed environment. Maintain the simple. Therefore, shock waves and / or mechanical shocks should be avoided in a closed, sterile container. It is generated on the outside and transmitted to the inside through one or more walls of the container, The sample is positioned inside.

[0051] By maintaining an ESW supply source at a predetermined distance from the sample of interest, the supply of ESW The power source (the device that generates ESW) does not make physical contact with the sample. This is because The sterile state of the sample, and the sterile state of any cell fraction isolated from the sample, To improve. From the perspective of healthcare providers, this also greatly improves the requirements for sterility, and Sun Helps ensure that the cell fraction of interest derived from the pull is sterile for patient use. Additionally, maintaining the ESW source from the sample of interest means that the generator is energy - Or adding heat to the sample of interest (this may damage the cells or (This does not mean that it could kill cells, damage DNA, and drastically reduce yield.) This guarantees that the device provided by this disclosure is capable of such as ultrasound. This is an improvement over other publicly known systems.

[0052] The methods and devices provided in this disclosure, in some embodiments, take less than 30 minutes. Within this system, the desired cell fraction can be rapidly isolated from a tissue sample. The methods and devices used may be the speed at which the desired cell image is isolated from the sample. This offers a significant advantage in that minutes may be available for immediate use. .

[0053] method Figure 1 shows the cell fraction of interest being obtained from a tissue sample according to embodiments provided in this disclosure. The document then presents a method for isolation.

[0054] In the embodiments shown, the method begins by first extracting tissue from the subject. (100). Under the condition that tissue will be destroyed when exposed to external shock waves. The organization can be of any type. In some embodiments, the organization This is adipose tissue or lipids. In some embodiments, the tissue is an internal organ, for example Brain, pharynx, larynx, heart, arteries, muscles, liver, gallbladder, kidneys, small intestine, large intestine, lymph nodes, lungs, From the spleen, bone marrow, stomach, veins, pancreas, bladder, bone, teeth, dentin, gums, or skin, etc. In some embodiments, the tissue is a component of the endocrine system, for example, the pineal It originates from the glands, pituitary gland, thyroid gland, adrenal gland, pancreas, ovaries, or testicles.

[0055] Extraction can be carried out by any number of known methods. Several implementations In this configuration, tissue extraction is performed via syringe. In some embodiments, tissue extraction is performed The procedure is performed surgically, and once the tissue sample is removed from the subject, it is placed in the first container. It is designed that way.

[0056] The tissue is then transferred into a processing container or cartridge. The device provided by the disclosure is a fast and easy-to-use ESW cell bioprocessing device. Provides all the components required for the disclosed method and / or Even though some of the device's components are separable from one another They are configured to connect to each other in a completely sterile manner, thereby, Transferring samples from one component to another during processing. This makes it possible. In that respect, the entire sample is processed within a single system. This ensures a sterile environment.

[0057] Aseptic transport is carried out without exposing the sample to the external environment. The transport can be carried out via any number of methods that maintain a closed, sterile environment. It is possible. In some embodiments, the transfer contains the sample to be extracted from the subject. Between container 1 and the processing container or cartridge, male and female connectors This is achieved by attaching Arlock connectors to each other. Several embodiments Then, the transfer is performed using a sterile connection device to the first container and the processing container. This is achieved by aseptically docking the cartridge. The sample is mechanically... By either gravity or flow, the first container is discharged from the processing container or cartridge. They will be transferred to the ledge.

[0058] In some embodiments, the first container is inserted into the cartridge and is sterile It is aseptically docked using a connecting device and into the processing container inside the cartridge. Yes. The first container, which serves as a processing chamber, already contains the sample. .

[0059] In that respect, in this disclosure, as something that can be moved from one container to another, Each time a tissue and / or cell sample is disclosed, such movement is a closed system It is possible to do so in a way that maintains the integrity of the sample, thereby allowing the sample to be removed from the outside. Do not expose it to the environment.

[0060] Once the sample is transferred to the processing container or cartridge, the tissue sample is processed as follows: Washing is performed once or multiple times. Washing is done by using sterile saline solution. This can be done as described herein. Washing is performed so that the sample is processed We ensure that as many impurities as possible are removed before slicing, and therefore, there This improves the purity level of the cell fraction that will be isolated.

[0061] In some embodiments, cleaning is performed mechanically on the processing container or cartridge. This is carried out by stirring. Multiple washing steps are desired and / or required. Sterile saline solution should be placed in a processing container or carton after a single wash has been completed. Drain is discharged from the ridge, and fresh sterile saline solution is used for subsequent washing. It is introduced into a container or cartridge. In this respect, sterile saline solution is drained out. And each time new sterile saline solution is introduced into the container or cartridge, it This is done to maintain a closed, sterile environment, as described herein.

[0062] After washing, the tissue sample is processed as shown. Sessing involves ESW, mechanical shock, vibration, and shear, or a combination thereof. It is possible to do so through use.

[0063] In some embodiments, processing is performed via ESW, and the tissue sample is It is broken down into multiple smaller cell groups. In some embodiments, the processing is performed on ES This process is carried out via W, and the tissue sample is broken down into multiple small cell groups and multiple individual cells. In some embodiments, processing is carried out via ESW and the organization Sun The pull is completely broken down into multiple individual cells.

[0064] In some embodiments, processing is carried out via mechanical impact and shear. The tissue sample is broken down into several smaller groups of cells. In some embodiments, the process Sing is performed via mechanical shock and shearing, and the tissue sample is subjected to multiple small cell groups. and is broken down into multiple individual cells. In some embodiments, the processing is performed by This process is carried out via mechanical shock and shearing, and the tissue sample is completely broken down into multiple individual cells. It can be done.

[0065] In some embodiments, the processing involves a combination of ESW and mechanical impact and shear. This is done via a grafting process, in which the tissue sample is broken down into several smaller groups of cells. In this embodiment, the processing involves a combination of ESW and mechanical impact and shear. This process is carried out, and the tissue sample is broken down into multiple small cell groups and multiple individual cells. In some embodiments, the processing involves ESW and mechanical impact and shear. This process is carried out through combination, and the tissue sample is completely broken down into multiple individual cells.

[0066] After processing, a cell suspension (which represents what remains of the sample) is formed. A group of cells, multiple individual cells, or both, are filtered and separated. 120 is separated from the heart. Filtration is performed in a separate device from the processing container or cartridge. This is done inside the chair, or inside the processing container or cartridge itself. It is possible to do so. In some embodiments, filtration is performed in a separate device. In that case, the cell suspension is transferred via sterile means as described herein. It is transferred to a filter. In some embodiments, the cell suspension is transferred to a container or cartridge. Filtration is performed when the material is being transferred from the jar into one or more centrifuge tubes. .

[0067] In some embodiments, transfer to the filtration device is performed using a processing container or cart. Male and female Luer lock connectors are used between the ridge and the filtration device. This is achieved by attaching it to a sterile connection. In some embodiments, the transfer is performed by attaching it to a sterile connection. Using a vise, sterile processing containers or cartridges and filtration devices are used. This is achieved by locking. The sample is either mechanically or by gravity flow. This process transfers the contents from the processing container or cartridge to the filtration device.

[0068] In some embodiments, filtration is performed as described herein, processing This is done in a container or cartridge.

[0069] In various embodiments, centrifugation is performed outside the processing vessel or cartridge. In some embodiments, the cell suspension is placed in a processing container or cartridge. It is transferred to one or more centrifuge tubes located inside the container. It is then filled. Sometimes, centrifuge tubes are used in processing vessels or as described herein. The material is transferred from the cartridge to the centrifuge using sterile means.

[0070] In some embodiments, the cell suspension is processed as described herein. One or more centrifugal tubes located on the outside of the single container or cartridge It is then transferred via sterile means. When filled, the centrifuge tube is specified in this specification. As described, sterile processing is performed from the processing container or cartridge to the centrifuge. It can be moved by means of a means.

[0071] The duration and rate of centrifugation vary depending on the type of tissue being processed. It is possible to convert it. In some embodiments, the cell suspension is converted for a period of 3 to 30 minutes. The cells are then centrifuged at a rate of 500g to 2,000g. In some embodiments, the cells are suspended. The liquid is centrifuged at a rate of 1,000g to 1,800g for 10 to 25 minutes. In one embodiment, the cell suspension is centrifuged at 1,200 g for 10 minutes. In the application method, the cell suspension is centrifuged at 1,200 g for 7 minutes. Several embodiments The cell suspension is then centrifuged at 1,200 g for 5 minutes. In some embodiments, The cell suspension is centrifuged at 1,200 g for 3 minutes. In some embodiments, the cell suspension The turbidity is centrifuged at 900g for 10 minutes. In some embodiments, the cell suspension is The cell suspension is centrifuged at 900g for 7 minutes. In some embodiments, the cell suspension is centrifuged at 90 for 5 minutes. It is centrifuged at 0g. In some embodiments, the cell suspension is centrifuged at 900g for 3 minutes. They are separated. In some embodiments, the cell suspension is centrifuged at 600 g for 10 minutes. In some embodiments, the cell suspension is centrifuged at 600g for 7 minutes. In one embodiment, the cell suspension is centrifuged at 600 g for 5 minutes. In this state, the cell suspension is centrifuged at 600g for 3 minutes.

[0072] Multiple cell types present in a cell suspension can be separated from each other by centrifugation. Numerous methods exist. For example, a density gradient can be used, and during centrifugation, cells However, according to their inherent density, they separate into bands. Additionally, the size exclusion protocol , can be used to separate cells by size. Additionally, for example, process By allowing the tissue sample to settle, and by using a remote method, Before heart separation, unwanted cell debris is separated from the desired cell fraction. This can be started before centrifugation. The type of separation technique used is isolated. It is possible to change based on the desired cell fraction.

[0073] Once the desired cell fraction is isolated, the cell fraction is used for administration to the target from which the cell fraction was derived. 125 can be resuspended for this purpose. In some embodiments, the desired cell fraction is placed in a centrifuge tube. It is resuspended in a small volume of fluid obtained from. In some embodiments, the desired The cell fraction is resuspended in sterile saline solution.

[0074] In one embodiment, the desired cell fraction is stem cells, and the tissue from which the stem cells are derived is lipid-rich. That is the case.

[0075] The first embodiment of the method for processing stem cells from lipids is as follows:

[0076] Lipids are obtained from the patient via liposuction. Liposuction is a syringe. This can be carried out via a liposuction machine, where the lipid is directly absorbed through a needle or liposuction machine. It is removed from the patient via the suction. The amount of lipids removed during liposuction is desired. It can change depending on the number of stem cells. In some embodiments, liposacral The suction is a mini-liposuction system, and with a mini-liposuction system, you can get approximately 30cc to approximately 50cc cc of lipids are removed from the patient. In some embodiments, liposuction is performed by a microphone. This is liposuction, and in microliposuction, approximately 5cc to 29cc of lipids are transferred. It is removed from the patient. In some embodiments, liposuction is performed in a typical clinical manner. This is a posuction procedure, in which case more than 300cc of lipids are removed from the patient.

