Methods for separating, dissociating and / or lysing cells using shock waves or mechanical impact

Abstract

The method provided by the present disclosure utilizes extracorporeal shock waves, mechanical shocks and / or lithotripsy principles to break tissue samples into smaller cell fragments, cell clusters and / or single cells, after which the desired cell fractions can be separated from the sample. The device provided by the present disclosure utilizes concentrated and / or directed shock waves and / or concentrated and directed mechanical shocks to separate tissue samples. During exposure to shock waves or mechanical shocks, the device maintains the sample in a sterile, closed environment. Therefore, shock waves and / or mechanical shocks are generated outside the closed device and transmitted through one or more walls of the device to the interior of the device where the sample is located.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of March 17, 2017, application number 201780030309.7, and invention name “Method for separating, dissociating and / or lysing cells using shock waves or mechanical shock”. Technical Field

[0002] The present invention relates to methods and devices for separating, dissociating and / or lysing cells using shock waves or mechanical impact Background Art

[0003] Cell bioprocessing is a form of biopharmaceutical production that aims to establish reproducible and stable manufacturing methods for the isolation and / or production of therapeutic cells.

[0004] Current cell production processes are highly user-dependent, requiring human intervention at many points. Due to this dependency, current methods are cumbersome, highly variable, expensive, and produce low yields.

[0005] As an example, many current manufacturing methods are performed as follows: tissue samples (lipoaspirates, whole blood, bone marrow, umbilical cord blood, etc.) are obtained from patients. The obtained biological samples are transferred to a laboratory for bioprocessing. In the laboratory, cell separation techniques are used to obtain the desired cell fractions. The cell fractions are then separated and transferred back to the point-of-care facility for use by the patient, or further processed by introduction into a bioreactor for cell expansion. Some known cell separation methods include the use of mechanical separation by ultrasonic cavitation, enzymatic digestion or other mechanical methods, which include contacting the sample with a foreign object (e.g., beads) to obtain the desired cell fraction. The use of enzymes leads to an increased regulatory compliance burden because the enzymes must be in direct contact with the sample to produce the desired results. Enzymes can also destroy the integrity of harvested cells and may change their properties. There are also drawbacks to using high-power ultrasonic processing. The sonicator tip frequency is fixed and must be introduced into a cell or tissue suspension, which may have a negative impact on sterility. In addition, the use of such high-energy sound waves introduces a large amount of energy into the sample, which manifests as heat. This destroys the cells, thereby reducing the yield of usable cells.

[0006] Once obtained, the expanded cell product is then removed from the bioreactor where it is purified and concentrated. A test sample is taken from the washed and concentrated cell product for quality testing, and if the test results indicate an acceptable product, the final engineered cell product is prepared for long-term storage, cryopreservation, administration to the patient from whom the sample was derived, or some combination of the foregoing.

[0007] As can be appreciated, several of these steps require moving the sample from one container to another, requiring extensive user intervention, which not only increases the risk of mislabeled or misprocessed samples during processing, but can also negatively impact the sterility, identity, purity, or potency of the sample. Additionally, the time required to process the sample in this manner means that the desired cell fraction is not ready for delivery to the patient for hours, if not days, which can negatively impact the patient's health. The potential for compromised sterility and unreasonably long time periods required to process cell samples is unacceptable to patients, particularly those who are immunocompromised or require immediate care, and is also unacceptable to many patients who require timely, high-quality care. Summary of the invention

[0008] Therefore, there is a need for an optimized and scalable method to obtain the desired cell fractions directly at the point of care in a short time and without multiple user interventions. There is also a need for a device capable of implementing the method. The present disclosure provides such methods and devices. The disclosed methods and devices eliminate the need to transfer cell samples to a laboratory for processing and do not expose cell samples to foreign matter during separation, resulting in processed samples providing the desired cell samples in a very short time frame with little contact with the user, thereby allowing optimization of expensive technical resources.

[0009] The apparatus and methods disclosed herein have several features, no single one of which is solely responsible for their desirable attributes. Without limiting the scope of the claims that follow, certain features of the disclosed apparatus and methods will be briefly discussed. After considering this discussion, and particularly after reading the section heading "Detailed Description," one of ordinary skill in the art will understand how the features of the apparatus and methods provide several advantages over conventional systems and methods.

[0010] In one aspect, the present disclosure provides a fully closed system suitable for bioprocessing of cell samples, such as processing adipose tissue samples for autologous stem cell therapy. The system is not open to air, thus allowing sterile sample processing and sample transfer throughout the bioprocessing process.

[0011] In a first aspect, a method for separating a cell fraction from a tissue sample is provided, the method comprising: obtaining a tissue sample from a subject; contacting the tissue sample with a shock wave, a mechanical shock force, or both; and separating the cell fraction from the tissue sample; wherein the source of the shock wave and / or the source of the mechanical shock force does not come into physical contact with the tissue sample.

[0012] In some embodiments, 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, vein tissue, pancreatic tissue, bladder tissue, bone, tooth, dentin tissue, gum tissue, skin tissue, pineal gland tissue, pituitary gland tissue, thyroid gland tissue, adrenal gland tissue, pancreatic tissue, ovarian tissue and testicular tissue.

[0013] In some embodiments, the tissue is adipose tissue.

[0014] In some embodiments, the tissue is contacted with shock waves and the source of the shock waves is a shock wave applicator driven by a shock wave generator.

[0015] In some embodiments, the tissue sample is contacted with a mechanical impact force, and the source of the mechanical impact force is an impact arm driven by a motor.

[0016] In some embodiments, the tissue sample is washed one or more times prior to contacting with the shock wave and / or mechanical impact force.

[0017] In some embodiments, the shock waves fragment the tissue sample into a plurality of small cell clusters, a plurality of single cells, or both.

[0018] In some embodiments, the mechanical impact force breaks the tissue sample into a plurality of small cell clusters, a plurality of single cells, or both.

[0019] In some embodiments, the cell fraction comprises a plurality of small cell clusters, a plurality of single cells, or both, and separating the cell fraction is performed by centrifugation.

[0020] In some embodiments, centrifugation is performed at a speed of 500 g to 2,000 g for 3 to 30 minutes.

[0021] In some embodiments, centrifugation is performed at 1,200 g for 10 minutes.

[0022] In some embodiments, the separated cell fraction is resuspended following centrifugation.

[0023] In some embodiments, the cell fraction is separated in 30 minutes or less.

[0024] In a second aspect, a method for isolating stem cells from adipose tissue is provided, the method comprising: obtaining an adipose tissue sample from a subject; placing the tissue sample in a container or cartridge; subjecting the tissue sample to a mechanical impact force to release the stem cells; separating the stem cell fraction from the adipose tissue; and centrifuging the stem cell fraction; wherein the source of the mechanical impact force is not in physical contact with the adipose tissue.

[0025] In some embodiments, the adipose tissue is washed one or more times and then subjected to mechanical impact.

[0026] In some embodiments, the source of the mechanical impact force is an impact arm driven by an electric motor that includes gears that are capable of reducing the speed of the impact arm when the motor is running at full speed.

[0027] In some embodiments, the mechanical impact force is delivered to the adipose tissue through the wall of the container or cartridge.

[0028] In some embodiments, the gear allows the speed in the impact arm to be reduced from 10 to 1.

[0029] In some embodiments, the motor comprises a gear diameter of 0.75 inches and at a motor speed of 3000 rpm, the impact arm has a speed of 117.8 inches per second.

[0030] In some embodiments, isolating the stem cell fraction comprises allowing the adipose tissue to separate from the aqueous layer, the aqueous layer comprising the stem cells.

[0031] In some embodiments, stem cells are isolated from adipose tissue in 30 minutes or less.

[0032] In some embodiments, centrifugation is performed at 500 g - 1,600 g for 3-10 minutes.

[0033] In a third aspect, a method for isolating stem cells from adipose tissue is provided, the method comprising: obtaining an adipose tissue sample from a subject; placing the tissue sample in a container or cartridge; subjecting the tissue sample to shock waves to release the stem cells; separating the stem cell fraction from the adipose tissue; and centrifuging the stem cell fraction; wherein the source of the shock waves is not in physical contact with the adipose tissue.

[0034] In some embodiments, the adipose tissue is washed one or more times prior to being subjected to the shock waves.

[0035] In some embodiments, the shock wave source is a shock wave applicator, which is driven by a shock wave generator.

[0036] In some embodiments, the shock waves are delivered to the adipose tissue through the wall of the container or cartridge.

[0037] In some embodiments, the adipose tissue is subjected to shock waves having a force of 0.5 to 5.0 bars.

[0038] In some embodiments, the container is a vinyl bag having a wall 0.25 mm thick, and the adipose tissue is subjected to a shock wave having a force of 2.0 to 2.5 bar.

[0039] In some embodiments, the total area of ​​adipose tissue subjected to the shock waves at any one time is 1 cm 2 -100cm 2 .

[0040] In some embodiments, the container is a 19 oz vinyl bag with a wall thickness of 0.25 mm, the total volume of the adipose tissue sample is 30 cc, and the total area of ​​adipose tissue exposed to the shock wave at any one time is 5 cm 2 .

[0041] In some embodiments, the adipose tissue is subjected to a plurality of shock waves between 5,000 and 100,000.

[0042] In some embodiments, isolating the stem cell fraction comprises allowing the adipose tissue to separate from the aqueous layer, the aqueous layer comprising the stem cells.

[0043] In some embodiments, centrifugation is performed at 500 g - 1,600 g for 3-10 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Features and advantages of the present disclosure may be further explained by reference to the following detailed description and accompanying drawings, which set forth exemplary embodiments.

[0045] Figure 1 An overview of methods of isolating a cellular fraction from a tissue sample according to embodiments provided by the present disclosure is provided.

[0046] Figure 2 Representative examples of extracorporeal shock wave devices suitable for use with the systems and methods provided by the present disclosure are depicted.

[0047] Figure 3 Representative examples of mechanical impact devices suitable for use with the systems and methods provided by the present disclosure are depicted.

[0048] Figure 4 A first representative example of a rigid, test-per-unit cartridge suitable for use with the extracorporeal shock wave and / or mechanical percussion devices provided by the present disclosure is depicted. In the illustrated embodiment, several of the internal components are flexible containers.

[0049] Figure 5 Depicted are representative examples of rigid, in-house test cartridges suitable for use with the extracorporeal shock wave and / or mechanical impact devices provided by the present disclosure. DETAILED DESCRIPTION

[0050] Before describing the embodiments of the present disclosure, it should be understood that these embodiments are provided by way of example only, and various alternatives of the embodiments of the present disclosure described herein can be used to implement the present disclosure. Without departing from the present disclosure, those skilled in the art will now think of many variations, changes and substitutions.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of the disclosure, suitable methods and materials are described below. In case of conflict, patent specifications (including definitions) will be controlled. In addition, materials, methods and embodiments are exemplary only and not restrictive. Without departing from the disclosure, those skilled in the art will expect many variations, changes and substitutions.

[0052] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0053] As used herein, "extracorporeal shock waves" and / or "ESW" means a sudden, high-amplitude pulse of mechanical energy, similar to a sound wave, which is generated outside a target sample and then transmitted to the sample. In this regard, the source of the ESW does not make physical contact with the sample; the shock wave itself contacts the sample, but the source does not. Thus, the extracorporeal shock wave is generated by a source at a distance from the sample and then transmitted to the sample. The transmission can occur through the air and / or through the wall of the container in which the sample is located. The ESW can be concentrated and / or directed so that, for example, the ESW is concentrated in a target area that can cover the entire sample or only a portion of the entire sample.

[0054] As used herein, "subject" or "patient" refers to a mammal, such as a human.

[0055] The present disclosure provides methods that utilize extracorporeal shock waves, mechanical shock, vibration and / or lithotripsy principles to break tissue samples into smaller fragments - cell clusters and / or single cells - after which desired cell fractions can be isolated from the sample. In various aspects, the method includes isolating a sample from a subject, the sample containing a target cell type, contacting the sample with extracorporeal shock waves, but not with a device for generating ESW, to break the sample into cell clusters and / or single cells, and then isolating the target cell type from the broken sample.

[0056] The device provided by the present disclosure utilizes concentrated and / or directional shock waves, and / or focused and directional mechanical shocks to break up the target sample. In different aspects, the device provided by the present disclosure keeps the sample in a sterile closed environment during exposure to shock waves or mechanical shocks. Therefore, shock waves and / or mechanical shocks are generated outside the closed sterile container and are transmitted to the inside thereof by one or more walls of the container, and the sample is located inside thereof.

[0057] By maintaining the ESW source at a distance from the target sample, the ESW source (the device that produces them) is not in physical contact with the sample. This greatly improves the sterility of the sample and any cell fractions separated from the sample. From the perspective of a healthcare provider, this also greatly improves the requirements for sterility and helps ensure that the target cell fraction from the sample is sterile for patient use. In addition, maintaining the ESW generation source at a distance from the target ensures that the generator does not add energy or heat to the target sample, which may damage or kill cells, damage DNA and significantly reduce yield. In this regard, the device provided by the present disclosure is an improvement over other known systems such as ultrasound.

[0058] The method and device provided by the present disclosure can quickly separate the desired cell fraction from the tissue sample, in some embodiments, within 30 minutes or less. A certain speed (at which the disclosed method and device can be used) gives a significant advantage, which is that the desired cell fraction separated from the sample can be used immediately.

[0059] method

[0060] Figure 1 Methods of isolating a target cell fraction from a tissue sample according to embodiments provided by the present disclosure are presented.

[0061] In the depicted embodiment, the method begins by first extracting tissue from a subject (100). The tissue can be of any type, as long as the tissue is fragmented when subjected to an extracorporeal shock wave. In some embodiments, the tissue is adipose tissue or fat. In some embodiments, the tissue is from an internal organ, such as the brain, pharynx, larynx, heart, artery, muscle, liver, gallbladder, kidney, small intestine, large intestine, lymph node, lung, spleen, bone marrow, stomach, vein, pancreas, bladder, bone, tooth, dentin, gums or skin. In some embodiments, the tissue is from a component of the endocrine system, such as the pineal gland, pituitary gland, thyroid gland, adrenal gland, pancreas, ovary or testicle.

[0062] Extraction can be performed by many known methods. In some embodiments, tissue extraction is performed by a syringe. In some embodiments, tissue extraction is performed by surgery so that the tissue sample is placed in the first container after being removed from the subject.

[0063] The tissue is then transferred to a processing container or cartridge 105. The apparatus provided by the present disclosure provides all the components required for rapid and easy-to-use ESW cell bioprocessing. Even though some components of the disclosed methods and / or apparatus may be separable from one another, they are configured to be connected to one another in a completely sterile manner, thereby allowing the sample to be transferred from one component to another during processing. In this regard, the sample is processed as a whole in a single system, thereby ensuring sterility.

[0064] Aseptic transfer is performed without exposing the sample to the external environment. In this regard, transfer can be performed by any number of modes, and these modes will maintain a closed aseptic environment. In some embodiments, transfer is achieved by connecting male and female Luer lock connectors (male and female Luer lock connectors) to each other between the first container and the processing container or the cartridge containing the sample extracted from the subject. In some embodiments, transfer is completed by using an aseptic connection device to carry out aseptic docking of the first container and the processing container or the cartridge. Sample is transferred to the processing container or the cartridge mechanically or by gravity flow from the first container.

[0065] In some embodiments, the first container is inserted into the cartridge and aseptically docked using aseptic devices so that the processing container is located within the cartridge.The first container, which is a processing chamber, already contains a sample.

[0066] In this regard, each time a tissue and / or cell sample is disclosed in the present disclosure as being transferred from one container to another, such movement may be performed so as to maintain the integrity of the closed system, thereby not exposing the sample to the external environment.

[0067] Once the sample is transferred to the processing container or cartridge, the tissue sample is washed one or more times 110. Washing can be performed as described herein using a sterile saline solution. Washing ensures that the sample is free of as many impurities as possible prior to processing, thereby increasing the purity level of the cell fraction isolated therefrom.

[0068] In some embodiments, washing is performed by mechanically agitating the processing container or cartridge. If multiple washing steps are desired and / or required, sterile saline is drained from the processing container or cartridge after a single wash is completed, and new sterile saline is introduced into the container or cartridge for each subsequent wash. In this regard, each time sterile saline is drained and new sterile saline is introduced into the container or cartridge, as described herein to maintain a closed sterile environment.

[0069] After washing, the tissue sample is processed 115. As indicated, processing can be performed by using ESW, mechanical impact, vibration and shearing, or a combination thereof.

[0070] In some embodiments, the tissue sample is processed by ESW and broken into multiple small cell clusters. In some embodiments, the tissue sample is processed by ESW and broken into multiple small cell clusters and multiple single cells. In some embodiments, the tissue sample is processed by ESW and completely broken into multiple individual cells.

[0071] In some embodiments, the tissue sample is processed by mechanical impact and shearing, and is broken into multiple small cell clusters. In some embodiments, the tissue sample is processed by mechanical impact and shearing, and is broken into multiple small cell clusters and multiple single cells. In some embodiments, the tissue sample is processed by mechanical impact and shearing, and is completely broken into multiple individual cells.

[0072] In some embodiments, the tissue sample is processed by a combination of ESW and mechanical impact and shearing, and is broken into multiple small cell clusters. In some embodiments, the tissue sample is processed by a combination of ESW and mechanical impact and shearing, and is broken into multiple small cell clusters and multiple single cells. In some embodiments, the tissue sample is processed by a combination of ESW and mechanical impact and shearing, and is completely broken into multiple individual cells.

[0073] After processing, the cell suspension representing the remainder of the sample (which may be a plurality of small cell clusters, a plurality of single cells, or both) is filtered and centrifuged 120. Filtration may be performed in a device separate from the processing container or cartridge, or within the processing container or cartridge itself. In some embodiments, filtration is performed in a separate device, in which case the cell suspension is transferred to the filter by a sterile device, as described herein. In some embodiments, filtration is performed while the cell suspension is moved from the container or cartridge into one or more centrifuge tubes.

[0074] In some embodiments, the transfer to the filtration device is accomplished by connecting male and female Luer lock connectors to each other between the process container or cartridge and the filtration device. In some embodiments, the transfer is accomplished by aseptically docking the process container or cartridge and the filtration device using a sterile connection device. The sample is transferred from the process container or cartridge to the filtration device mechanically or by gravity flow.

[0075] In some embodiments, filtration is performed inside a process vessel or cartridge, as described herein.

[0076] In various aspects, centrifugation is performed outside of the processing vessel or cartridge. In some embodiments, the cell suspension is transferred to one or more centrifuge tubes located inside the processing vessel or cartridge. When filled, the centrifuge tubes are moved from the processing vessel or cartridge to the centrifuge by sterile means, as described herein.

[0077] In some embodiments, the cell suspension is transferred to one or more centrifuge tubes located outside the processing container or cartridge by sterile means, as described herein. When filled, the centrifuge tubes are moved from the processing container or cartridge to a centrifuge by sterile means, as described herein.

[0078] The duration and speed of centrifugation can vary depending on the type of tissue being processed. In some embodiments, the cell suspension is centrifuged for 3 to 30 minutes and the speed is 500g to 2,000g. In some embodiments, the cell suspension is centrifuged at a speed of 1,000g-1,800g for 10-25 minutes. In some embodiments, the cell suspension is centrifuged at 1,200g for 10 minutes. In some embodiments, the cell suspension is centrifuged at 1,200g for 7 minutes. In some embodiments, the cell suspension is centrifuged at 1,200g for 5 minutes. In some embodiments, the cell suspension is centrifuged at 1,200g for 3 minutes. In some embodiments, the cell suspension is centrifuged at 900g for 10 minutes. In some embodiments, the cell suspension is centrifuged at 900g for 7 minutes. In some embodiments, the cell suspension is centrifuged at 900g for 5 minutes. In some embodiments, the cell suspension is centrifuged at 900g for 3 minutes. In some embodiments, the cell suspension is centrifuged at 600g for 10 minutes. In some embodiments, the cell suspension is centrifuged at 600 g for 7 minutes. In some embodiments, the cell suspension is centrifuged at 600 g for 5 minutes. In some embodiments, the cell suspension is centrifuged at 600 g for 3 minutes.

[0079] There are many methods to separate the various cell types present in a cell suspension from each other by centrifugation. For example, a density gradient can be used so that during centrifugation, cells will be separated into bands according to their specific density. In addition, size exclusion schemes can be used to separate cells by size. In addition, separation can be started before centrifugation, such as by precipitating a processed tissue sample before centrifugation and separating unwanted cell debris from the desired cell fraction. The type of separation technique used can vary depending on the desired cell fraction to be separated.

[0080] Once the desired cell fraction is isolated, it can be resuspended for administration to the subject from which it originated 125. In some embodiments, the desired cell fraction is resuspended in a small amount of fluid obtained from a centrifuge tube. In some embodiments, the desired cell fraction is resuspended in sterile saline.

[0081] In one aspect, the desired cell fraction is stem cells and the tissue from which the stem cells are derived is adipose.