[0077] Once the lipids are removed from the patient, they are stored in a sterile processing container or cartridge. It is transferred into the container. The transfer is carried out by sterile means as described herein. In some embodiments, lipids are removed from the patient via a syringe. Male and female Luer locks are used between the processing container or cartridge. By attaching the connectors to each other, the syringe can be used to transfer the contents from a processing container or a syringe. It is transferred to a cartridge. In some embodiments, the lipids are transferred from the patient via syringe. Once removed, the syringe is separated from the container or cartridge by the male and female connectors. By attaching the type of Luer lock connector to each other, processing containers and It is sterile docked in the cartridge. In some embodiments, lipids are Removed from the patient via the syringe, and into the processing container in the syringe and cartridge. Alternatively, male and female Luer lock connectors can be attached to each other between the cartridge and the other. By attaching it, the liquid is transferred from the syringe to the processing container or cartridge. In some embodiments, lipids are removed from the patient via a liposuction device. Using a sterile connection device, liposuction devices and processing containers By sterile docking the cartridge, the liposuction device is professional It is transferred to a sessing container or cartridge.

[0078] In some embodiments, transport can be carried out via gravity flow. In that embodiment, the transfer can be carried out mechanically, for example, by using a syringe. By physically pressing the plunger, and from inside the syringe the process This can be done by pushing lipids into the inside of a sizing container or cartridge. In some embodiments, the transfer is carried out via a device such as a pump. It is possible to do so.

[0079] The volume of the container or cartridge used to receive lipids is obtained from the patient. It can change with the amount of lipids being absorbed. In some embodiments, minilipos 30cc to 50cc of lipids removed via the action, 9 flow ounces to 19 flow It is transferred to a container or cartridge with an ounce volume.

[0080] Next, a volume of sterile saline equal to the amount of lipids removed from the patient is used to wash away the lipids. To do this, it is transferred to a container or cartridge. For example, 30cc of lipids is given to the patient. If removed, then 30 mL of sterile saline solution is used to wash the sample. It is used for the purpose of removing more lipids from the patient than the amount of lipids removed from the patient. In some embodiments, the amount of lipids removed from the patient is then greater than the amount of lipids removed from the patient. A large volume of sterile saline solution is transferred to a container or cartridge for lipid washing. It is sent. For example, if 30cc of lipids are removed from the patient, then 35mL Sterile saline solution is used to wash the sample. In some embodiments, Next, a volume of sterile saline solution smaller than the amount of lipids removed from the patient will then remove the lipids. For cleaning, it is transferred to a container or cartridge. For example, 30cc of lipids is used. If removed from the body, the sample is then washed with 25 mL of sterile saline solution. It is used for the following purposes. In some embodiments, the combination of volumes of sterile saline is To wash away lipids, physiological salt is added to the amount of lipids. Water transfer is carried out in a manner that maintains the sterility of the system, as described herein. In some embodiments, a container for holding sterile saline solution and processing are performed. Male and female Luer lock connectors are connected to each other between the container or cartridge. By attaching it, sterile saline solution is transferred to a container or cartridge. In some embodiments, a sterile connection device is used to connect a sterile saline container and By aseptically docking the processing container or cartridge, sterile The saline solution is transferred to a container or cartridge.

[0081] Washing involves gently shaking the container or cartridge containing lipids and sterile saline solution. This is done by turning or rotating.

[0082] Next, the container or cartridge is cleaned of (now washed) lipids in sterile saline solution. They are placed in a way that allows them to be separated. Since lipids have a lower density than physiological saline, lipids are separated from physiological food. It will rise to the top of the saltwater and float. The amount of time required for separation to occur is This will vary depending on the individual composition of the lipid sample. In some embodiments, Separation can occur between 1 and 30 minutes. In some embodiments, separation occurs between 1 and 15 minutes. It can occur. In some embodiments, separation can occur between 1 and 10 minutes. In the application method, separation can occur in 1 to 5 minutes. In some embodiments, separation occurs in 10 minutes. It can occur in seconds to one minute.

[0083] Next, the saline solution is drained from the container or cartridge as completely as possible. Therefore, only lipids remain. Drain discharge is performed using the container or ca in this method. Depending on the type of cartridge, this can be done through various means. In one embodiment, the physiological saline solution is located at the bottom of the container or cartridge. The drain is discharged from the container or cartridge to the outside via a tube. Either actively advance using either a syringe or a pump, or use gravity flow. It can proceed passively through this. In some embodiments, drain discharge is performed by this As described in the specification, this is done to maintain the integrity of the sterile system.

[0084] After washing is complete, repeat washing until the lipids turn golden and the saline solution is only slightly cloudy. It is repeated. In some embodiments, washing is performed 1 to 5 times, and in some embodiments This is performed 1 to 4 times, and in some embodiments, 1 to 3 times, and in some embodiments This is performed one to two times, and in some embodiments, it is performed once.

[0085] Once washing is complete, the lipids are prepared for processing.

[0086] Optionally, although not required, sterile saline solution can be used for processing. It is added to the bag. Adding saline solution helps cells and / or small groups of cells process This provides a space where they can move freely away from each other during the session, and therefore This helps to break down lipids into individual cells. If physiological saline is added, In a manner that maintains the integrity of the sterile system, as described herein Then, saline solution is added.

[0087] The volume of saline solution added at will can be varied. Morphologically, the volume of saline solution added is 0 to 2 times the volume of lipids removed from the patient. It is within a double range. In some embodiments, equal amounts of saline solution are used (even (For example, 30 mL of physiological saline solution for 30 cc of lipids.)

[0088] Excess air is removed from the container or cartridge using a pump or syringe. In some embodiments, excess air drains from the sterile saline solution. Removed from the same tube located at the bottom of the container or cartridge from which it is discharged. In some embodiments, air is provided as described herein in a sterile, enclosed space. The drain is discharged in a manner that maintains the integrity of the system.

[0089] Next, the processing container or cartridge is moved to the processing device. An example of a suitable processing apparatus 200 is shown in Figure 2. In form, the processing container or cartridge 205 is a processing unit for processing It is fixed onto the rig platform 210. Platform 210 is ES During W processing, the movement of the container or cartridge 205 is controlled.

[0090] Platform 210 includes wood, metal, plastic, and acrylic, It can be made from any suitable rigid material. In some embodiments, the platform The Mu210 is made from wood.

[0091] The processing device 200 delivers shock waves into the container or cartridge 205. It houses a shock wave applicator 215 that can perform shock wave applications. 5 is on the processing unit 200 via the applicator platform 220 It is housed in the applicator platform 220 and shockwave applicator 2. 15 is separated by a predetermined distance from the lipids contained in the container or cartridge 205. The shock wave applicator 215 is kept in a state where it is never in direct contact with lipids. It is designed so that it will not be placed there. This is because the shock waves delivered to the lipids are external, or It is guaranteed to be ESW. In some embodiments, shock wave applicator 2 15 is placed in direct contact with the processing container or cartridge 205. In such embodiments, the wall portion of the container or cartridge 205 is impact-resistant. We guarantee that the wave applicator 215 never comes into direct contact with lipids.

[0092] Movement of the applicator platform 220, and consequently, the shockwave applicator 21 Movement 5 can be performed within the three directional planes (X, Y, and Z). Each of them has its own motor: X-plane motor 230, Y-plane motor 235, and controlled by the Z-plane motor 240. In some embodiments, these motors The motor is a NEMA 17 stepper motor. Motors 230, 235, and Each of the 240 is controlled by a motor controller 225, and the motor controller The roller 225 is positioned above and around the container or cartridge 205, and the applicator This enables three-dimensional movement of the platform 220 and the shock wave applicator 215. In some embodiments, the motor is programmed by Marlin firmware. It is controlled by a modified Arduino v6 microprocessor board. In this embodiment, the motor is connected to an Arduino v with Marlin firmware. It is controlled by 6 boards. In some embodiments, only the X-plane motor 230 is used. It is used. In some embodiments, only the Y-plane motor 235 is used. In this embodiment, only the Z-plane motor 240 is used. In some embodiments, X Only the planar motor 230 is used. In some embodiments, the X planar motor 230 And only the Y-plane motor 235 is used. In some embodiments, the X-plane motor - Only the 230 and Z-plane motors 240 are used. In some embodiments, the Y-plane motors are used. Only the plane motor 235 and the Z-plane motor 240 are used. In some embodiments The X-plane motor 230, Y-plane motor 235, and Z-plane motor 240 are used. It will be done.

[0093] This third stage of the applicator platform 220 and shockwave applicator 215 The original movement was due to all the lipids in the sample being extracted being subjected to external shock during processing. Allows exposure to waves.

[0094] Next, in order to break down the lipids, shock waves are emitted from the shock wave applicator 215. Through the transmission gel, or a membrane containing the transmission gel, the container Alternatively, it is delivered to the lipid sample through the wall of cartridge 205, and small cell populations and The individual cells are separated from the lipids. In some embodiments, the transmission gel ,DJO,LLC,1430 Decision Street,Vista,CA 9 REF 4248 manufactured by Chattanooga in 2081, USA This is Conductor Transmission Gel. Before processing. In those embodiments, sterile saline solution is added to the lipids, and small amounts are separated from the lipids. The rhizoid cell group and / or individual cells will migrate into the physiological saline solution.

[0095] The shock wave applicator 215 is powered by the shock wave generator 245, and shock waves The generator 245 produces ESW, and the ESW is transmitted via the shock wave applicator 215. It is transmitted to quality. In some embodiments, the shock wave generator is Storz Medica l AG,8274 Tagerwilen, Switzerland Master This is the Puls MP100. In some embodiments, the shock wave generator is Lumsai l Industrial Inc.,4 / F,No. 9Yi,Lane 2,Sui BS-SWT2X, manufactured in China, de Road, Shanghai, 200331. be.

[0096] The power of ESW delivered to lipids can vary. For example, in thick walls... To penetrate the enclosed container or the walls of cartridge 205, increase the power of the ESW. It may be necessary to do so. Generally, the power of ESW delivered to lipids is The thickness of the wall of the container or cartridge 205, and / or the container or cartridge Depending on the material from which the 205 is fabricated, it can be modified. In terms of morphology, the power of ESW delivered to lipids ranges from 0.5 bar to 5.0 bar. Yes. In some embodiments, the power of the ESW delivered to the lipids is from 1.0 bar. It is in the range of 4.5 bar. In some embodiments, the power of ESW delivered to lipids The pressure range is from 1.5 bar to 4.0 bar. In some embodiments, it is delivered to lipids. The power of the ESW is in the range of 2.0 bar to 3.5 bar. In terms of form, container 205 is a vinyl bag with walls 0.25 mm thick, ESW The power level is in the range of 2.0 bar to 2.5 bar.

[0097] The area covered by the ESW delivered by the shock wave applicator 215 is The area covered by the shock wave applicator 215 can be changed. The larger the size, the greater the surface area of ​​lipids exposed to the ESW. In some embodiments, the total area covered by the shock wave applicator 215 is from 1 cm 2 to 100 cm 2 In some embodiments, the total area covered by the shock wave applicator 215 is from 1 cm 2 to 90 cm 2 In some embodiments, the total area covered by the shock wave applicator 215 is from 1 cm to 80 cm 2 In some embodiments, the total area covered by the shock wave applicator 215 is from 1 cm 2 to 70 cm In some embodiments, the total area covered by the shock wave applicator 215 is from 1 cm 2 to 60 cm 2 In some embodiments, the total area covered by the shock wave applicator 215 is from 1 cm 2 to 50 cm 2 In some embodiments, the total area covered by the shock wave applicator 215 is from 1 cm 2 to 40 cm 2 In some embodiments, the total area covered by the shock wave applicator 215 is from 1 cm to 30 cm 2 In some embodiments, the total area covered by the shock wave applicator 215 is from 1 cm 2 to 20 cm In some embodiments, the total area covered by the shock wave applicator 215 is from 1 cm 2 to 10 cm 2 In some embodiments, the total area covered by the shock wave applicator 215 is from 1 cm 2 to 5 cm 2 In some embodiments, the total area covered by the shock wave applicator 215 is from 1 cm 2 to 5 cm 2 In some embodiments, the total area covered by the shock wave applicator 215 is from 5 cm to 5 cm 2 In one embodiment, the container 205 is a 19 ounce vinyl bag having a wall thickness of 0.25 mm, the total amount of lipid obtained from the patient is 30 cc, and optionally 2 In some embodiments, the total area covered by the shock wave applicator 215 is 5 cm In some embodiments, the total area covered by the shock wave applicator 215 is 5 cm 2 or less.

[0098] In one embodiment, the container 205 is a 19 ounce vinyl bag having a wall thickness of 0.25 mm, the total amount of lipid obtained from the patient is 30 cc, and optionally In one embodiment, the container 205 is a 19 ounce vinyl bag having a wall thickness of 0.25 mm, the total amount of lipid obtained from the patient is 30 cc, and optionally An additional 30 mL of saline solution (equal volume to the lipid sample) is added before processing. It is added to the vinyl bag. In this embodiment, the total surface of the bag exposed to the ESW The volume, and therefore the total volume of the lipid samples, is approximately 5 cm³. 2 That is the case.

[0099] The number of shock waves delivered to the lipid sample can be varied. In terms of morphology, the total number of shock waves ranges from 5,000 to 100,000, and several In some embodiments, the range is 10,000 to 50,000, and in some embodiments, It ranges from 10,000 to 25,000, and in some embodiments, 10,000 or The range is 20,000. In some embodiments, the total number of shock waves is 25,000. It is 0.

[0100] In this embodiment, stem cells are isolated from a lipid sample obtained from the patient. 5cm above the wave applicator 215 2 Using the applicator chip, from 2.0 The shock wavenumber range of 10,000 to 50,000 in the bar range of 2.5 is for stem cells. It is sufficient to separate both the lipid and interstitial vascular cell fractions. As such, the "interstitial vascular cell fraction" is a cell fraction (which, in some embodiments, is lipid Processed cells obtained from separating and dissociating (the quality) This refers to the cell fraction obtained from a tissue sample. The interstitial vascular cell fraction is limited to... In particular, it includes stem cells, growth factors, and progenitor cells.

[0101] After ESW is applied to the lipid sample, the container or cartridge will remain in place for a short period of time. It is subjected to mechanical stirring over time. In some embodiments, the mechanical stirring is shaken. In some embodiments, mechanical stirring is performed once or multiple times in the container or This involves turning the cartridge 205 upside down. In some embodiments, a short period of time The interval is in the range of 1 to 30 seconds, and in some embodiments, it is in the range of 1 to 15 seconds. Yes, and in some embodiments, it is in the range of 1 to 10 seconds. The short duration is approximately 10 seconds. Stirring releases stem cells and lipids. The interstitial vascular cell fraction may still be present in the container or cartridge 205. This makes it possible to separate cell debris and any persistent lipid tissue.

[0102] Next, the (now processed) lipids were separated from the lipids by ESW. Stem cell and stromal vascular cell fractions can now be isolated from sterile saline containing them. The container or cartridge 205 is placed there. Lipids are greater than those in physiological saline containing stem cells. Because of its low density, the lipids will rise to the top of the saline solution and float. Separation is performed. The amount of time required to process varies depending on the individual composition of the lipid sample. In some embodiments, separation can occur between 1 and 30 minutes. In this state, separation can occur in 1 to 15 minutes. In some embodiments, separation occurs in 1 minute or It can occur in 10 minutes. In some embodiments, separation can occur in 1 to 5 minutes. In some embodiments, separation can occur in 10 seconds to 1 minute. In some embodiments, Departure can be completed in less than one minute.

[0103] Stem cells are isolated here from the lipid sample, and here in a sterile saline layer. It is suspended inside.

[0104] Next, the saline layer containing stem cells is placed as completely as possible in the container or cartridge. 205 is removed, and therefore only lipids remain. Removal is utilized in this method. Depending on the type of container or cartridge 205, this may be done by various means. This is possible. In some embodiments, the physiological saline solution is placed in a container or cartridge 205 Through the tube located at the bottom, it exits the container or cartridge 205. Drain is discharged. Drain discharge is performed actively using either a syringe or a pump. It is possible to proceed either by force or passively by gravity flow. In the embodiment, the layer containing stem cells is a closed, sterile layer as described herein. It will be removed in a manner that preserves the integrity of the environment.

[0105] In some embodiments, the saline layer containing stem cells is in a container or cartridge. During the removal process from 205, the material is passed through a filter. The size of the filter changes. This is possible. In some embodiments, the filter size ranges from 40 μm to 100 μm. It is in the range of μm. In some embodiments, the filter size is 70 μm. In various embodiments, the filter is made of nylon.

[0106] Either after or during removal from the container or cartridge 205, the stem cells The contained physiological saline is transferred into one or more centrifuge tubes. In this embodiment, the movement is carried out in a closed, sterile environment as described herein. This is done in a manner that maintains the following: At the bottom of the centrifuge tube, the stems are introduced into the pellet. Centrifugation continues to concentrate the cells.

[0107] Stem cells are concentrated by centrifugation at 500g to 1600g for 3 to 10 minutes. They are reduced. In some embodiments, stem cells are centrifuged at 1,200 g for 10 minutes. It is concentrated by separation.

[0108] The fluid is removed from the tube, leaving only the stem cells at the bottom. The fluid is decanted. It can be removed by suction or similar means.

[0109] A small amount of fluid is placed in a centrifuge tube to resuspend the stem cells. In this embodiment, the amount of fluid added to the centrifuge tube is in the range of 1 mL to 5 mL. In some embodiments, the fluid is physiological saline, platelet-rich plasma, hyaluronic acid, and fixative. Gel (fixing gel), hydrogel, scaffold, fibrin, glue, or tip It is any combination of the above. In some embodiments, the cells are centrifuged and absorbed. It is resuspended in the material.

[0110] The stem cells are suspended in a suspension and are ready for use. Such uses include, without limitation, the reintroduction and freezing of stem cells into patients from whom they were derived. This can include preservation and expansion. In some embodiments, it can be concentrated and re-concentrated. The suspended stem cells are transferred into a syringe via a subcutaneous injection needle for patient use. In some embodiments, concentrated and resuspended stem cells are used in the heart, muscle, bone, and soft tissue. Tissue scaffolds for growth into bone, liver, kidney, or other tissue and organ structures. Seeds are sown in a container. In some embodiments, concentrated and resuspended stem cells are This leads to the expression of pluripotency transcription factors in stem cells. Through this process, they are converted into induced pluripotent stem cells.

[0111] A second embodiment of the method for processing stem cells from lipids is as follows:

[0112] Lipids are obtained from the patient via liposuction. Liposuction is a syringe. This can be carried out via a liposuction machine, where the lipid is directly absorbed through a needle or liposuction machine. It is removed from the patient via the suction. The amount of lipids removed during liposuction is desired. It can change depending on the number of stem cells. In some embodiments, lipids are It is removed from the patient via the syringe, and the syringe is between the syringe and the cartridge. By attaching the female and female Luerlock connectors to each other, cartridges They are sterile docked in the jar. In some embodiments, the lipids are delivered via syringe. The processing container or carton is removed from the patient and placed in the syringe and cartridge. Attaching male and female Luer lock connectors to each other between the ridge and the other. This is used to transfer the contents from the syringe to a processing container or cartridge. In this embodiment, the liposuction is a miniliposuction, and the miniliposuction is Approximately 30cc to 50cc of lipids are removed from the patient. In some embodiments, Liposuction is microliposuction, and in microliposuction, approximately 5 Approximately 29 cc of lipids are removed from the patient from cc. In some embodiments, liposuccinate This is a typical clinical case of liposuction, in which case more than 300cc of lipids were lost. It is removed from the patient.

[0113] Once the lipids are removed from the patient, they are stored in a sterile processing container or cartridge. It is transferred into the container. The transfer is carried out by sterile means as described herein. In some embodiments, lipids are removed from the patient via a syringe. Male and female Luer locks are used between the processing container or cartridge. By attaching the connectors to each other, the syringe can be used to transfer the contents from a processing container or a syringe. The lipids are transferred to a cartridge. In some embodiments, the lipids are transferred to a liposuction device. It is removed from the patient via a sterile connection device and the liposuction device and By aseptically docking the processing container or cartridge, liposac It is transferred from the processing device to a processing container or cartridge.

[0114] In some embodiments, transport can be carried out via gravity flow. In that embodiment, the transfer can be carried out mechanically, for example, by using a syringe. By physically pressing the plunger, and from inside the syringe the process This can be done by pushing lipids into the inside of a sizing container or cartridge. In some embodiments, the transfer is carried out via a device such as a pump. It is possible to do so.

[0115] The volume of the container or cartridge used to receive the lipid can vary with the amount of lipid obtained from the patient. In some embodiments, 30 cc to 50 cc of lipid removed via a mini-liposuction is transferred to a container or cartridge having a volume of 9 fluid ounces to 19 fluid ounces. Next, a volume of sterile saline equal to the amount of lipid removed from the patient is transferred to the container or cartridge to wash the lipid. As an example, if 30 cc of lipid is removed from the patient, then 30 mL of sterile saline is used to wash the sample. In some embodiments, the lipid is removed from the patient via a syringe, and the syringe is sterilely docked in the cartridge by attaching male and female Luer lock connectors to each other between the syringe and the cartridge. In some embodiments, the lipid is removed from the patient via a syringe, and is transferred from the syringe to a processing container or cartridge in the cartridge by attaching male and female Luer lock connectors to each other between the syringe and the processing container or cartridge in the cartridge. The transfer of saline to the container or cartridge is performed to maintain the sterility of the system as described herein. In some embodiments, sterile saline is transferred to the container or cartridge by attaching male and female Luer lock connectors to each other between the container holding the sterile saline and the processing container or cartridge

[0116] ​​​​​​​​​​​​​​In one embodiment, a sterile connection device is used to connect a sterile saline container and a pro By aseptically docking a sessing container or cartridge, sterile physiological food can be prepared. The saltwater is transferred to a container or cartridge.

[0117] Washing involves gently shaking the container or cartridge containing lipids and sterile saline solution. This is done by turning or rotating.

[0118] Next, the container or cartridge is cleaned of (now washed) lipids in sterile saline solution. They are placed in a way that allows them to be separated. Since lipids have a lower density than physiological saline, lipids are separated from physiological food. It will rise to the top of the saltwater and float. The amount of time required for separation to occur is This will vary depending on the individual composition of the lipid sample. In some embodiments, Separation can occur between 1 and 30 minutes. In some embodiments, separation occurs between 1 and 15 minutes. It can occur. In some embodiments, separation can occur between 1 and 10 minutes. In this application method, separation can occur within 1 to 5 minutes.