[0082] Method for processing stem cells from fat The first one The implementation plan is as follows:

[0083] Obtain fat from the patient by liposuction.Liposuction can be performed by a syringe, wherein by a needle or by a liposuction machine directly remove fat from the patient's body. The amount of fat removed during liposuction can vary according to the number of desired stem cells. In some embodiments, liposuction is miniature (mini) liposuction, wherein about 30cc to about 50cc of fat is removed from the patient's body. In some embodiments, liposuction is micro (micro) liposuction, wherein about 5cc to about 29cc of fat is removed from the patient's body. In some embodiments, liposuction is typical clinical liposuction, in which case 300cc or more of fat is removed from the patient's body.

[0084] Once fat is removed from the patient, it is transferred to aseptic processing container or cartridge. As described herein, transfer is carried out in an aseptic manner. In some embodiments, fat is removed from the patient by a syringe, and fat is transferred from the syringe to the processing container or cartridge by connecting male and female Luer lock connectors to each other between syringe and processing container or cartridge. In some embodiments, fat is removed from the patient by a syringe, and by connecting male and female Luer lock connectors to each other between syringe and container or cartridge, the syringe is aseptically docked in processing container or cartridge. In some embodiments, fat is removed from the patient by a syringe, and fat is transferred from the syringe to the processing container or cartridge by connecting male and female Luer lock connectors to each other between syringe and processing container or cartridge in cartridge. In some embodiments, fat is removed from the patient by a liposuction device, and fat is transferred from the liposuction device to the processing container or cartridge by using an aseptic connection device to aseptically dock the liposuction device and processing container or cartridge.

[0085] In some embodiments, the transfer can be performed by gravity flow. In some embodiments, the transfer can be performed mechanically, such as by physically depressing the plunger of a syringe and forcing the fat from the interior of the syringe into the interior of a processing container or cartridge. In some embodiments, the transfer can be performed by a device such as a pump.

[0086] The volume of the container or cartridge for receiving fat can vary with the amount of fat obtained from the patient. In some embodiments, 30cc to 50cc of fat removed by micro-liposuction is transferred to a container or cartridge having a volume of 9 fluid ounces to 19 fluid ounces.

[0087] Then the sterile saline of a certain volume equal to the amount of fat removed from the patient is transferred to a container or cartridge to wash fat. For example, if 30cc fat is taken out from the patient, 30mL sterile saline is used to wash the sample. In some embodiments, then the sterile saline of a certain volume greater than the amount of fat removed from the patient is transferred to a container or cartridge to wash fat. For example, if 30cc fat is removed from the patient, 35mL sterile saline is used to wash the sample. In some embodiments, then the sterile saline of a certain volume less than the amount of fat removed from the patient is transferred to a container or cartridge to wash fat. For example, if 30cc fat is removed from the patient, 25mL sterile saline is used to wash the sample. In some embodiments, the combination of sterile saline volume is added to a certain amount of fat to wash fat. As described herein, saline is transferred to a container or cartridge to keep the sterility of the system. In some embodiments, by connecting male and female Luer lock connectors to each other between the container and processing container or cartridge that hold sterile saline, sterile saline is transferred to a container or cartridge. In some embodiments, the sterile saline is transferred to the container or cartridge by aseptically docking the sterile saline container and the processing container or cartridge using a sterile connection device.

[0088] Washing is performed by gently shaking or swirling the container or cartridge containing the fat and sterile saline.

[0089] The container or cartridge is then positioned in such a way that the (now washed) fat is separated from the sterile saline. Because the fat is less dense than the saline, it will rise and float on top of the saline. The amount of time required for the separation will vary depending on the individual makeup of the fat sample. In some embodiments, the separation can be carried out for 1 to 30 minutes. In some embodiments, the separation can be carried out for 1 to 15 minutes. In some embodiments, the separation can be carried out for 1 to 10 minutes. In some embodiments, the separation can be carried out for 1 to 5 minutes. In some embodiments, the separation can be carried out for 10 seconds to 1 minute.

[0090] The saline is then drained from the container or cartridge as completely as possible so that only fat remains. The draining can be done in a variety of ways, depending on the type of container or cartridge used in the method. In some embodiments, the saline is drained from the container or cartridge by a tube located at the bottom of the container or cartridge. The draining can be done actively using a syringe or pump, or passively by gravity flow. In some embodiments, the draining is done to maintain the integrity of the sterile system, as described herein.

[0091] The washes are repeated until the fat is golden yellow and the brine is only slightly cloudy after the washes are complete. In some embodiments, the washes are performed 1-5 times, in some embodiments 1-4 times, in some embodiments 1-3 times, in some embodiments 1-2 times, and in some embodiments 1 time.

[0092] Once washed, the fat is ready for processing.

[0093] Optionally, sterile saline is added to the bag for processing, but this is not required. Adding saline will provide space for cells and / or small cell clusters to move freely away from each other during processing, thereby facilitating the breaking of the fat into individual cells. If saline is added, as described herein, the saline is added in a manner that maintains the integrity of the sterile system.

[0094] The volume of the optional saline added can vary. In some embodiments, the volume of saline added ranges from none to twice the volume of fat removed from the patient. In some embodiments, an equal amount of saline is used (e.g., 30 mL of saline is used for 30 cc of fat).

[0095] Excess air is removed from the container or cartridge using a pump or syringe. In some embodiments, excess air is removed from the same tube located at the bottom of the container or cartridge from which the sterile saline wash is expelled. In some embodiments, air is expelled in a manner that maintains the integrity of the sterile closed system, as described herein.

[0096] The process container or cartridge is then moved to the processing unit. Figure 2 An example of a suitable processing apparatus 200 is shown in . In the illustrated embodiment, a processing container or cartridge 205 is secured to a rigid platform 210 for processing. The platform 210 controls the movement of the container or cartridge 205 during ESW processing.

[0097] The platform 210 may be made of any suitable rigid material, including wood, metal, plastic, acrylic, etc. In some embodiments, the platform 210 is made of wood.

[0098] The processing device 200 houses a shock wave applicator 215 capable of delivering shock waves to the interior of the container or cartridge 205. The shock wave applicator 215 is housed on the processing device 200 via an applicator platform 220. The applicator platform 220 and the shock wave applicator 215 are kept at a distance from the fat contained in the container or cartridge 205 so that the shock wave applicator 215 never comes into direct contact with the fat. This ensures that the shock waves delivered to the fat are extracorporeal or ESW. In some embodiments, the shock wave applicator 215 can be placed in direct contact with the processing container or cartridge 205. In such an embodiment, the wall of the container or cartridge 205 ensures that the shock wave applicator 215 does not come into direct contact with the fat.

[0099] The movement of the applicator platform 220 and the shock wave applicator 215 can be performed in three directional planes X, Y and Z, each plane being controlled by its own motor: X-plane motor 230, Y-plane motor 235 and Z-plane motor 240. In some embodiments, these motors are NEMA 17 stepper motors. Each of the motors 230, 235 and 240 is controlled by a motor controller 225, which enables the applicator platform 220 and the shock wave applicator 215 to move in three dimensions over and around the container or cartridge 205. In some embodiments, the motors are controlled by an Arduino microprocessor v6 board with Marlin firmware. In some embodiments, the motors are controlled by an Arduino v6 board with Marlin firmware. In some embodiments, only the X-plane motor 230 is used. In some embodiments, only the 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. In some embodiments, only X-plane motor 230 and Z-plane motor 240 are used. In some embodiments, only Y-plane motor 235 and Z-plane motor 240 are used. In some embodiments, X-plane motor 230, Y-plane motor 235, and Z-plane motor 240 are used.

[0100] This three-dimensional movement of applicator platform 220 and shock wave applicator 215 allows all of the fat in the extracted sample to be exposed to the extracorporeal shock waves during processing.

[0101] Shock waves are then delivered from the shock wave applicator 215, through a transmissive gel or membrane containing a transmissive gel, through the wall of the container or cartridge 205, to the fat sample to disrupt the fat and release cell clusters and single cells from the fat. In some embodiments, the transmissive gel is a REF 4248 conductor transmissive gel manufactured by DJO, LLC, 1430 Decision Street, Vista, CA 92081, USA for Chattanooga. In those embodiments in which sterile saline is added to the fat prior to processing, small clusters of cells and / or single cells released from the fat will move into the saline.

[0102] The shock wave applicator 215 is driven by a shock wave generator 245, which generates an ESW that is transmitted to the fat through the shock wave applicator 215. In some embodiments, the shock wave generator is a device manufactured by Storz Medical AG, 8274 In some embodiments, the shock wave generator is a BS-SWT2X manufactured by Lumsail Industrial Inc., 4 / F, No. 9Yi, Lane 2, Suide Road, Shanghai, 200331, China.

[0103] The force of the ESW delivered to fat can vary. For example, the force of the ESW may need to be increased to penetrate the wall of a thick-walled container or cartridge 205. Generally, the force of the ESW delivered to fat can vary depending on the thickness of the wall of the container or cartridge 205 and / or the material from which the container or cartridge 205 is made. In some embodiments, the force of the ESW delivered to fat ranges from 0.5 to 5.0 bar. In some embodiments, the force of the ESW delivered to fat ranges from 1.0 to 4.5 bar. In some embodiments, the force of the ESW delivered to fat ranges from 1.5 to 4.0 bar. In some embodiments, the force of the ESW delivered to fat ranges from 2.0 to 3.5 bar. In some embodiments, the container 205 is a vinyl bag with a wall 0.25 mm thick, and the level of force of the ESW is in the range of 2.0 to 2.5 bar.

[0104] The area covered by the ESW delivered by the shock wave applicator 215 can vary. The larger the area covered by the shock wave applicator 215, the greater the surface area of ​​fat exposed to the ESW. In some embodiments, the total area covered by the shock wave applicator 215 is within 1 cm 2 -100cm 2 , in some embodiments 1 cm 2 -90cm 2 , in some embodiments, 1 cm2 -80cm 2 , in some embodiments, 1 cm 2 -70cm 2 , in some embodiments, 1 cm 2 -60cm 2 , in some embodiments, 1 cm 2 -50cm 2 , in some embodiments, 1 cm 2 -40cm 2 , in some embodiments, 1 cm 2 -30cm 2 , in some embodiments, 1 cm 2 -20cm 2 , in some embodiments, 1 cm 2 -10cm 2 , in some embodiments, 1 cm 2 -5cm 2 And in some embodiments, the total area covered by the shock wave applicator 215 is 5 cm 2 .

[0105] In one embodiment, container 205 is a 19 oz vinyl bag with a 0.25 mm thick wall, the total amount of fat obtained from the patient is 30 cc, and 30 mL of saline (equal volume to the fat sample) is optionally added to the vinyl bag prior to processing. In this embodiment, the total area of ​​the bag exposed to the ESW, and therefore the total amount of fat sample, is approximately 5 cm 2 .