[0119] Next, the saline solution is drained from the container or cartridge as completely as possible. Therefore, only lipids remain. Drain discharge is performed using the container or ca in this method. Depending on the type of cartridge, this can be done through various means. In one embodiment, the physiological saline solution is located at the bottom of the container or cartridge. The drain is discharged from the container or cartridge to the outside via a tube. Either actively advance using either a syringe or a pump, or use gravity flow. It is possible to proceed passively through the medium. In some embodiments, the drain discharge is performed to maintain the integrity of the aseptic system as described in this specification.

[0120] After the completion of the washing, the washing is repeated until the lipid turns golden and the saline becomes slightly turbid. In some embodiments, the washing is performed 1 to 5 times, in some embodiments, 1 to 4 times, in some embodiments, 1 to 3 times, in some embodiments, 1 to 2 times, and in some embodiments, 1 time. After the completion of the washing, the lipid is prepared for processing. Optionally, although not required, sterile saline is added to the bag for processing. The addition of saline will provide a space for the cells and / or small cell groups to move freely away from each other during processing, thereby

[0121] helping to break down the lipid into individual cells. When saline is added, the saline is added in a manner that will maintain the integrity of the aseptic system as described in this specification.

[0122] Optionally, although not required, sterile saline is added to the bag for processing. The addition of saline will provide a space for the cells and / or small cell groups to move freely away from each other during processing, thereby helping to break down the lipid into individual cells. When saline is added, the saline is added in a manner that will maintain the integrity of the aseptic system as described in this specification. helping to break down the lipid into individual cells. When saline is added, the saline is added in a manner that will maintain the integrity of the aseptic system as described in this specification. helping to break down the lipid into individual cells. When saline is added, the saline is added in a manner that will maintain the integrity of the aseptic system as described in this specification. helping to break down the lipid into individual cells. When saline is added, the saline is added in a manner that will maintain the integrity of the aseptic system as described in this specification. helping to break down the lipid into individual cells. When saline is added, the saline is added in a manner that will maintain the integrity of the aseptic system as described in this specification.

[0123] The volume of the optionally added saline can vary. In some embodiments, the volume of the added saline ranges from 0 times to 2 times the volume of the lipid removed from the patient. In some embodiments, an equal amount of saline is used (for example, 30 mL of saline for 30 cc of lipid). embodiments, an equal amount of saline is used (for example, 30 mL of saline for 30 cc of lipid). embodiments, an equal amount of saline is used (for example, 30 mL of saline for 30 cc of lipid).

[0124] Excess air is removed from the container or cartridge using a pump or syringe. In some embodiments, excess air drains from the sterile saline solution. Removed from the same tube located at the bottom of the container or cartridge from which it is discharged. In some embodiments, air is provided as described herein in a sterile, enclosed space. The drain is discharged in a manner that maintains the integrity of the system.

[0125] Next, the processing container or cartridge is moved to the processing device. An example of a suitable processing apparatus 300 is shown in Figure 3. In form, the processing container or cartridge 205 is a processing unit for processing It is fixed on top of platform 315. Platform 315 is pro During the cessation process, the movement of the container or cartridge 205 is controlled.

[0126] Platform 315 can be made of any suitable rigid material, including wood and plastic. It can be made from wood or other materials. In some embodiments, the platform 315 is made of wood or other materials. They are being manufactured.

[0127] In this embodiment, mechanical impact is used to pulverize the lipid tissue and release the stem cells.

[0128] The mechanical shock is generated by motor 305, and motor 305 is impact The arm 310 is driven, and the impact arm 310 will process the oil. Physical contact is made with the container or cartridge 205 containing the substance. In this embodiment, Motor 305 drives the impact arm 310, causing the impact arm 310 to move up and down. It is designed to allow joint movement, and at the bottom of the range of motion, the container or cartridge It makes contact with the 205. In some embodiments, the motor is Black and De cker,1000 Stanley Drive,New Britain,CT 0 This is a 4.5Amp motor from the 6053, USA-made model BDEJS300C.

[0129] In some embodiments, the motor 305 is a variable speed motor, and this variable speed motor The turbocharger has a rate ranging from 0 rpm to 30,000 rpm, and in some embodiments... This is a rate in the range of 0 rpm to 20,000 rpm, and in some embodiments, With rates ranging from 0 rpm to 10,000 rpm, in some embodiments, 0r The impact arm is driven at a rate ranging from 5,000 rpm to 5,000 rpm. It is scalable.

[0130] In some embodiments, the motor 305 includes a gearing, and the gearing is more In order to generate a lot of torque, the motor 305 is at full speed (in some embodiments, When operating at 30,000 rpm, reduce the speed of the impact arm 310. This can be done, when generated by the impact arm 310. The momentum is transferred to the tissue in the container or cartridge 205. This is lipid tissue. This process breaks down the cells, greatly increasing the rate at which stem cells are released from fatty tissue.

[0131] In one embodiment, the motor 305 operates from 30,000 rpm to 3,000 rpm. In terms of speed, the gears are installed to allow for a reduction of 1 / 10. In terms of form, the motor 305 includes a gear diameter of 0.75 inches, which is 1 / 10th of this. This enables deceleration at 3,000 rpm and a gear diameter of 0.75 inches. The Impact Arm 310 reaches a speed of 117.8 inches per second, and at that speed, The impact arm 310 articulates back and forth, and therefore, the container or cartridge 2 Contact with 05. The impact arm 310 comes into contact with the container or cartridge 205. The speed at which this can be done allows for a significant reduction in processing time. This is very For known or existing tissue processing techniques that require long processing times And it has significant advantages.

[0132] The tissue will continue until the lipids are broken down to the desired level, or until the desired amount of stem cells are released from the lipids. They are exposed to mechanical shock until they are released. In some embodiments, the lipid sample is Processing takes place within 30 minutes. In some embodiments, the lipid sample is processed within 30 seconds. The processing takes place between 30 and 30 minutes, and in some embodiments, between 30 seconds and 20 minutes. The processing is performed, and in some embodiments, the processing takes place between 30 seconds and 10 minutes. The data is then processed, and in some embodiments, between 30 seconds and 5 minutes.

[0133] The movement of platform 315 is performed in three directional planes (X, Y, and Z). Each of them has its own motor: X-plane motor 230, Y-plane motor 23 5, and controlled by the Z-plane motor 240. In some embodiments, the X-plane Only motor 230 is used. In some embodiments, only Y-plane motor 235 is used. In some embodiments, only the Z-plane motor 240 is used. In some embodiments, only the X-plane motor 230 is used. In some embodiments, Only the X-plane motor 230 and the Y-plane motor 235 are used. Several implementations In this configuration, only the X-plane motor 230 and the Z-plane motor 240 are used. In that embodiment, only the Y-plane motor 235 and the Z-plane motor 240 are used. In some embodiments, the X-plane motor 230, the Y-plane motor 235, and the Z-plane motor are used. A surface motor 240 is used. Motors 230, 235, and 240 are each, It is controlled by the motor controller 225, and the motor controller 225 controls 3 This enables the movement of platform 315 in dimensions.

[0134] This maximum three-dimensional movement of platform 315 allows for the extraction of lipids within the sample. All of this allows the sample to be exposed to mechanical shock during processing. This enables optimal positioning of the processing device.

[0135] Length, impact surface area, number of impact surfaces, and impact arm 310 The shape can be changed. In some embodiments, the total impact surface Increasing the area and / or the total number of impact surfaces allows the lipid sample to... Reduce the amount of time required for sorting.

[0136] Next, the (now processed) lipids are separated from the lipids by mechanical shock. The stem cells are now isolated from sterile saline solution containing them, in a container or carton. Ridge 205 is placed. Since lipids have a lower density than physiological saline, lipids are less dense than physiological saline. It will rise to the top of the water and float. The amount of time required for separation to occur is This will vary depending on the individual composition of the lipid sample. In some embodiments, separation This can occur in 1 to 30 minutes. In some embodiments, separation occurs in 1 to 15 minutes. In some embodiments, separation can occur in 1 to 10 minutes. In some embodiments, separation can occur within 1 to 5 minutes. In some embodiments, separation occurs within 1 minute. This can be done in full. In some embodiments, separation occurs within 10 seconds to 1 minute. It is possible. In some embodiments, separation can be performed in less than one minute.

[0137] The stem cell and stromal vascular cell fractions are isolated here from the lipid sample, and here It is suspended in physiological saline.

[0138] Next, the saline layer containing stem cells is placed as completely as possible in the container or cartridge. 205 is removed, and therefore only lipids remain. Removal is utilized in this method. Depending on the type of container or cartridge 205, this may be done by various means. This is possible. In some embodiments, the physiological saline solution is placed in a container or cartridge 205 Through the tube located at the bottom, it exits the container or cartridge 205. Drain is discharged. Drain discharge is performed actively using either a syringe or a pump. It is possible to proceed either by force or passively by gravity flow. In the embodiment, the layer containing stem cells is a closed, sterile layer as described herein. It will be removed in a manner that preserves the integrity of the environment.

[0139] In some embodiments, the saline solution containing stem cells is placed in a container or cartridge 2 During the removal process from step 05, the material is passed through a filter. The size of the filter can change. This is possible. In some embodiments, the filter size ranges from 40 μm to 100 μm. It is in the range of m. In some embodiments, the filter size is 70 μm. In various embodiments, the filter is made of nylon.

[0140] Either after or during removal from the container or cartridge 205, the stem cells The contained physiological saline is transferred into one or more centrifuge tubes. In this embodiment, the movement is carried out in a closed, sterile environment as described herein. This is done in a manner that maintains the following: At the bottom of the centrifuge tube, the stems are introduced into the pellet. Centrifugation continues to concentrate the cells.

[0141] Stem cells are concentrated by centrifugation at 500g to 1600g for 3 to 10 minutes. They are reduced. In some embodiments, stem cells are centrifuged at 1,200 g for 10 minutes. It is concentrated by separation.

[0142] The fluid is removed from the tube, leaving only the stem cells at the bottom. The fluid is decanted. It can be removed by suction or similar means.

[0143] A small amount of fluid is placed in a centrifuge tube to resuspend the stem cells. In this embodiment, the amount of fluid added to the centrifuge tube is in the range of 1 mL to 5 mL. In some embodiments, the fluid is physiological saline, platelet-rich plasma, hyaluronic acid, and fixative. Gel, hydrogel, scaffold, fibrin glue, glue, or the aforementioned It is one of the combinations. In some embodiments, the cells are in the aspirated material from centrifugation. It is resuspended.

[0144] When stem cells are in a suspension, they are ready for use. The use of stem cells is not limited to reintroduction into patients from whom stem cells have been extracted, cryopreservation, etc. It may include expansion and other processes. In some embodiments, it is concentrated and resuspended. The stem cells are then transferred into a syringe via a subcutaneous injection needle for patient use. In some embodiments, concentrated and resuspended stem cells are used in the heart, muscle, bone, cartilage, and liver. , kidneys, or tissue scaffolds for growth into other tissues and organ structures Seeds are sown inside. In some embodiments, concentrated and resuspended stem cells are used as stem cells. This causes the expression of pluripotent transcription factors, which in turn leads to conversion into induced pluripotent stem cells. ru.

[0145] The first and second methods described above involve a container or cartridge, and The mechanical energy source is portable. In some embodiments, lipids are used in ES It is pumped through a chamber located beneath the W or impact surface. In these embodiments, both the bag and the mechanical energy source are fixed in appropriate locations. The tissue is defined and passed through the chamber, and ESW and / or mechanical energy It will be exposed to energy sources.

[0146] A third embodiment of the method for processing stem cells from lipids is as follows:

[0147] Lipids are obtained from the patient via liposuction. Liposuction is a syringe. This can be carried out via a liposuction machine, where the lipid is directly absorbed through a needle or liposuction machine. It is removed from the patient via the suction. The amount of lipids removed during liposuction is desired. It can change depending on the number of stem cells. In some embodiments, liposacral The suction is a mini-liposuction system, and with a mini-liposuction system, you can get approximately 30cc to approximately 50cc cc of lipids are removed from the patient. In some embodiments, liposuction is performed by a microphone. This is liposuction, and in microliposuction, approximately 5cc to 29cc of lipids are transferred. It is removed from the patient. In some embodiments, liposuction is performed in a typical clinical manner. This is a posuction procedure, in which case more than 300cc of lipids are removed from the patient.

[0148] Once the lipids are removed from the patient, they are transferred into a sterile processing cartridge. The transfer is carried out by sterile means as described herein. In one embodiment, lipids are removed from the patient via a syringe, and the syringe and process Male and female Luer lock connectors are attached to each other between the single cartridge. By attaching it, the liquid is transferred from the syringe to the processing cartridge. In that embodiment, lipids are removed from the patient via a syringe, and the syringe is a syringe Male and female Luer lock connectors are attached to each other between the cartridge and the other. By doing so, it is sterile docked within the cartridge. Several embodiments Then, the lipids are removed from the patient via the syringe, and the pro in the syringe and cartridge is removed. Male and female Luer lock connectors between the cession container or cartridge. By attaching the cartridges together, the syringe can be used to transfer the contents from a processing container or cartridge. It is transferred to the jar. In some embodiments, the lipids are transferred via a liposuction device. Removed from the patient and connected using a sterile connection device, the liposuction device and process By sterile docking the suction cartridge, the liposuction device It is then transferred to the processing cartridge.

[0149] In some embodiments, transport can be carried out via gravity flow. In that embodiment, the transfer can be carried out mechanically, for example, by using a syringe. By physically pressing the plunger, and from inside the syringe the process This is done by pushing lipids into the internal chamber of the cartridge. It is possible. In some embodiments, the transfer is carried out via a device such as a pump. It is possible to break it.

[0150] The volume of the internal chamber of the lipid-receiving cartridges 400 and 500 (for example, Figure) (See the cartridge embodiment provided in Figures 4 and 5) The lipids obtained from the patient It is possible for quality to change along with quantity. In some embodiments, miniliposuction 30cc to 50cc of lipids are removed via the filter, while the lipids in cartridges 400 and 500 are removed. The fat is transferred to fat chambers 405 and 505.

[0151] In this embodiment, lipids are contained in self-contained cartridges 400, 500 of specific components. The solution is injected into cartridges 405 and 505, and the sample is removed from cartridges 400 and 500. Processing of lipid samples without exposure to the surrounding environment using cartridge 400, 5 It is designed to be completely contained within 00. In some embodiments, it receives lipids The fat chambers 405 and 5 are located inside the cartridges 400 and 500. 05 is a flexible bag with a volume ranging from 9 flow ounces to 19 flow ounces.

[0152] In various forms, cartridges 400 and 500 are ESW or mechanical shock devices. Designed for use with any of the methods disclosed herein. In some embodiments, the processing is as described below. Some of the steps are performed automatically by a machine. In this embodiment, the processing is performed by the user, and the user is described below. Perform the steps that are revealed manually. In both embodiments, the processing is Externally controlled, the internal processing of cartridges 400 and 500 is controlled, but the sample is external. It is carried out in a sterile manner, without exposure to the environment.

[0153] In the embodiment shown, the lipid sample is passed through the first one-way valves 410 and 510. By passing through the sample, it is introduced into the fat chambers 405 and 505. The direction of the directional flow valves 410, 510 is as shown, and the direction of the fat chambers 405, 5 It is oriented toward the interior of 05. In some embodiments, after removal from the patient A container for holding lipids and the first one-way valve of processing cartridges 400 and 500. Male and female Luer lock connectors are attached to each other between 410 and 510. By doing so, the lipids are introduced into the internal chambers of fat chambers 405 and 505. In some embodiments, lipids are removed from the patient via a liposuction device. Removed and using a sterile connection device, liposuction device and processing By sterile docking the cartridge, the process from the liposuction device It is transferred to the thievery cartridge.

[0154] Once inside fat chambers 405 and 505, the lipids are washed before processing. Cartridges 400 and 500 contain fluid reservoirs 415 and 515. Fluid reservoirs 415 and 515 are filled with sterile saline solution, and several implementations are carried out. Morphologically, it is filled with sterile 1x phosphate-buffered saline, which is used to wash the tissue. It is used to introduce sterile saline solution into the fat chambers 405 and 505. Therefore, saline solution is passed through the second one-way valves 420 and 520 to the fat chamber 405. It is placed inside 505. The flow direction is as indicated. Physiological saline Water is supplied to the fat chambers 405, 5 via the use of external actuators 425, 525. Moved into the interior of 05, the external actuators 425 and 525 control the first one-way valve 4 To push saline solution through 20, 520, the first plunger 430, 530 Moves. In some embodiments, external actuators 425, 525 are used The system is manually operated by the user, and the desired amount of saline solution is dispensed through a second one-way valve. Until it is inserted into the fat chambers 405 and 505 through 420 and 520, the acti Press the inverters 425 and 525. In some embodiments, an external actuator The movement of 425 and 525 is automated, and an external machine controls the external actuator 425. , causing 525 to move in the direction indicated, thereby in the second direction The desired amount of physiological food enters the fat chambers 405 and 505 through valves 420 and 520. Measure out the saltwater.

[0155] Washing is done gently using cartridges 400 and 500 containing lipids and sterile saline solution. This is done by swinging or rotating it.

[0156] Next, cartridges 400 and 500 contain (now washed) lipids and sterile physiological salt. They are placed so as to be separated from water. In this embodiment, cartridges 400 and 500 are There are two variations. In some embodiments, cartridges 400, 500 are It has a fixed vertical orientation and is contained within fat chambers 405, 505. The lipids and saline wash solution will always naturally separate from each other via gravity. As a result, the lipids float to the top of the fat chambers 405 and 505. In some embodiments, Cartridges 400 and 500 have a horizontal orientation and are rotated vertically. This causes the lipids to float, making it possible to separate them from the saline wash. Because it has a lower density, lipids will rise to the top of the saline solution and float. The amount of time required to perform the procedure varies depending on the individual composition of the lipid sample. This is the result. In some embodiments, separation can occur in 1 to 30 minutes. In the application, separation can occur in 1 to 15 minutes. In some embodiments, separation occurs in 1 It can occur between 1 and 10 minutes. In some embodiments, separation can occur between 1 and 5 minutes. In some embodiments, separation may occur between 10 seconds and 1 minute. The separation can be completed in less than one minute.

[0157] Next, the saline solution is drained as completely as possible from the fat chambers 405 and 505. It is discharged, and therefore only lipids remain. Drain discharge is carried out using the cart used in this method. Depending on the type of Ridge 400 or 500, this can be done through various means. Yes. In some embodiments, saline solution is placed on the outside of cartridges 400 and 500. Through the tube located at the bottom of the flowing fat chambers 405 and 505, the fat Drain the fat from the fat chambers 405 and 505 away from the cartridges 400 and 500. The drain is discharged. Drain discharge is actively carried out using either a syringe or a pump. It is possible to either move or proceed passively via gravity flow. Several implementations In terms of form, drain discharge maintains the integrity of the sterile system as described herein. It is done to maintain it.

[0158] After washing is complete, repeat washing until the lipids turn golden and the saline solution is only slightly cloudy. It is repeated. In some embodiments, washing is performed 1 to 5 times, and in some embodiments This is performed 1 to 4 times, and in some embodiments, 1 to 3 times, and in some embodiments This is performed one to two times, and in some embodiments, it is performed once.

[0159] In some embodiments, sterile saline solution is used from fluid reservoirs 415 and 515. Washing is repeated until all is removed. In some embodiments, fat chamber 405 The saline solution in 505 becomes sufficiently clear, and after washing, 50% to 100% Washing is repeated until light passes through the saline solution and it shows a high degree of transparency. In that embodiment, cleaning is repeated until a set number of cleaning cycles are completed, and the set number In some embodiments, the number of washes is 1 to 5, and in some embodiments, it is 1 wash The process involves four washes, and in some embodiments, one to three washes, and several In some embodiments, there are one to two washes, and in some embodiments, there is one wash. In some embodiments, there are two washes, and in some embodiments, there are three washes. That is the case.

[0160] Once washing is complete, the lipids are ready for processing.

[0161] The lipids in fat chambers 405 and 505 release stem cells from the lipids. By exposing the quality to ESW and / or mechanical shock, in methods 1 and 2 It is processed according to one of the methods described in the document. In the application configuration, cartridges 400 and 500 are located above the fat chambers 405 and 505. It has a mouth section, enabling optimal processing and protection against ESW and / or mechanical shock. These forces do not pass through the rigid plastic exterior of cartridges 400 and 500. In such embodiments, the walls of the flexible fat chambers 405 and 505 are , exposed for processing.

[0162] After tissue processing is complete, lipids separate from the fluid and rise to the top. This is permitted. As described above, in this embodiment, cartridge 400, 500 has two variations. In some embodiments, cartridge 400, 500 has a fixed vertical orientation, and within fat chambers 405, 505 The processed lipids and saline contained in it are always naturally affected by gravity. As a result, they separate from each other, and the lipids float to the top of the fat chambers 405 and 505. In some embodiments, the cartridges 400, 500 have a horizontal orientation. By rotating it vertically, the lipids float to the surface, making it possible to separate them from saline solution. Since lipids are less dense than saline solution, they rise to the top and float above the saline solution. This is what happens. Here, the stem cells released from the lipids are contained in the physiological saline layer. .

[0163] Next, the saline solution containing stem cells is taken from fat chambers 405 and 505, and then from the carton. Lipids are transferred to cell reservoirs 435 and 535, which are located within ridges 400 and 500. It is moved away from the third one-way valve 440, 540. The cell reservoir 435 is accessed via the use of second external actuators 445, 545. , moved into the interior of 535, the second external actuator 445, 545 is the third Second plunger 45 to push saline solution through one-way valves 440 and 540 Move 0 and 550. Inside cell reservoirs 435 and 535, the second plunger 450 and 550 are oriented in a completely closed configuration, and cell reservoirs 435 and 535 The second actuator 445 is completely closed. Moving 545 away from the third one-way valve 440, 540, the second Move plungers 450 and 550 to the inside of cell reservoirs 435 and 535. A vacuum is created, and this vacuum allows the saline layer containing stem cells to move into the fat chamber 405. , 505, through the third one-way valve 440, 540, cell reservoir 435, 535 It is pulled into the interior. In some embodiments, a second external actuator 445, 54 5 is operated manually by the user, and the user controls the saline solution containing stem cells. The entire thing enters the cell reservoirs 435, 535 through the third one-way valves 440, 540. Pull the second actuators 445 and 545 until they are retracted. Several implementations In this configuration, the movement of the second external actuators 445, 545 is automated, and external The machine pulls the second external actuators 445, 545, and the living stem cells are released. Transfer the saline solution into cell reservoirs 435 and 535.