[0106] The number of shock waves delivered to the fat sample can vary. In some embodiments, the total number of shock waves ranges from 5,000 to 100,000, in some embodiments from 10,000 to 50,000, in some embodiments from 10,000 to 25,000, and in some embodiments from 10,000 to 20,000. In some embodiments, the total number of shock waves is 25,000.

[0107] In this embodiment, stem cells are separated from a fat sample obtained from a patient. A 5 cm 2The applicator tip of the invention has a shock wave number ranging from 10,000 to 50,000 in the range of 2.0 to 2.5 bar, which is sufficient to separate stem cells and stromal vascular fractions from fat. As used herein, "stromal vascular fraction" refers to a cell fraction obtained from a tissue sample process, which contains cells obtained from the separation and dissociation of cell fractions, and in some embodiments, the cell fraction is fat. The stromal vascular fraction includes, but is not limited to, stem cells, growth factors, and progenitor cells, among others.

[0108] After the ESW is applied to the fat sample, the container or cartridge is mechanically stirred for a short period of time. In some embodiments, the mechanical stirring is shaking. In some embodiments, the mechanical stirring causes the container or cartridge 205 to flip over once or multiple times. In some embodiments, the short period of time is 1 to 30 seconds, in some embodiments 1 to 15 seconds, and in some embodiments 1 to 10 seconds. In some embodiments, the short period of time is about 10 seconds. Stirring allows the stem cells and stromal vascular fractions released from the fat to be separated from the adipocyte fragments and any residual adipose tissue that may still be present in the container or cartridge 205.

[0109] The container or cartridge 205 is then positioned in such a way that the (now processed) fat can be separated from the sterile saline that now contains the stem cells and stromal vascular fraction, which have been separated from the fat by the ESW. Because the fat is less dense than the saline containing the stem cells, it will rise and float on the top of the saline. The amount of time required for separation will vary depending on the individual composition of the fat sample. In some embodiments, the separation can be carried out for 1 to 30 minutes. In some embodiments, the separation can be carried out for 1 to 15 minutes. In some embodiments, the separation can be carried out for 1 to 10 minutes. In some embodiments, the separation can be carried out for 1 to 5 minutes. In some embodiments, the separation can be carried out for 10 seconds to 1 minute. In some embodiments, the separation can be carried out for less than 1 minute.

[0110] The stem cells have now been separated from the fat sample and are now suspended in a layer of sterile saline.

[0111] The saline layer containing the stem cells is then removed from the container or cartridge 205 as completely as possible so that only the fat remains. Depending on the type of container or cartridge 205 used in the method, the removal can be performed in a variety of ways. In some embodiments, the saline is drained from the container or cartridge 205 through a tube located at the bottom of the container or cartridge 205. The draining can be done actively using a syringe or pump, or passively by gravity flow. In some embodiments, the layer containing the stem cells is removed in a manner that maintains the integrity of the closed sterile environment, as described herein.

[0112] In some embodiments, the saline layer containing stem cells is passed through a filter during removal from the container or cartridge 205. The size of the filter can vary. In some embodiments, the filter size ranges from 40 μm to 100 μm. In some embodiments, the filter size is 70 μm. In various embodiments, the filter is nylon.

[0113] After or during removal from the container or cartridge 205, the saline containing the stem cells is moved into one or more centrifuge tubes. In some embodiments, the movement is performed in a manner that maintains the integrity of the closed sterile environment, as described herein. Centrifugation is then performed to concentrate the stem cells into a mass at the bottom of the centrifuge tube.

[0114] The stem cells are concentrated by centrifugation at 500 g to 1,600 g for 3 to 10 minutes. In some embodiments, the stem cells are concentrated by centrifugation at 1,200 g for 10 minutes.

[0115] The fluid is removed from the tube, leaving only the dry cells at the bottom. The fluid may be removed by decanting, aspirating, or the like.

[0116] A small amount of fluid is placed in a centrifuge tube to resuspend the stem cells. In some embodiments, the amount of fluid added to the centrifuge tube is 1 mL to 5 mL. In some embodiments, the fluid is saline, platelet-rich plasma, hyaluronan, fixed gel, hydrogel, scaffold, fibrin, glue, or any combination of the foregoing. In some embodiments, the cells are resuspended in a centrifugal aspirate.

[0117] With the stem cells floating in the suspension, they are ready for use. Such uses may include, but are not limited to, reintroduction into the patient from whom they came, cryopreservation, expansion, etc. In some embodiments, the concentrated resuspended stem cells are moved into a syringe through a hypodermic needle for use by the patient. In some embodiments, the concentrated resuspended stem cells are seeded into a tissue scaffold to grow into myocardium, muscle, bone, cartilage, liver, kidney or other tissue and organ structures. In some embodiments, the concentrated, resuspended stem cells are converted into induced pluripotent stem cells by expressing pluripotency transcription factors.

[0118] Method for processing stem cells from fat The second The implementation plan is as follows:

[0119] Obtain fat from the patient by liposuction.Liposuction can be carried out by a syringe, wherein fat is directly removed from the patient's body by a needle, or by a liposuction machine. The amount of fat removed during liposuction can vary according to the number of desired stem cells.In some embodiments, fat is removed from the patient's body by a syringe, and by connecting male and female Luer lock connectors to each other between the syringe and the cartridge, the syringe is aseptically docked in the cartridge.In some embodiments, fat is removed from the patient's body by a syringe, and fat is transferred from the syringe to a processing container or cartridge by connecting male and female Luer lock connectors to each other between the syringe and the cartridge.In some embodiments, liposuction is micro liposuction, wherein about 30cc to about 50cc of fat is removed from the patient's body.In some embodiments, liposuction is micro liposuction, wherein about 5cc to about 29cc of fat is removed from the patient's body.In some embodiments, liposuction is typically clinical liposuction, in which case 300cc or more of fat is removed from the patient's body.

[0120] Once the fat is removed from the patient, it is transferred to a sterile processing container or cartridge. As described herein, the transfer is performed in an aseptic manner. In some embodiments, fat is removed from the patient by a syringe, and the fat is transferred from the syringe to the processing container or cartridge by connecting the male and female Luer lock connectors to each other between the syringe and the processing container or cartridge. In some embodiments, fat is removed from the patient by a liposuction device, and the liposuction device and the processing container or cartridge are aseptically docked and transferred from the liposuction device to the processing container or cartridge using a sterile connection device.

[0121] In some embodiments, the transfer can be performed by gravity flow. In some embodiments, the transfer can be performed mechanically, such as by physically depressing the plunger of a syringe and forcing the fat from the interior of the syringe into the interior of a processing container or cartridge. In some embodiments, the transfer can be performed by a device such as a pump.

[0122] The volume of the container or cartridge for receiving fat can vary with the amount of fat obtained from the patient. In some embodiments, 30cc to 50cc of fat removed by micro-liposuction is transferred to a container or cartridge having a volume of 9 fluid ounces to 19 fluid ounces.

[0123] Then the sterile saline of the volume equal to the fat amount removed from the patient is transferred to a container or cartridge to wash fat. For example, if 30cc fat is taken out from the patient, 30mL sterile saline washing sample is used. In some embodiments, fat is removed from the patient by a syringe, and by connecting male and female Luer lock connectors to each other between a syringe and a cartridge, the syringe is aseptically docked in the cartridge. In some embodiments, fat is removed from the patient by a syringe, and fat is transferred from the syringe to a processing container or cartridge by connecting male and female Luer lock connectors to each other between a processing container or cartridge in a syringe and a cartridge. As described herein, saline is transferred to a container or cartridge to keep the sterility of the system. In some embodiments, by connecting male and female Luer lock connectors to each other between a container and a processing container or a cartridge that holds sterile saline, sterile saline is transferred to a container or a cartridge. In some embodiments, sterile saline container and a processing container or a cartridge are aseptically docked by using an aseptic connection device, sterile saline is transferred to a container or a cartridge.

[0124] Washing is performed by gently shaking or swirling the container or cartridge containing the fat and sterile saline.

[0125] The container or cartridge is then positioned in such a way that the (now washed) fat can be separated from the sterile saline. Because the fat is less dense than the saline, it will rise and float on top of the saline. The amount of time required for the separation will vary depending on the individual makeup of the fat sample. In some embodiments, the separation can take place for 1 to 30 minutes. In some embodiments, the separation can take place for 1 to 15 minutes. In some embodiments, the separation can take place for 1 to 10 minutes. In some embodiments, the separation can take place for 1 to 5 minutes.

[0126] The saline is then drained from the container or cartridge as completely as possible so that only fat remains. The draining can be done in a variety of ways, depending on the type of container or cartridge used in the method. In some embodiments, the saline is drained from the container or cartridge by a tube located at the bottom of the container or cartridge. The draining can be done actively using a syringe or pump, or passively by gravity flow. In some embodiments, the draining is done to maintain the integrity of the sterile system, as described herein.

[0127] The washes are repeated until the fat is golden yellow and the brine is only slightly cloudy after the washes are complete. In some embodiments, the washes are performed 1-5 times, in some embodiments 1-4 times, in some embodiments 1-3 times, in some embodiments 1-2 times, and in some embodiments 1 time.

[0128] Once washing is complete, the fat is ready for processing.

[0129] Optionally, sterile saline is added to the bag for processing, but this is not required. Adding saline will provide space for cells and / or small cell clusters to move freely away from each other during processing, thereby aiding in breaking the fat into individual cells. If saline is added, it is added in a manner that maintains the integrity of the sterile system, as described herein.

[0130] The volume of saline that is optionally added can vary. In some embodiments, the volume of saline added ranges from none to twice the volume of fat removed from the patient. In some embodiments, an equal amount of saline is used (e.g., 30 mL of saline is used for 30 cc of fat).

[0131] Excess air is removed from the container or cartridge using a pump or syringe. In some embodiments, excess air is removed from the same tube located at the bottom of the container or cartridge from which the sterile saline wash is discharged. In some embodiments, air is discharged in a manner that maintains the integrity of the sterile closed system as described herein.

[0132] The process container or cartridge is then moved to the processing unit. Figure 3 An example of a suitable processing apparatus 300 is shown in FIG. 2. In the illustrated embodiment, the processing container or cartridge 205 is secured to a rigid platform 315 for processing. The platform 315 controls the movement of the container or cartridge 205 during processing.

[0133] The platform 315 can be made of any suitable rigid material, including wood, plastic, etc. In some embodiments, the platform 315 is made of wood.

[0134] In this embodiment, mechanical impact is used to disrupt the adipose tissue and release the stem cells.

[0135] The mechanical impact is generated by a motor 305, which drives an impact arm 310, which is in physical contact with the container or cartridge 205 containing the fat to be processed. In this embodiment, the motor 305 drives the impact arm 310 so that the impact arm 310 is hinged up and down and contacts the container or cartridge 205 at the bottom of its range of motion. In some embodiments, the motor is a 4.5 amp motor model BDEJS300C manufactured by Black and Decker, 1000 Stanley Drive, New Britain, CT 06053, USA.

[0136] In some embodiments, the motor 305 is a variable speed motor that is adjustable to drive the impact arm at a rate of 0 to 30,000 rpm, in some embodiments 0 to 20,000 rpm, in some embodiments 0 to 10,000 rpm, and in some embodiments 0 to 5,000 rpm.

[0137] In some embodiments, the motor 305 includes gears that can reduce the speed of the impact arm 310 when the motor 305 is operating at full speed (in some embodiments, 30,000 rpm) so as to generate more torque. When doing so, the momentum generated by the impact arm 310 is transferred to the tissue in the container or cartridge 205. This greatly increases the rate at which the adipose tissue is disrupted and stem cells are released from the adipose tissue.

[0138] In one embodiment, the motor 305 is adapted to allow a 10 to 1 reduction in speed from 30,000 rpm to 3,000 rpm. In this embodiment, the motor 305 includes a 0.75 inch gear diameter, which allows for a 10 to 1 reduction. At 3,000 rpm and a 0.75 inch gear diameter, the impact arm 310 reaches a speed of 117.8 inches per second - the speed at which the impact arm 310 articulates back and forth and, therefore, contacts the container or cartridge 205. The speed at which the impact arm 310 is able to contact the container or cartridge 205 allows for a significant reduction in processing time. This provides a significant advantage over known or existing tissue processing techniques that require very long processing times.

[0139] The tissue is exposed to mechanical impact until the fat is broken down to the desired level, or until the desired amount of stem cells has been released from the fat. In some embodiments, the fat sample is processed in 30 minutes or less. In some embodiments, the fat sample is processed between 30 seconds and 30 minutes, in some embodiments between 30 seconds and 20 minutes, in some embodiments between 30 seconds and 10 minutes, and in some embodiments between 30 seconds and 5 minutes.

[0140] The movement of the platform 315 is performed in three directional planes X, Y and Z, each of which is controlled by its own motor: X-plane motor 230, Y-plane motor 235 and Z-plane motor 240. In some embodiments, only X-plane motor 230 is used. In some embodiments, only Y-plane motor 235 is used. In some embodiments, only Z-plane motor 240 is used. In some embodiments, only X-plane motor 230 is used. In some embodiments, only X-plane motor 230 and Y-plane motor 235 are used. In some embodiments, only X-plane motor 230 and Z-plane motor 240 are used. In some embodiments, only Y-plane motor 235 and Z-plane motor 240 are used. In some embodiments, X-plane motor 230, Y-plane motor 235 and Z-plane motor 240 are used. Each of the motors 230, 235 and 240 is controlled by a motor controller 225, which enables the platform 315 to move in three dimensions.

[0141] This up to three-dimensional movement of the platform 315 allows all fat in the extracted sample to be exposed to mechanical shock during processing and for optimal positioning of the sample and processing equipment.

[0142] The length of the impact arm 310, the area of ​​the impact surface, the number of impact surfaces, and the shape of the impact arm 310 can vary. In some embodiments, increasing the total area of ​​the impact surface and / or the total number of impact surfaces reduces the amount of time required to process the fat sample.

[0143] The container or cartridge 205 is then positioned in such a way as to allow the (now processed) fat to separate from the sterile saline that now contains the stem cells that have been separated from the fat by mechanical impact. Because the fat is less dense than the saline, it will rise and float on top of the saline. The amount of time required for the separation will vary depending on the individual makeup of the fat sample. In some embodiments, the separation can be carried out for 1 to 30 minutes. In some embodiments, the separation can be carried out for 1 to 15 minutes. In some embodiments, the separation can be carried out for 1 to 10 minutes. In some embodiments, the separation can be carried out for 1 to 5 minutes. In some embodiments, the separation can be carried out in less than 1 minute. In some embodiments, the separation can be carried out for 10 seconds to 1 minute. In some embodiments, the separation can be carried out in less than 1 minute.

[0144] The stem cells and stromal vascular fraction have now been separated from the fat sample and are now suspended in saline.

[0145] The saline layer containing the stem cells is then removed from the container or cartridge 205 as completely as possible so that only the fat remains. Depending on the type of container or cartridge 205 used in the method, the removal can be performed in a variety of ways. In some embodiments, the saline is drained from the container or cartridge 205 through a tube located at the bottom of the container or cartridge 205. The draining can be done actively using a syringe or pump, or passively by gravity flow. In some embodiments, the layer containing the stem cells is removed in a manner that maintains the integrity of the closed sterile environment, as described herein.

[0146] In some embodiments, the saline containing stem cells is passed through a filter during removal from the container or cartridge 205. The size of the filter can vary. In some embodiments, the filter size ranges from 40 μm to 100 μm. In some embodiments, the filter size is 70 μm. In various embodiments, the filter is nylon.

[0147] After or during removal from the container or cartridge 205, the saline containing the stem cells is moved into one or more centrifuge tubes. In some embodiments, the movement is performed in a manner that maintains the integrity of the closed sterile environment, as described herein. Centrifugation is then performed to concentrate the stem cells into a mass at the bottom of the centrifuge tube.

[0148] The stem cells are concentrated by centrifugation at 500 g to 1,600 g for 3 to 10 minutes. In some embodiments, the stem cells are concentrated by centrifugation at 1,200 g for 10 minutes.

[0149] The fluid is removed from the tube, leaving only the dry cells at the bottom. The fluid may be removed by decanting, aspirating, or the like.

[0150] A small amount of fluid is placed in the centrifuge tube to resuspend the stem cells. In some embodiments, the amount of fluid added to the centrifuge tube is 1 mL to 5 mL. In some embodiments, the fluid is saline, platelet-rich plasma, hyaluronan, a fixed gel, a hydrogel, a scaffold, a fibrin glue, a glue, or a combination of any of the foregoing. In some embodiments, the cells are resuspended in a centrifugal aspirate.

[0151] When the stem cells are in suspension, they can be used for later use. Such uses may include, but are not limited to, reintroduction into the patient from whom they came, cryopreservation, expansion, etc. In some embodiments, the concentrated resuspended stem cells are moved into a syringe through a hypodermic needle for use by the patient. In some embodiments, the concentrated resuspended stem cells are seeded into a tissue scaffold to grow into myocardium, muscle, bone, cartilage, liver, kidney or other tissue and organ structures. In some embodiments, the concentrated, resuspended stem cells are converted into induced pluripotent stem cells by expressing pluripotency transcription factors.

[0152] In the first and second methods described above, the container or cartridge and the source of mechanical energy are removable. In some embodiments, the fat is pumped through a chamber placed below the ESW or impact surface. In these embodiments, the bag and the source of mechanical energy are both fixed in place, and the tissue passes through the chamber to be exposed to the ESW and / or the source of mechanical energy.

[0153] Method for processing stem cells from fat The third The implementation plan is as follows:

[0154] Obtain fat from the patient by liposuction. Liposuction can be performed by a syringe, wherein the fat is removed directly from the patient's body by a needle, or by a liposuction machine. The amount of fat removed during liposuction can vary according to the amount of desired stem cells. In some embodiments, liposuction is micro liposuction, wherein about 30cc to about 50cc of fat is removed from the patient's body. In some embodiments, liposuction is micro liposuction, wherein about 5cc to about 29cc of fat is removed from the patient's body. In some embodiments, liposuction is typical clinical liposuction, in which case 300cc or more of fat is removed from the patient's body.

[0155] Once fat is removed from the patient, it is transferred to the aseptic processing cartridge. As described herein, transfer is carried out in an aseptic manner. In some embodiments, fat is removed from the patient by a syringe, and fat is transferred from the syringe to the processing cartridge by connecting male and female Luer lock connectors to each other between the syringe and the processing cartridge. In some embodiments, fat is removed from the patient by a syringe, and by connecting male and female Luer lock connectors to each other between the syringe and the cartridge, the syringe is aseptically docked in the cartridge. In some embodiments, fat is removed from the patient by a syringe, and fat is transferred from the syringe to the processing container or cartridge by connecting male and female Luer lock connectors to each other between the syringe and the cartridge. In some embodiments, fat is removed from the patient by a liposuction device, and fat is transferred from the liposuction device to the processing cartridge by using an aseptic connection device to aseptically dock the liposuction device and the processing cartridge.

[0156] In some embodiments, the transfer can be performed by gravity flow. In some embodiments, the transfer can be performed mechanically, such as by physically depressing the plunger of a syringe and forcing the fat from the interior of the syringe into the interior chamber of the processing cartridge. In some embodiments, the transfer can be performed by a device such as a pump.

[0157] The volume of the lumen of the cartridge 400, 500 that receives the fat (see, e.g., Figure 4 and 5 The cartridge embodiments provided in the present invention may vary with the amount of fat obtained from the patient. In some embodiments, 30cc to 50cc of fat removed by micro-liposuction is transferred to the fat chambers 405, 505 of the cartridges 400, 500.

[0158] In this embodiment, the fat is injected into a specific component 405, 505 of the cartridge 400, 500 within the test itself, and the cartridge 400, 500 is designed to accommodate the processing of the fat sample completely within the cartridge 400, 500 without exposing the sample to the outside environment. In some embodiments, the fat chamber 405, 505 located within the cartridge 400, 500 that receives the fat is a flexible bag having a volume of 9 fluid ounces to 19 fluid ounces.

[0159] In various aspects, the cartridges 400, 500 are designed for use with any of the ESW or mechanical impact devices and methods disclosed herein. In some embodiments, processing is performed automatically by a machine that runs several of the processing steps described below. In some embodiments, processing is performed by a user who manually performs the steps described below. In both embodiments, processing is controlled externally so that the internal processing of the cartridges 400, 500 is performed in a sterile manner without exposing the sample to the external environment.

[0160] In the depicted embodiment, the fat sample is introduced into the fat chamber 405, 505 by passing the fat sample through the first one-way valve 410, 510. The first one-way flow valve 410, 510 is oriented as shown - into the interior of the fat chamber 405, 505. In some embodiments, the fat is introduced into the lumen of the fat chamber 405, 505 by connecting the male and female Luer lock connectors to each other between the container that retains the fat after extrusion from the patient and the first one-way valve 410, 510 of the processing cartridge 400, 500. In some embodiments, the fat is removed from the patient by a liposuction device, and the fat is transferred from the liposuction device to the processing cartridge by aseptically docking the liposuction device and the processing cartridge using a sterile connection device.