[0164] In some embodiments, saline solution containing stem cells is used to remove unwanted cell damage. To filter and remove the flakes from the processed fatty tissue, pull them through a mesh. It is inserted. In some embodiments, the mesh is placed inside the fat chambers 405, 505. It is positioned, and within fat chambers 405 and 505, the mesh is the third It covers the openings of the one-way valves 440 and 540. In some embodiments, a mesh is used. It is located inside cell reservoirs 435 and 535, and cell reservoir 435 Inside 535, the mesh covers the outlet of the third one-way valve 440, 540. In some embodiments, the mesh is positioned inside the cell reservoir. Furthermore, inside the cell reservoir, the mesh opens the fourth one-way valve 455, 555. It covers the opening. In some embodiments, the pores in the mesh are 40 in size. The pore size ranges from μm to 100 μm, and in some embodiments, the pore size is 70 μm In some embodiments, the mesh is made of nylon.

[0165] In the embodiment shown in Figure 4, the fraction containing stem cells is in the cell reservoir 435. From the inside, through the fourth one-way valve 455, the far-infrared gas contained in the cartridge 400 The stem cell fraction is transferred into cardiac tube 460. The stem cell fraction is then transferred to the second external actuator 445. Through the use of the second external actuator, it is moved into the centrifugal tube 460. 445 pushes saline solution into the centrifuge tube 460 through the third one-way valve 440. To do so, the second plunger 450 is moved. As described herein, This movement can be performed manually or it can be automated. Then centrifugal force Tube 460 is removed from cartridge 400 for centrifugation. Centrifugation is performed. The process proceeds as described in either of the two embodiments described above, and the stem cells The pellet remains at the bottom of centrifugal tube 460.

[0166] In the embodiment described in Figure 5, the fraction containing stem cells is located in the cell reservoir 535 From the inside, through the fourth one-way valve 555, the contents contained in the cartridge 500 It is transferred into the centrifugal separation component 560. The centrifugal separation component 560 is three The rectangular chamber and the triangular chamber act as centrifuge components. This causes the cells to spin out of the saline solution, and the cells move towards the outer edge of the chamber. It is designed to concentrate into a single point. Access port 565 allows users This allows access to the stem cell pellet, resuspends the pellet, and / or Alternatively, the pellets are removed from the centrifugal separation component 560. In that embodiment, the operation of the centrifugal separation component 560 is automated by an external device. It is controlled, and here the external device is in the center of the chamber inside the cartridge It engages with the spin chamber through the motor and gears inserted into it. In that respect, The centrifugal separation component 560 can be either direct drive or geared. This is possible. The layer / substrate containing stem cells is centrifuged as described above. It is loaded into the centrifugation component, and the other side is balanced before centrifugation. Once introduced into the centrifugation component, the stem cells are centrifugated for 3 to 10 minutes at a rate of 500g or Then it is centrifuged at 1600g. In some embodiments, centrifugation is performed at 120 for 10 minutes. It will be done with 0g.

[0167] A small amount of fluid is added to the tube to resuspend the cells. This is typical. Typically, the volume is between 1 mL and 5 mL. The fluid is physiological saline, platelet-rich plasma, hyaluronic acid, and Alternatively, fixation gels, hydrogels, scaffolds, or fibrin or glue, It could be any other compound.

[0168] With the cells in suspension, they can be administered through a subcutaneous needle for further use. It is drawn into the syringe. This is done automatically by the processing machine. Alternatively, it may be done manually by an operator.

[0169] A small amount of fluid is used to resuspend the stem cells. In some embodiments, The amount of fluid added to resuspend the cells ranges from 1 mL to 5 mL. In that embodiment, the fluid is physiological saline, platelet-rich plasma, hyaluronic acid, fixation gel, hyaluronic acid Drogel, scaffold, fibrin, glue, or any combination of the above. This is a combination. In some embodiments, the cells are resuspended in the centrifugal aspirate. .

[0170] The stem cells are suspended in a suspension and are ready for use. Such uses include, without limitation, the reintroduction and freezing of stem cells into patients from whom they were derived. This can include preservation and expansion. In some embodiments, it can be concentrated and re-concentrated. The suspended stem cells are transferred into a syringe via a subcutaneous injection needle for patient use. In some embodiments, concentrated and resuspended stem cells are used in the heart, muscle, bone, and soft tissue. Tissue scaffolds for growth into bone, liver, kidney, or other tissue and organ structures. Seeds are sown in a container. In some embodiments, concentrated and resuspended stem cells are This leads to the expression of pluripotent transcription factors in stem cells, thereby creating induced pluripotent stem cells. It will be converted.

[0171] device As described above, this disclosure provides a method for bioprocessing tissue samples and The system is provided. In addition, in various aspects, this disclosure is provided in the manner disclosed. It provides an easy-to-use device suitable for use together, and such a device is external Such devices are enclosed from the environment and designed for single use. Includes single-use cartridges, and single-use cartridges are complete within a single device. This enables advanced processing.

[0172] container The embodiments described above include withered tissue samples, lipid samples, A fraction containing the desired cell fraction, a fraction containing stem cells, or any of the above. One or more containers are used as the receiving part for the combination. Such containers are shown in Figure Container used in step 105; container 205; fat chambers 405, 50 5; fluid reservoirs 415, 515; and cell reservoirs 435, 535. These containers are suitable for use with the systems and methods provided by this disclosure. Yes, it can be flexible, rigid, or semi-rigid. In some embodiments, the container is a syringe.

[0173] Container used in step 105 of Figure 1; container 205; fat chamber 405, 505; fluid reservoirs 415, 515; and cell reservoirs 435, 535 respectively This, either by itself or as part of a sealed cartridge, protects against the external environment. It is sealed and disposable.

[0174] In some embodiments, the container used in step 105 of Figure 1; container 205 Fat chambers 405, 505; fluid reservoirs 415, 515; and cell reservoirs Each of the 435 and 535 is a flexible container, such as a flexible bag. In such embodiments, each container is made of ethylene vinyl acetate (EVA), poly( Vinyl chloride (PVC), ethylene vinyl acetate (EVA), nylon, or other materials. It can be made from plastic.

[0175] Each of the flexible containers can be blow-molded. In some embodiments, the flexible Each of the containers is either radio frequency welding, high-frequency welding, or induction heating welding. It can be made by blow molding.

[0176] In some embodiments, each of the flexible containers is an internal chamber of the flexible container. It is a three-dimensional bag having at least one outlet that is fluidly connected to several In this embodiment, a single container has two or more outlets that are fluid-connected to the internal chamber of the container. It is possible to have a part. Such an outlet part may be, for example, an internal chamber of the container. To drain the fluid, or to move the fluid from one container to another. It can be used for that purpose.

[0177] In some embodiments, the container is positioned on the outside of the container along the rim. Alternatively, it may include multiple holes, one or more of which suspend the container in space. It can be used to lower something.

[0178] The total volume of the container can range from 5 flow ounces to 50 flow ounces, and how many In that embodiment, the flow rate can range from 9 flow ounces to 19 flow ounces.

[0179] In some embodiments, a tissue sample, sterile saline solution, or other form is used. To receive input, the container receives an inlet line or connection at a separate point. It is configured to allow fluid in, and the inlet line or connection point is fluidly connected to the inside of the container. Connect to the inlet. To maintain the integrity of a closed, sterile environment, the inlet line or connection The connecting part includes a female lure connector, and the female lure connector allows the container to connect to an external device. The container allows for connection, and the syringe containing the tissue sample can be used to release the substance from there. This is another container, or something else, into which the contents will be transferred. In other embodiments The inlet line or connection point connects to the external device using a sterile connection device. This includes sterile docks.

[0180] In some embodiments, the container can be a flat bag. The g has an upper edge, a bottom edge, and two substantially similar side edges. The edges are fluid-connected to the inside of the bag to allow fluid to drain out of the bag. Includes an exit section. Furthermore, the flat bag allows for the sterile introduction of materials into the bag. It has an entrance section.

[0181] In some embodiments, the container is a three-dimensional bag with a rectangular shape. The three-dimensional bag has an upper edge, a bottom edge, and two substantially similar side edges. The bottom edge is connected to the inside of the container to allow fluid to drain out of the container. It includes an outlet section. The upper edge is for introducing material into the container and flowing into the container. Including the entrance portion to which the body is connected.

[0182] In some embodiments, the container is connected to one or more other containers via a line. The line is made of poly(vinyl) chloride (PVC) and ethylene vinyl acetate (EV). A) or tubing that may be made from other materials.

[0183] In some embodiments, the container can be semi-rigid. In the application form, the container includes a rigid plastic housing, and the rigid plastic The woving maintains its shape and can move inside the housing during tissue processing. It can hold a flexible container. The housing is also on platform 210. It is configured to include a mounting point to 315.

[0184] Rigid plastic housings are made of, for example, high-density polyethylene (HDPE) or It is made from any suitable rigid plastic material, including polypropylene (PP). Obtain. Rigid plastic materials do not exhibit elastic deformation, and also rigid plastics The material exhibits either elastic behavior, which is typically represented by flexible plastics. Not necessarily. Rigid plastic housings can be injection molded or die-cut.

[0185] In some embodiments, the flexible container is contained within a rigid plastic housing. The rigid plastic housing is provided, and the flexible container is not completely sealed. or may not be done. In some embodiments, the flexible container is rigid plus It is not completely sealed inside the tick housing. Rather, the housing is flexible. The flexible container is partially sealed and held in place during tissue processing. ru.

[0186] cartridge In various embodiments, cartridges suitable for use with this disclosure are among them. It is a three-dimensional container that can accommodate one or more containers inside. The internal container is They can be connected by one or more lines and / or valves. Suitable cartridge Examples of this are cartridges 400 and 500, shown in Figures 4 and 5.

[0187] In some embodiments, the cartridge is made from a rigid plastic material. Therefore, rigid plastic materials maintain their shape during tissue processing. The cartridge holds at least one, preferably more, flexible containers inside. The containers can be attached to platform 210 or 315. It is configured to include one or more points.

[0188] Rigid plastics include, for example, high-density polyethylene (HDPE) or polypropylene. It can be made from any suitable rigid plastic material, including pyrene (PP). Rigid plastic materials do not exhibit elastic deformation, and rigid plastic materials do not. It does not exhibit any of the elastic behaviors typically represented by flexible plastics. Rigid plastic cartridges can be injection molded or die-cut.

[0189] Other forms of the container are described above.

[0190] Centrifuge tubes Suitable centrifuge tubes for use with the methods and devices disclosed herein are centrifuge rotors. - Designed to fit precisely, made of glass or plastic with a precise finish. This is a high-strength tube. The volume of the centrifuge tube will be processed. It can be varied depending on the total volume of the tissue sample. In some embodiments The centrifuge tubes have capacities ranging from 1 mL to 50 mL, in some embodiments, Volumes ranging from 0.5 mL to 20 mL, and in some embodiments, 250 μL It has a volume ranging from 2.0 mL.

[0191] In some embodiments, the centrifuge tube is an Eppendorf® tube. In some embodiments, it is a microfuse tube, and in some implementations In this context, it is a microcentrifuge tube.

[0192] The material of the centrifugal tube can be varied. In some embodiments, the centrifugal tube The tube is made from glass. In some embodiments, the centrifuge tube is made of plastic. It is made of stick. In each embodiment, the centrifuge tube is designed for single use. They are disposable. In some embodiments, the centrifuge tube is made of polyethylene. It is made from a flexible, transparent plastic such as N, and has a semi-conical shape. It includes an integrated hinged sealing cap.