[0161] Once inside the fat chamber 405, 505, the fat is washed before processing. The cartridge 400, 500 includes a fluid reservoir 415, 515 filled with sterile saline, in some embodiments, sterile 1x phosphate buffered saline, which is used to wash the tissue. In order to introduce sterile saline into the interior of the fat chamber 405, 505, the saline enters the interior of the fat chamber 405, 505 through the second one-way valve 420, 520. The flow direction is shown in the figure. The saline is moved to the interior of the fat chamber 405, 505 by using an external actuator 425, 525 that moves the first plunger 430, 530 to force the saline through the first one-way valve 420, 520. In some embodiments, the external actuator 425, 525 is manually operated by the user, and the user presses the actuator 425, 525 until the desired amount of saline enters the interior of the fat chamber 405, 505 through the second one-way valve 420, 520. In some embodiments, movement of the external actuator 425 , 525 is automated, and an external machine moves the external actuator 425 , 525 in the direction shown, thereby metering a desired amount of saline through the second one-way valve 420 , 520 and into the interior of the fat chamber 405 , 505 .

[0162] Washing is performed by gently shaking or rotating the cartridge 400, 500 containing the fat and sterile saline.

[0163] The cartridges 400, 500 are then positioned in such a way as to allow the (now washed) fat to be separated from the sterile saline. In this embodiment, the cartridges 400, 500 have two variations. In some embodiments, the cartridges 400, 500 have a fixed vertical orientation so that the fat and saline wash contained in the fat chambers 405, 505 will always be naturally separated from each other by gravity, with the fat floating to the top of the fat chambers 405, 505. In some embodiments, the cartridges 400, 500 have a horizontal orientation and rotate vertically to allow the fat to float and separate from the saline wash. Because the fat is less dense than the saline, it will rise and float on the saline. The amount of time required for separation will vary depending on the individual composition of the fat sample. In some embodiments, the separation can be carried out for 1 to 30 minutes. In some embodiments, the separation can be carried out for 1 to 15 minutes. In some embodiments, the separation can be carried out for 1 to 10 minutes. In some embodiments, the separation can be carried out for 1 to 5 minutes. In some embodiments, the separation can be carried out for 10 seconds to 1 minute. In some embodiments, the separation can be carried out for less than 1 minute.

[0164] The saline is then drained from the fat compartment 405, 505 as completely as possible so that only fat remains. Depending on the type of cartridge 400, 500 used in the method, draining can be done in a variety of ways. In some embodiments, saline is drained from the fat compartment 405, 505 and away from the cartridge 400, 500 through a tube located at the bottom of the fat compartment 405, 505 that leads to the exterior of the cartridge 400, 500. The draining can be done actively using a syringe or pump, or passively by gravity flow. In some embodiments, draining is done so as to maintain the integrity of the sterile system, as described herein.

[0165] The washes are repeated until the fat is golden yellow and the brine is only slightly cloudy after the washes are complete. In some embodiments, the washes are performed 1-5 times, in some embodiments 1-4 times, in some embodiments 1-3 times, in some embodiments 1-2 times, and in some embodiments 1 time.

[0166] In some embodiments, washing is repeated until the sterile saline is exhausted from the fluid reservoir 415, 515. In some embodiments, washing is repeated until the saline wash in the fat compartment 405, 505 is sufficiently clear that 50%-100% of the light passes through the saline after washing, indicating high clarity. In some embodiments, washing is repeated until a certain number of washing cycles are completed, which in some embodiments is 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, in some embodiments 1, in some embodiments 2 times, and in some embodiments, 3 times.

[0167] Once washed, the fat is ready for processing.

[0168] The fat in the fat chamber 405, 505 is processed according to one of the methods described in methods 1 and 2 above by subjecting the fat to ESW and / or mechanical impact to release stem cells from the fat. In some embodiments, the cartridge 400, 500 has an opening above the fat chamber 405, 505 to allow for optimal processing, so that the ESW and / or mechanical impact forces do not pass through the rigid plastic exterior of the cartridge 400, 500. In such embodiments, the walls of the flexible fat chamber 405, 505 are exposed for processing.

[0169] After the tissue processing is complete, the fat is allowed to separate from the fluid and rise to the top. As described above, in this embodiment, the cartridge 400, 500 has two variations. In some embodiments, the cartridge 400, 500 has a fixed vertical orientation so that the processed fat and saline contained in the fat chamber 405, 505 will always naturally separate from each other by gravity, with the fat floating to the top of the fat chamber 405, 505. In some embodiments, the cartridge 400, 500 has a horizontal orientation and rotates vertically to allow the fat to float and separate from the saline. Because the fat is less dense than the saline, it will rise and float on top of the saline. The stem cells that have been released from the fat are now contained in the saline layer.

[0170] The saline containing the stem cells is then moved from the fat chamber 405, 505 through the third one-way valve 440, 540 to the cell reservoir 435, 535 located within the cartridge 400, 500, away from the fat. The saline is moved into the interior of the cell reservoir 435, 535 by using a second external actuator 445, 545 that moves the second plunger 450, 550 to force the saline through the third one-way valve 440, 540. In the cell reservoir 435, 535, the second plunger 450, 550 is oriented in a fully closed configuration, so that the cell reservoir 435, 535 is fully closed. In this manner, moving the second actuator 445, 545 moves the second plunger 450, 550 away from the third one-way valve 440, 540, creating a gentle vacuum inside the cell reservoir 435, 535, which pulls the layer of saline containing stem cells out of the fat chamber 405, 505, through the third one-way valve 440, 540 and into the interior of the cell reservoir 435, 535. In some embodiments, the second external actuator 445, 545 is manually operated by a user who pulls the second actuator 445, 545 until the entire saline containing stem cells is pulled through the third one-way valve 440, 540 and into the interior of the cell reservoir 435, 535. In some embodiments, the movement of the second external actuator 445, 545 is automated, and an external machine pulls the second external actuator 445, 545 to move the saline containing stem cells into the cell reservoir 435, 535.

[0171] In some embodiments, the saline containing stem cells is drawn through a mesh to filter out unwanted cell debris left over from the processed adipose tissue. In some embodiments, the mesh is located in the fat chamber 405, 505, where it covers the opening of the third one-way valve 440, 540. In some embodiments, the mesh is located inside the cell reservoir 435, 535, where it covers the outlet of the third one-way valve 440, 540. In some embodiments, the mesh is located inside the cell reservoir, where it covers the opening of the fourth one-way valve 455, 555. In some embodiments, the size of the holes in the mesh ranges from 40 μm to 100 μm, and in some embodiments, the size of the holes is 70 μm. In some embodiments, the mesh is nylon.

[0172] exist Figure 4 In the embodiment shown, the fraction containing stem cells is transferred from the interior of the cell reservoir 435 through the fourth one-way valve 455 to the centrifuge tube 460 contained within the cartridge 400. The stem cell fraction is moved into the centrifuge tube 460 by using a second external actuator 445, which moves the second plunger 450 to force saline through the third one-way valve 440 and into the centrifuge tube 460. As described herein, this movement can be performed manually, or it can be automated. The centrifuge tube 460 is then removed from the cartridge 400 for centrifugation. Centrifugation is performed as described in either of the above two embodiments; the stem cell mass remains at the bottom of the centrifuge tube 460.

[0173] exist Figure 5 In the embodiment shown, the fraction containing stem cells is transferred from the interior of the cell reservoir 535 through the fourth one-way valve 555 to the centrifugal assembly 560 contained in the cartridge 500. The centrifugal assembly 560 is a triangular chamber that serves as a centrifugal component; it spins cells out of saline so that the cells are concentrated at a single point at the outer edge of the chamber. The entry port 565 allows the user to enter the stem cell mass so as to resuspend the mass and / or remove it from the centrifugal assembly 560. In some embodiments, the operation of the centrifugal assembly 560 is automated and controlled by an external device. Wherein the external device is engaged with the rotating chamber by a motor and a gear, which is inserted into the cartridge in the center of the chamber. In this regard, the centrifugal assembly 560 can be a direct drive or a gear drive. The layer / infranatant containing stem cells is loaded into the centrifugal component as described above, and the other side is balanced before centrifugation. Once the centrifugal component is introduced, the stem cells are centrifuged at 500g to 1600g for 3 to 10 minutes. In some embodiments, centrifugation is performed at 1200g for 10 minutes.

[0174] A small amount of fluid is placed into the tube to resuspend the cells. This is usually 1mL to 5mL. The fluid can be saline, platelet-rich plasma, hyaluronan or a fixed gel, a hydrogel, a scaffold or fibrin or glue, or other compounds.

[0175] In case of cell suspension, they are drawn up through a hypodermic needle into a syringe for further use. This is done automatically by a processing machine, or manually by an operator.

[0176] The stem cells are resuspended using a small amount of fluid. In some embodiments, the amount of fluid added to resuspend the cells is 1 mL to 5 mL. In some embodiments, the fluid is saline, platelet-rich plasma, hyaluronan, a fixed gel, a hydrogel, a scaffold, fibrin, a glue, or a combination of any of the foregoing. In some embodiments, the cells are resuspended in a centrifugal aspirate.

[0177] With the stem cells floating in the suspension, they are ready for use. Such uses may include, but are not limited to, reintroduction into the patient from whom they came, cryopreservation, expansion, etc. In some embodiments, the concentrated resuspended stem cells are moved into a syringe through a hypodermic needle for use by the patient. In some embodiments, the concentrated resuspended stem cells are seeded into a tissue scaffold to grow into myocardium, muscle, bone, cartilage, liver, kidney or other tissue and organ structures. In some embodiments, the concentrated, resuspended stem cells are converted into induced pluripotent stem cells by expressing pluripotency transcription factors.

[0178] Device

[0179] As described above, the present disclosure provides methods and systems for bioprocessing tissue samples. In different aspects, the present disclosure also provides easy-to-use devices suitable for the disclosed methods, which are closed to the external environment and designed for single use. Such devices include single-use cartridges that allow complete processing in a single device.

[0180] container

[0181] The above embodiments utilize one or more containers as receptacles for degenerative tissue samples, fat samples, fractions containing desired cell fractions, fractions containing stem cells, or any combination of the foregoing. Such containers are included in Figure 1 Container used in step 105; container 205; fat chamber 405, 505; fluid reservoir 415, 515; cell reservoir 435, 535. These containers suitable for use in the systems and methods provided by the present disclosure can be flexible, rigid or semi-rigid. In some embodiments, the container is a syringe.

[0182] exist Figure 1The container used in step 105; container 205; fat chambers 405, 505; fluid reservoirs 415, 515; cell reservoirs 435, 535, each of which is closed to the external environment by itself or as part of a closed cartridge, and is disposable.

[0183] In some embodiments, Figure 1 The container used in step 105; container 205; fat chamber 405, 505; fluid reservoir 415, 515; cell reservoir 435, 535, each of which is a flexible container, such as a flexible bag. In such an embodiment, each container can be made of ethylene vinyl acetate (EVA), poly (vinyl) chloride (PVC), ethylene-vinyl acetate (EVA), nylon or other plastics.

[0184] Each flexible container can be blow molded. In some embodiments, each flexible container can be radio frequency, high frequency or dielectric welded and can be blow molded.

[0185] In some embodiments, each flexible container is a three-dimensional bag having at least one outlet that is fluidly connected to the lumen of the flexible container. In some embodiments, a single container may have more than one outlet that is fluidly connected to the lumen of the container. Such outlets may be used, for example, to discharge fluid from the lumen of a container or to move fluid from the interior of a container to another container.

[0186] In some embodiments, the container may include one or more holes along the edge on the outside of the container that can be used to suspend the container in space.

[0187] The total volume of the container may be from 5 to 50 fluid ounces, and in some embodiments, from 9 to 19 fluid ounces.

[0188] In some embodiments, in order to receive input in the form of tissue samples, sterile saline, or other forms, the container is configured to receive an inlet line or connection at a discrete point that connects to an inlet that is fluidically connected to the interior of the container. In order to maintain the integrity of the closed sterile environment, the inlet line or connection includes a female Luer connection that allows the container to be connected to an external device, such as a syringe containing a tissue sample, another container from which material is transferred to the container, or other aspects. In other embodiments, the inlet line or connection includes a sterile docking object for connecting to an external device using a sterile connection device.

[0189] In several embodiments, the container can be a flat bag having a top edge, a bottom edge, and two substantially similar side edges. The bottom edge includes an outlet that is fluidly connected to the interior of the bag for draining fluid out of the bag. The flat bag also has an inlet that allows material to be aseptically introduced into the interior of the bag.

[0190] In several embodiments, the container is a three-dimensional bag having a rectangular shape with a top edge, a bottom edge, and two substantially similar side edges. The bottom edge includes an outlet connected to the interior fluid of the container for draining fluid out of the container. The top edge includes an inlet connected to the interior fluid of the container for introducing material into the container.

[0191] In some embodiments, a container is connected to one or more other containers via a line. A line is a tube that can be made of poly(vinyl) chloride (PVC), ethylene-vinyl acetate (EVA), or other materials.

[0192] In some embodiments, the container can be semi-rigid. In such an embodiment, the container includes a rigid plastic shell that maintains its shape and can hold a flexible container inside the shell during tissue processing. The housing is configured so that it contains a connection point with the platform 210 or 315.

[0193] The rigid plastic housing can be made of any suitable rigid-plastic material, including, for example, high-density polyethylene (HDPE) or polypropylene (PP). Rigid plastic materials do not deform elastically, nor do they exhibit any elastic properties that flexible plastics typically exhibit. The rigid plastic housing can be injection molded or die cut.

[0194] In some embodiments, the flexible container is contained within a rigid plastic shell, which may or may not completely surround the flexible container. In some embodiments, the flexible container is not completely enclosed within the rigid plastic shell; instead, the shell partially surrounds the flexible container, keeping it in place during tissue processing.

[0195] Cartridge

[0196] In various aspects, a cartridge suitable for use with the present disclosure is a three-dimensional container that is capable of housing one or more containers within it. The internal containers may be connected by one or more lines and / or valves. Examples of suitable cartridges are Figure 4 and 5 The cartridges 400, 500 shown in FIG.

[0197] In some embodiments, the cartridge is made of a rigid plastic material that retains its shape and is capable of retaining at least one, preferably multiple, flexible containers within the cartridge during tissue processing. The containers are configured so that they contain one or more connections to the platform 210 or 315.

[0198] The rigid plastic can be made of any suitable rigid-plastic material, including, for example, high density polyethylene (HDPE) or polypropylene (PP). Rigid plastic materials do not deform elastically, nor do they exhibit any elastic properties that flexible plastics typically exhibit. Rigid plastic cartridges can be injection molded or die cut.

[0199] Further aspects of the container are as described above.

[0200] Centrifuge tubes

[0201] Centrifuge tubes suitable for the methods and devices of the present disclosure are precisely manufactured high-strength glass or plastic tubes designed to fit precisely in a centrifuge rotor. The capacity of the centrifuge tube can vary depending on the total volume of the tissue sample to be processed. In some embodiments, the capacity of the centrifuge tube ranges from 1 mL to 50 mL, from 0.5 mL to 20 mL in some embodiments, and from 250 μL to 2.0 mL in some embodiments.

[0202] In some embodiments, the centrifuge tube is The tube, in some embodiments, is a microfuge tube, and in some embodiments, is a microcentrifuge tube.

[0203] The material of the centrifuge tube can vary. In some embodiments, the centrifuge tube is made of glass. In some embodiments, the centrifuge tube is plastic. In each embodiment, the centrifuge tube is designed to be disposable and is disposable after use. In some embodiments, the centrifuge tube is made of flexible transparent plastic (e.g., polyethylene), is semi-conical, and includes an integral hinged sealing cover.

[0204] Check valve

[0205] A check valve is a valve that allows fluid to flow through it in a single direction only.

[0206] In various aspects, one-way valves suitable for use with the apparatus and methods of the present disclosure include dual-port valves having two openings - one for fluid entry and another for fluid exit. One-way valves are used to automatically provide one-way fluid flow without any user intervention or control. In some embodiments, one-way valves useful with the methods and apparatus of the present disclosure are made of a rigid plastic, such as poly(propylene).

[0207] In some embodiments, the one-way valve suitable for the apparatus and method of the present disclosure is a ball check valve, wherein the component preventing the backflow of fluid is a spherical ball. In some embodiments, the ball is spring loaded to help keep the one-way valve closed.

[0208] In some embodiments, a one-way valve suitable for use in the apparatus and methods of the present disclosure is a diaphragm check valve, which includes a curved rubber diaphragm positioned to produce valve closure. In such embodiments, pressure generated on the upstream side by user intervention or by an automatic process causes the diaphragm to open and fluid to flow through the valve. When the positive pressure ceases, the diaphragm closes, terminating fluid flow.

[0209] In some embodiments, the one-way valve suitable for the apparatus and method of the present disclosure is a swing check valve or a tilting disc check valve. In such embodiments, the disc is the movable part of the valve, which allows fluid to flow in one direction and prevents fluid from flowing back in the opposite direction.

[0210] Example

[0211] Materials, methods and embodiments described herein are further defined in the following examples. Certain embodiments are also defined in the examples herein. It should be understood that these examples, although indicating certain embodiments, are provided only by way of example. According to the disclosure herein and these examples, those skilled in the art can determine the essential features of the disclosure, and, without departing from its spirit and scope, can make various changes and modifications to the present invention to adapt it to various uses and conditions.

[0212] Example 1

[0213] General Protocol for ESW Tissue Processing

[0214] Obtain lipoaspirate from the subject, the volume of which is approximately 30 cc. Prior to processing, transfer the lipoaspirate to a flexible, sterile ethylene vinyl acetate (EVA), poly(vinyl) chloride (PVC), ethylene-vinyl acetate (EVA), or nylon bag having side walls approximately 0.25 inches thick. A sterile catheter bag is sufficient.

[0215] The lipoaspirate was washed with an equal volume of sterile saline introduced into the flexible bag by connecting the male Luer lock connector from the saline bag to the female Luer lock connector on the flexible bag and manually dispensing saline into the flexible bag. The lipoaspirate and saline wash were then mechanically agitated by gently shaking the flexible bag.

[0216] The tissue sample / saline wash mixture was allowed to settle for at least 10 minutes to allow the lipoaspirate to float to the top of the saline.

[0217] The lower layer or saline wash is removed from the flexible bag through a drain line located at the bottom of the flexible bag. Removal can be performed by connecting a syringe to the line and drawing the lower layer material out of the bag or by gravity flow.

[0218] Washing was repeated 3-4 times, leaving the sterile saline wash in the bag for the final wash.

[0219] The next step is to apply extracorporeal shock waves to the lipoaspirate using a Masterpuls MP100 extracorporeal shock wave generator.

[0220] The generator may be in contact with the walls of the flexible bag, but care should be taken to ensure that the shock wave generator does not contact the lipoaspirate itself.

[0221] If necessary, ultrasound gel may be applied to the sides of the flexible bag before applying shock waves to the lipoaspirate.

[0222] Apply ESW to the lipoaspirate for 1-30 minutes.

[0223] Thereafter, the flexible bag was moderately agitated by shaking or light vortexing.

[0224] The currently processed lipoaspirate is allowed to settle so that the remaining adipose tissue can float to the top of the saline.

[0225] Remove the lower layer from the flexible bag by connecting a sterile syringe to the drain tube as described above and aspirate as much of the lower layer as possible. Do not remove any remaining adipose tissue.

[0226] During removal, the lower layer was passed through one of three nylon mesh filters with a pore size of 40 μm, 70 μm or 100 μm.

[0227] Dispense the lower layer into one or more sterile centrifuge tubes. If using an odd number of tubes, make sure to place a balance tube containing sterile saline into the rotator to maintain balance.

[0228] Centrifuge at 1,200 g for 10 min.

[0229] Optional: Resuspend the pellet in 1 mL RBC lysis buffer and incubate in a water bath at 37°C for 10 min. Neutralize the RBC buffer.

[0230] If RBC lysis buffer is not desired, resuspend the cell pellet in 1 mL sterile saline.

[0231] Count cells

[0232] Example 2

[0233] Comparison of liposuction processing methods

[0234] Abstract: The purpose of the tests conducted in this example was to compare the nucleated cell yield of mesenchymal stem cells extracted from adipose tissue using collagenase digestion and extracorporeal shock wave (ESW) disruption.

[0235] Material:

[0236] 1,500 mL 1x phosphate buffered saline (PBS)

[0237] 35mL 1x Red Blood Cell (RBC) Lysis Buffer

[0238] 200 mL 0.1% collagenase type I

[0239] 15 mL 10% fetal bovine serum (FBS) in Dulbecco's modified eagle's medium (DMEM)

[0240] method:

[0241] Comparison

[0242] Use a sterile 60 mL catheter to transfer approximately 250 mL of lipoaspirate into a 100 mL beaker. Take care to ensure that little to no excess fluid is introduced into the beaker.