[0193] One-way valve A one-way valve is a valve that allows a fluid to flow through it in only one direction.

[0194] In various embodiments, a unidirectional orientation suitable for use with the devices and methods of this disclosure. The valve includes a two-port valve, which has two openings, one of which allows fluid to flow through. One is for letting the fluid in, and the other is for letting the fluid out. A one-way valve is for the fluid It functions to automatically provide a constant-direction flow, without any user intervention or control. Not required. In some embodiments, a one-way valve useful in the methods and devices of this disclosure. They are made from rigid plastics such as poly(propylene).

[0195] In some embodiments, a suitable device for use with the devices and methods of this disclosure The directional valve is a ball check valve, and in a ball check valve, a component prevents backflow of fluid. The Nent is a spherical ball. In some embodiments, the ball closes a one-way valve. It is spring-loaded to help maintain its state.

[0196] In some embodiments, a suitable device for use with the devices and methods of this disclosure The directional valve is a diaphragm check valve, and the diaphragm check valve generates valve closure. It includes a flexible rubber diaphragm positioned at such a location. In such embodiments, The pressure generated upstream is caused by user intervention or by automated processes. This causes the diaphragm to open and the fluid to flow through the valve. When the pressure in the lath stops, the diaphragm closes, ending the fluid flow.

[0197] In some embodiments, a suitable device for use with the devices and methods of this disclosure The directional valve is a swing check valve or an inclined disc check valve. Such implementations In this configuration, the disk allows fluid flow in one direction and in the opposite direction. This is a movable part of a valve used to prevent fluid backflow.

[0198] example The materials, methods, and embodiments described herein are further defined in the following examples. It is defined as such. Furthermore, specific embodiments are defined in the examples herein. These examples illustrate specific embodiments, but are given for illustrative purposes only. It should be understood that, from the disclosures herein and these examples, those skilled in the art will understand this. It is possible to confirm the essential characteristics of the disclosure, and to avoid deviations from the spirit and scope of this disclosure. Without escaping, the present invention is designed to adapt to various uses and conditions. Various transformations and modifications are possible.

[0199] Example 1 General Protocols for ESW Tissue Processing The liposuctioned material was obtained from the subject, and its volume is approximately 30cc. Before liposuction, the liposuctioned tissue has side walls approximately 0.25 inches thick. Polyethylene vinyl acetate (EVA), poly(vinyl) chloride (PVC), ethylene vinyl acetate It is transferred to a sterile, flexible bag made of EVA or nylon. Sterile catheter bag The bag is sufficient.

[0200] Position the male Luer lock connector from the saline bag onto the flexible bag. By connecting to the installed female Luerlock connector, and, By manually dispensing saline solution into a flexible bag, the liposuction material is removed. The bag is washed with an equal volume of sterile saline solution introduced into the flexible bag. Next, the liposuction material and saline solution are gently shaken in a flexible bag. It's then mechanically stirred.

[0201] The tissue sample / saline solution mixture should be allowed to stand for at least 10 minutes before liposuction. This allows objects to float on top of saline solution.

[0202] The supernatant or saline solution is located in the drain at the bottom of the flexible bag. It is removed from the flexible bag via the line. By connecting the syringe to the line... And, by drawing the supernatant out of the bag, or by gravity flow Removal can be performed.

[0203] The washing process is repeated 3-4 times, and in the final wash, the sterile saline solution inside the bag is washed away. leave.

[0204] The next step is to use the Masterpuls MP100 extracorporeal shock wave generator to This involves applying external shock waves to the liposuctioned tissue.

[0205] The generator may be in contact with the wall of the flexible bag, but the shock wave generator may be in contact with the liposuctioned tissue itself. Care should be taken to ensure that there is no contact with it.

[0206] If necessary, the ultrasound gel is applied to the flexible bag before applying shock waves to the liposuctioned tissue. It may also be applied to the sides.

[0207] Apply ESW to the liposuctioned tissue for 1 to 30 minutes.

[0208] Afterward, gently shake or vortex the flexible bag to release its shape. Stir.

[0209] Now the processed liposuction material is left to stand, and the remaining fatty tissue is left in saline solution. It is designed to float above it.

[0210] As explained above, by attaching a sterile syringe to the drain tube... And, by drawing out as much of the supernatant as possible, from the flexible bag Remove the clear tissue. Do not remove any remaining fatty tissue.

[0211] During removal, three nibs with pore sizes of 40 μm, 70 μm, or 100 μm are removed. The supernatant is passed through one of the long mesh filters.

[0212] Distribute the supernatant into one or more sterile centrifuge tubes. An odd number of tubes If used, a balance tube containing sterile saline solution is used for balance. It is guaranteed to be installed inside the rotor.

[0213] Centrifuge at 1,200g for 10 minutes.

[0214] Optional: Resuspend the pellet in 1 mL of RBC lysis buffer and leave for 10 minutes. Cultivate in a tank at °C. Neutralize the RBC buffer.

[0215] If RBC lysis buffer is not desired, the cells can be lysed in 1 mL of sterile saline solution. Resuspend the lett.

[0216] Count the cells.

[0217] Example 2 Comparison of liposuction processing methods Summary: The purpose of the test performed in this example is to use collagenase digestion to break down fat cells. The yield of nucleated cells from mesenchymal stem cells extracted from tissue and the yield of cells obtained by extracorporeal shock wave (ESW) disruption. The task was to compare the two.

[0218] material: 1,500 mL of 1x phosphate-buffered saline (PBS) 35 mL of 1x red blood cell (RBC) lysis buffer 200 mL of 0.1% collagenase type I 15 mL of 10% fetal bovine serum (FB) in Dulbecco's modified Eagle medium (DMEM) S)

[0219] method: control Approximately 250 mL of liposuction material was removed using a sterile 60 mL catheter, and then 100 mL of the liposuction material was removed. It was transferred into the L beaker. Almost no excess fluid was introduced into the beaker, or none at all. Care was taken to ensure that it would not be implemented.

[0220] The liposuctioned tissue was washed four times with approximately equal volumes of sterile 1xPBS. For washing, sterile 1x PBS is added to the beaker and stirred twice. The mixture was allowed to stand. The supernatant was removed from the beaker by aspirating. The pulleys were washed a total of four times.

[0221] After the fourth wash, the samples were allowed to stand for 10 minutes.

[0222] A sterile 10 mL serological pipette was used, and 45 mL of the supernatant was taken to the fourth final stage. From the washing solution, 40 μm, 70 μm, into three sterile 50 mL cone-shaped tubes. The supernatant was then transferred through a 100 μm tube sterile filter. A total of 135 mL of supernatant was obtained. However, it was transported for use as a control.

[0223] Collagenase digestion 200 mL of liposuction material was deposited into a sterile 500 mL glass bottle. 00 mL of 0.1% collagenase type I was added to the bottle. To ensure that the enzyme is evenly mixed, turn the bottle upside down several times. It was lightly stirred by this.

[0224] Next, the bottle was placed in a tank of water at 37°C. Every 10 minutes, the bottle was removed from the tank. It was removed, turned upside down several times, the liposuctioned material was redistributed, and the bottle was allowed to settle. 30 minutes After the culture, the bottles were agitated one last time and removed from the tank.

[0225] The outside of the bottle is sprayed with 70% ETOH and placed inside a fume hood. The liposuction material and collagenase mixture were allowed to stand for 10 minutes.

[0226] A sterile 10 mL serological pipette was used, along with three sterile 50 mL cone-shaped tubes. The tubes are passed through sterile filters containing 40μm, 70μm, and 100μm tubes. The supernatant was transferred. A total of 135 mL of supernatant was transferred. 45 mL for balance. Three tubes, including a fourth tube containing water, contain collagenase-digested cells. To obtain pellets, 1,200g was centrifuged at 37°C for 10 minutes.

[0227] The outside of the three tubes containing the collagenase suspension is cleared with 70% ETOH. It was rinsed and placed inside the fume hood, leaving the balance tube. The supernatant was aspirated. Then, the cell pellet was left in each tube. 25 mL serological pipette Using this method, 5 mL of 10% FBS is dispensed into each tube, Each tube was vortexed for 5 seconds to resuspend the pellet. FB S was used to neutralize any remaining collagenase activity.

[0228] The three tubes were centrifuged at 1,200g for 10 minutes at 37°C. The outside of the tube is cleaned with 70% ETOH, and the tube is a fume hood. It was placed inside. The supernatant was aspirated, leaving the cell pellet.

[0229] ESW Processing 200 mL of liposuction material was removed using a sterile 60 mL catheter syringe, with the remainder being removed below. The entire sample, along with all of it, was transferred into a sterile 500 mL leg catheter bag. 1x PBS containing bacteria was added to achieve a total fluid volume of 200 mL, and the total volume was It was 400 mL including liposuction material. Approximately 2 mL of ultrasound gel was used in the catheter. It was applied to the . Using a large tip, approximately at 2 bar and 21 Hz. 10,000 ESW pulses were administered to the sample inside the bag. This process was An additional 10,000 ESW pulses were repeated on the other side of the bag. The outside of the catheter bag was cleaned with 70% ETOH, and it was then... It was placed inside the hood.

[0230] The bag is stirred, poured into a 500 mL beaker and emptied, and the suspension is left for 10 minutes. It was allowed to stand for a while. Using a sterile 10 mL serological pipette, the supernatant was collected in three sterile samples. 40μm, 70μm, and 100μm tubes into a 50mL cone-shaped tube. The solution was transferred through a sterile filter. A total of 135 mL of supernatant was transferred.

[0231] Concentration and dissolution Three tubes containing a control suspension, and a processed suspension. Three tubes containing the substance were centrifuged for 10 minutes at 1,200 g and 37°C. The supernatant was aspirated from all tubes, leaving each pellet, and 5 mL of 1xR was used. BC lysis buffer was added to each tube. Each tube was then filled with pellets. To resuspend the tube, vortex it for 5 seconds, then soak it in 37°C water for 10 minutes. It was placed inside the tank.

[0232] The outside of each tube is disinfected with 70% ETOH, and the tubes are then treated with a futile hose. It was placed in a hood. 25 mL of sterile 1xPBS was used to dilute the lysis buffer and To neutralize the effect, it was added to each tube.

[0233] cell counting Using a sterile micropipette tip, 300 μL of sterile 1x PBS is dispensed into six containers. It was transferred into sterile 0.5 mL microcentrifuge tubes. Also, 75 μL of each... Each 50 mL sample tube is used for cell counting, and each microcentrifuge tube is used for counting cells. They were transported inside the tube.

[0234] The number of cells in each sample is measured using both M-type and S-type cassettes. The cells were counted using a MoxiZ cell counter. To count, 100 μL of trypan blue was added to each microcentrifuge tube, and 10 μL of it was added. Each sample was loaded into a hemocytometer well, and the cells were counted. .

[0235] result: MoxiZ Cell Counter Table 1 shows the results of cell counting using the MoxiZ automated cell counter. Two types of cassettes were used for counting (M and S). Sample In the column, "c" represents collagenase digestion, and "e" represents ESW processing. And "0" represents control. Only counts with "y" in the "Completed" column. This is effective.

[0236] [Table 1-1]

[0237] [Table 1-2]

[0238] [Table 1-3]

[0239] Hemocytometer Table 2 details the counts obtained from the visual hemocytometer, and Cell count from each quadrant, total for the quadrants, dilution factor, and total sum. It is accompanied by counted cells. The number of cells refers to the cell count in the control group. This should be taken into consideration. The ratio of processed cells to control cells is It is an important indicator of the rate.

[0240] [Table 2]

[0241] overview Of the valid counts using the MoxiZ automated cell counter, control, co The overall mean cell counts for lagenease and ESW are shown in Table 3. That is correct.

[0242] [Table 3]

[0243] Collagenase consistently produced approximately 3.8E+05 cells / mL. ESW was The cell count ranged from 1.67e+05 to 7.08e+05 cells / mL.

[0244] Looking at the cell ratio in the hemocytometer count, the ESW process has the best ratio The ratio was generated, and that ratio ranged from 1.78 to 9.0 times higher than the control group. Only collagenase levels were 1.29 to 2.84 times higher than the control. A ratio was created.

[0245] Discussion These data suggest that ESW is effective in generating a high number of mesenchymal stem cells from adipose tissue. This demonstrates that it is effective. Based on this data, mesenchymal stem cells can be obtained. To achieve this, ESW performs ultrasound processing or collagenase processing of adipose tissue. It was concluded that this is a viable alternative to either of the peaks. In comparison, ESW has a collagenase peak of 7.08e+0 compared to 4.32e+05. This produced a cell yield of 5, which represented a 164% increase in yield.

[0246] Given the data presented above, the sample size of 50cc to 60cc of liposuction material is calculated. The pull process will generate approximately 5 million cells from ESW processing, and collagen It is reasonable to conclude that approximately 3 million cells are produced from the digestion of ze. That is the case.

[0247] Therefore, it is clear that using ESW to release stem cells from adipose tissue offers significant advantages over known techniques. The inventions described in the original claims of this application are listed below. [Invention 1] A method for isolating a cell fraction from a tissue sample, wherein the method is: The steps include obtaining tissue samples from the subject, The steps of bringing the tissue sample into contact with a shock wave, a force from a mechanical shock, or both, The steps include isolating the cell fraction from the tissue sample, Includes, The source of the force from the shock wave and / or the mechanical shock does not make physical contact with the tissue sample. [Invention 2] The method according to Invention 1, wherein the tissue is selected from adipose tissue, brain tissue, pharyngeal tissue, laryngeal tissue, heart tissue, arterial tissue, muscle tissue, liver tissue, gallbladder tissue, kidney tissue, small intestine tissue, large intestine tissue, lymph node tissue, lung tissue, spleen tissue, bone marrow tissue, stomach tissue, venous tissue, pancreatic tissue, bladder tissue, bone, tooth, dentin tissue, gingival tissue, skin tissue, pineal gland tissue, pituitary tissue, thyroid tissue, adrenal gland tissue, pancreatic tissue, ovarian tissue, and testicular tissue. [Invention 3] The method according to Invention 1 or Invention 2, wherein the tissue is adipose tissue. [Invention 4] The method according to any one of inventions 1 to 3, wherein the tissue sample is brought into contact with a shock wave, and the source of the shock wave is a shock wave applicator powered by a shock wave generator. [Invention 5] The method according to any one of inventions 1 to 3, wherein the tissue sample is brought into contact with a force from a mechanical impact, and the source of the force from the mechanical impact is an impact arm powered by a motor. [Invention 6] The method according to any one of inventions 1 to 5, wherein the tissue sample is washed once or more times before it comes into contact with the force from the shock wave and / or the mechanical shock. [Invention 7] The method according to any one of Inventions 1 to 4 or 6, wherein the shock wave decomposes the tissue sample into a plurality of small cell groups, a plurality of individual cells, or both. [Invention 8] The method according to any one of Inventions 1 to 3, 5, or 6, wherein the force from the mechanical impact decomposes the tissue sample into a plurality of smaller cell groups, a plurality of individual cells, or both. [Invention 9] The method according to Invention 7 or Invention 8, wherein the cell fraction comprises the plurality of small cell groups, the plurality of individual cells, or both, and the isolation of the cell fraction is performed by centrifugation. [Invention 10] The method according to Invention 9, wherein centrifugation is performed at a rate of 500g to 2,000g for 3 to 30 minutes. [Invention 11] The method according to Invention 10, wherein centrifugation is performed at 1,200g for 10 minutes. [Invention 12] The method according to any one of inventions 9 to 11, wherein the isolated cell fraction is resuspended after centrifugation. [Invention 13] The cell fraction is isolated within 30 minutes, according to any one of inventions 1 to 3, 5, 6, or 8 to 12. [Invention 14] A method for isolating stem cells from adipose tissue, wherein the method is The steps include obtaining adipose tissue samples from the subject, The steps include: placing the tissue sample in a container or cartridge; The steps include exposing the tissue sample to force from mechanical impact to release the stem cells, The steps include separating the stem cell fraction from the adipose tissue, The steps of centrifuging the aforementioned stem cell fraction, Includes, The source of the force from the mechanical impact does not physically contact the adipose tissue. [Invention 15] The method according to invention 14, wherein the adipose tissue is washed once or more times before being subjected to the force from the mechanical impact. [Invention 16] The method according to invention 14 or invention 15, wherein the source of the force of the mechanical impact is an impact arm powered by a motor, the motor includes a gearing, and the gearing can reduce the speed of the impact arm when the motor is operating at full speed. [Invention 17] The method according to invention 14 or invention 15, wherein the force from the mechanical impact is delivered to the adipose tissue through the wall of the container or cartridge. [Invention 18] The gearing allows for a reduction of 1 / 10 of the speed within the impact arm, according to any one of inventions 15 to 17. [Invention 19] The method according to any one of inventions 16 to 18, wherein the motor includes a gear diameter of 0.75 inches, and at a motor speed of 3,000 rpm, the impact arm has a speed of 117.8 inches per second. [Invention 20] The method according to any one of inventions 14 to 19, wherein the step of separating the stem cell fraction includes a step of enabling the adipose tissue to be separated from the aqueous layer, the aqueous layer comprising the stem cells. [Invention 21] The method according to any one of inventions 14 to 20, wherein the stem cells are isolated from the adipose tissue within 30 minutes. [Invention 22] The method according to any one of inventions 14 to 21, wherein centrifugation is performed for 3 minutes to 10 minutes with a volume of 500 g to 1,600 g. [Invention 23] A method for isolating stem cells from adipose tissue, wherein the method is The steps include obtaining adipose tissue samples from the subject, The steps include: placing the tissue sample in a container or cartridge; The steps include exposing the tissue sample to shock waves to release the stem cells, The steps include separating the stem cell fraction from the adipose tissue, The steps of centrifuging the aforementioned stem cell fraction, Includes, The method by which the source of the shock wave does not physically contact the adipose tissue. [Invention 24] The method according to invention 23, wherein the adipose tissue is washed once or more times before being exposed to the shock wave. [Invention 25] The method according to invention 23 or invention 24, wherein the source of the shock wave is a shock wave applicator powered by a shock wave generator. [Invention 26] The method according to any one of inventions 23 to 25, wherein the shock wave is delivered to the adipose tissue through the wall of the container or cartridge. [Discussion 27] The method according to any one of inventions 23 to 26, wherein the adipose tissue is exposed to shock waves having a power of 0.5 bar to 5.0 bar. [Invention 28] The method according to any one of Inventions 23 to 27, wherein the container is a vinyl bag having walls 0.25 mm thick, and the adipose tissue is subjected to shock waves having a power of 2.0 bar to 2.5 bar. [Invention 29] The total area of ​​the adipose tissue exposed to the shock wave at any one point in time is 1 cm². 2 From 100cm 2 A method according to any one of inventions 23 to 28, which falls within the scope of the invention. [Invention 30] The container is a 19-ounce vinyl bag with walls 0.25 mm thick, the total volume of the adipose tissue sample is 30 cc, and the total area of ​​the adipose tissue exposed to the shock wave at any one point in time is 5 cm². 2 The method according to invention 23. [Invention 31] The method according to any one of inventions 23 to 30, wherein the adipose tissue is exposed to several shock waves ranging from 5,000 to 100,000. [Invention 32] The method according to any one of inventions 23 to 31, wherein the step of separating the stem cell fraction includes a step of enabling the adipose tissue to be separated from the aqueous layer, the aqueous layer comprising the stem cells. [Invention 33] The method according to any one of inventions 23 to 32, wherein centrifugation is performed for 3 minutes to 10 minutes with a volume of 500 g to 1,600 g. [Explanation of Symbols]

[0248] 200 Processing Equipment 205 Processing container or cartridge 210 platforms 215 Shockwave Applicator 220 Applicator Platforms 225 Motor Controller 230 X Planar Motor 235 Y-plane motor 240 Z Planar Motor 245 Shock wave generator 300 Processing Equipment 305 Motor 310 Impact Arm 315 Platforms 400 cartridges 405 Fat Chamber 410 First one-way valve 415 Fluid Reservoir 420 Second one-way valve 425 External actuator 430 First plunger 435 Cell Reservoir 440 Third one-way valve 445 Second external actuator 450 Second plunger 455 Fourth one-way valve 460 Centrifuge tubes 500 cartridges 505 Fat Chamber 510 First one-way valve 515 Fluid Reservoir 520 Second one-way valve 525 External actuator 530 First plunger 535 Cell Reservoir 540 Third one-way valve 545 Second external actuator 550 Second plunger 555 Fourth one-way valve 560 Centrifugal Separation Components 565 Access Ports

Claims

1. A tissue processing apparatus, Processing container for holding tissue, The platform on which the processing container is attached; Impact arm; and A first motor drives the impact arm so that it articulates up and down at a maximum speed of 30,000 rpm to physically contact the processing container. Includes, The joints of the impact arm generate a mechanical impact applied to the outer surface of the processing vessel, which breaks down the tissue and isolates stem cells from the tissue. The first motor controls the speed at which the impact arm contacts the processing container. The aforementioned tissue processing apparatus.

2. The apparatus according to claim 1, wherein the motor drives the upper joint movement of the impact arm at a speed selected from the group consisting of a maximum of 20,000 rpm, a maximum of 10,000 rpm, a maximum of 5,000 rpm, and 3,000 to 30,000 rpm.

3. The apparatus according to claim 1, wherein the first motor is a variable speed motor.

4. The apparatus according to claim 1, further comprising a second motor for controlling the movement of the platform.

5. The apparatus according to claim 4, wherein the second motor controls the vertical movement of the platform.

6. The apparatus according to claim 1, wherein the processing container is a cartridge.

7. The apparatus according to claim 6, wherein the cartridge includes an outlet that is fluidly connected to the inside of the container.

8. The apparatus according to claim 6, wherein the cartridge includes a plurality of outlets that are fluidly connected to the inside of the container.

9. The apparatus according to claim 7, wherein the cartridge further includes an inlet that is fluidly connected to the inside of the container.

10. The apparatus according to claim 7, wherein the cartridge further includes a plurality of inlets that are fluidly connected to the inside of the container.

11. The apparatus according to claim 1, wherein the processing vessel does not contain enzymes other than those in the tissue.

12. The apparatus according to claim 1, wherein the impact does not cause mixing of the stem cells with the remaining tissue.

13. The apparatus according to claim 1, wherein the first motor is controlled by a microprocessor.

14. The apparatus according to claim 4, wherein the second motor is controlled by a microprocessor.

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