[0243] Wash the lipoaspirate four times with approximately equal volumes of sterile 1x PBS. For each wash, add sterile 1x PBS to the beaker, stir both, and allow the mixture to settle. Remove the lower layer from the beaker by aspiration. Wash the lipoaspirate a total of 4 times.

[0244] After the fourth wash, the samples were allowed to settle for 10 minutes.

[0245] Using a sterile 10 mL serological pipette, transfer 45 mL of the lower layer reagent from the fourth and final wash into 3 sterile 50 mL conical tubes and pass through 40 μm, 70 μm, and 100 μm tube sterile filters. Transfer a total of 135 mL of the lower layer as a control.

[0246] Collagenase digestion

[0247] Deposit 200 mL of lipoaspirate into a sterile 500 mL glass bottle. Add 200 mL of 0.1% collagenase type I to the bottle. Gently agitate the bottle by inverting several times to ensure that the lipoaspirate and collagenase are evenly mixed.

[0248] The bottles were then placed in a water bath at 37°C. Every 10 minutes, the bottles were removed from the water bath and inverted several times to redistribute the lipoaspirate that had settled. After 30 minutes of incubation, the bottles were stirred for the final time and removed from the water bath.

[0249] Spray the outside of the bottle with 70% ETOH and place in a fume hood. Allow the lipoaspirate and collagenase mixture to settle for 10 minutes.

[0250] The lower layer was transferred to three sterile 50 mL conical tubes using a sterile 10 mL serological pipette through 40 μm, 70 μm and 100 μm tube sterile filters. A total of 135 mL of the lower layer was transferred. The three tubes (including a fourth tube containing 45 mL of water for balance) were centrifuged at 1,200 g, 37° C. for 10 minutes to obtain a collagenase digested cell pellet.

[0251] Clean the outside of the 3 tubes containing the collagenase suspension with 70% ETOH and place in a fume hood with the balance tubes off. Aspirate the supernatant, leaving cell pellets in each tube. Using a 25 mL serological pipette, dispense 5 mL of 10% FBS into each tube and vortex each tube for 5 seconds to resuspend the pellets. The FBS is used to neutralize any remaining collagenase activity.

[0252] The three tubes were centrifuged at 1,200 g for 10 min at 37° C. The outside of the centrifuge tubes were cleaned with 70% ETOH and the tubes were placed in a fume hood. The supernatant was aspirated, leaving the cell pellet.

[0253] ESW Processing

[0254] Transfer 200 mL of lipoaspirate, along with all remaining substratum, to a sterile 500 mL leg-shaped catheter bag using a sterile 60 mL catheter syringe. Add sterile 1x PBS to reach a total fluid volume of 200 mL for a total volume of 400 mL including the lipoaspirate. Apply approximately 2 mL of ultrasound gel to the catheter bag. Using a large tip, apply approximately 10,000 ESW pulses at 2 bar and 21 Hz to the sample inside the bag. Repeat the process on the other side of the bag with another 10,000 ESW pulses. Clean the outside of the catheter bag with 70% ETOH and place it in a fume hood.

[0255] Stir the bag and pour all into a 500 mL beaker, allowing the suspension to settle for 10 minutes. Using a sterile 10 mL serological pipette, transfer the lower layer to 3 sterile 50 mL conical tubes and pass through 40 μm, 70 μm, and 100 μm tube sterile filters. Transfer a total of 135 mL of the lower layer.

[0256] Concentration and lysis

[0257] Centrifuge the 3 tubes containing the control suspension and the 3 tubes containing the processed suspension at 1,200 g and 37°C for 10 minutes. Aspirate the supernatant from all tubes and add each remaining pellet and 5 mL of 1x RBC lysis buffer to each tube. Vortex each tube for 5 seconds to resuspend the pellet and place the tubes in a 37°C water bath for 10 minutes.

[0258] Sterilize the outside of each tube with 70% ETOH and place the tubes in a fume hood. Add 25 mL of sterile 1x PBS to each tube to dilute and neutralize the lysis buffer.

[0259] Cell counting

[0260] Using a sterile micropipette tip, transfer 300 µL of sterile 1x PBS into 6 sterile 0.5 mL microcentrifuge tubes. Also transfer 75 µL of each 50 mL sample tube into each microcentrifuge tube for cell counting.

[0261] The number of cells in each sample was counted using a MoxiZ cell counter with cartridges type M and S. For counting, 100 μL of trypan blue was added to each microcentrifuge tube, 10 μL of each sample was loaded onto a hemocytometer and the cells were counted.

[0262] result:

[0263] MoxiZ Cell Counter

[0264] Table 1 shows the cell counting results using the MoxiZ automated cell counter. Two types of cartridges were used for counting, namely, M and S types. In the sample column, 'c' indicates collagenase digestion, 'e' indicates ESW processing, and '0' indicates control. Only the 'y' counts in the 'Finish' column are valid.

[0265] Table 1

[0266]

[0267]

[0268]

[0269] Blood cell counter

[0270] Table 2 details the counts obtained from the visual hemocytometer, with cell counts from each quadrant, the sum of the quadrants, the dilution factor, and the total counted cells. Cell numbers should be obtained with reference to the number of cells in the control population. The ratio of processed cells to control cells is a key indicator of yield.

[0271] Table 2

[0272]

[0273] Overview

[0274] In the efficient counting using the MoxiZ automated cell counter, the aggregated mean values ​​of cell counts for control, collagenase, and ESW are shown in Table 3:

[0275] Table 3

[0276] sample average value SD 0 2.24E+05 1.18E+05 C 3.81E+05 5.11E+04 E 3.15E+05 2.08E+05

[0277] Collagenase consistently yielded approximately 3.8E+05 cells / mL. ESW ranged from 1.67e+05 to 7.08e+05 cells / mL.

[0278] Looking at the ratios of the cells in the hemocytometer, the ESW process produced the highest ratios, with ratios ranging from 1.78 to 9.0 times higher than the control. Collagenase only produced ratios ranging from 1.29 to 2.84 times higher than the control.

[0279] discuss

[0280] These data indicate that ESW is effective in producing large numbers of mesenchymal stem cells from adipose tissue. Based on these data, it can be concluded that ESW is a viable alternative to ultrasound or collagenase processing of adipose tissue to obtain mesenchymal stem cells. When comparing peaks, ESW produced a cell yield of 7.08e+05 compared to the collagenase peak of 4.32e+05 - a 164% increase in yield.

[0281] Given the data presented above, it is reasonable to conclude that a 50cc to 60cc lipoaspirate sample will yield approximately 5 million cells from ESW processing and approximately 3 million cells from collagenase digestion.

[0282] It is therefore apparent that the use of ESW in releasing stem cells from adipose tissue confers significant advantages over known techniques.

[0283] In particular, the present invention also relates to the following items:

[0284] Item 1. A method for separating a cell fraction from a tissue sample, comprising:

[0285] obtaining a tissue sample from a subject;

[0286] exposing the tissue sample to shock waves, mechanical impact forces, or both; and

[0287] separating cell fractions from tissue samples;

[0288] The source of the shock wave and / or the source of the mechanical impact force does not come into physical contact with the tissue sample.

[0289] Item 2. The method of Item 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, gum tissue, skin tissue, pineal gland tissue, pituitary tissue, thyroid tissue, adrenal gland tissue, pancreatic tissue, ovarian tissue and testicular tissue.

[0290] Item 3. The method of Item 1 or Item 2, wherein the tissue is adipose tissue.

[0291] Item 4. The method of any one of Items 1-3, wherein the tissue sample is contacted with shock waves, and the shock wave source is a shock wave applicator driven by a shock wave generator.

[0292] Item 5. The method of any one of Items 1-3, wherein the tissue sample is contacted with a mechanical impact force, and the source of the mechanical impact force is an impact arm driven by a motor.

[0293] Item 6. The method of any one of Items 1-5, wherein the tissue sample is washed one or more times before contacting with the shock wave and / or mechanical impact force.

[0294] Item 7. The method of any one of Items 1-4 or 6, wherein the shock wave breaks the tissue sample into multiple small cell clusters, multiple single cells, or both.

[0295] Item 8. The method of any one of Items 1-3, 5 or 6, wherein the mechanical impact force breaks the tissue sample into a plurality of small cell clusters, a plurality of single cells, or both.

[0296] Item 9. The method of Item 7 or Item 8, wherein the cell fraction comprises a plurality of small cell clusters, a plurality of single cells, or both, and separation of the cell fraction is performed by centrifugation.

[0297] Item 10. The method of Item 9, wherein the centrifugation is performed at a speed of 500 g to 2,000 g for 3 to 30 minutes.

[0298] Item 11. The method of Item 10, wherein the centrifugation is performed at 1,200 g for 10 minutes.

[0299] Item 12. The method of any one of Items 9-11, wherein the separated cell fraction is resuspended after centrifugation.

[0300] Item 13. The method of any one of items 1-3, 5, 6 or 8-12, wherein the cell fraction is separated in 30 minutes or less.

[0301] Item 14. A method for isolating stem cells from adipose tissue, comprising:

[0302] obtaining an adipose tissue sample from the subject;

[0303] placing the tissue sample into a container or cartridge;

[0304] subjecting the tissue sample to mechanical shock forces to release the stem cells;

[0305] Isolating a stem cell fraction from adipose tissue; and

[0306] Centrifuge the stem cell fraction;

[0307] The source of the mechanical impact force does not physically contact the fat tissue.

[0308] Item 15. The method of Item 14, wherein the adipose tissue is washed one or more times before being subjected to the mechanical impact force.

[0309] Item 16. The method of Item 14 or Item 15, wherein the source of the mechanical impact force is an impact arm driven by an electric motor, the motor comprising a gear capable of reducing the speed of the impact arm when the motor is running at full speed.

[0310] Item 17. The method of Item 14 or Item 15, wherein the mechanical impact force is transmitted to the adipose tissue through the wall of the container or cartridge.

[0311] Item 18. The method of any of Items 15-17, wherein the gear allows the speed in the impact arm to be reduced from 10 to 1.

[0312] Item 19. The method of any of Items 16-18, wherein the motor comprises a gear diameter of 0.75 inches and, at a motor speed of 3000 rpm, the impact arm has a speed of 117.8 inches per second.

[0313] Item 20. The method of any one of Items 14-19, wherein isolating the stem cell fraction comprises allowing the adipose tissue to separate from an aqueous layer, wherein the aqueous layer contains the stem cells.

[0314] Item 21. The method of any one of Items 14-20, wherein the stem cells are isolated from the adipose tissue within 30 minutes or less.

[0315] Item 22. The method of any one of Items 14-21, wherein the centrifugation is performed at 500 g-1,600 g for 3-10 minutes.

[0316] Item 23. A method for isolating stem cells from adipose tissue, comprising:

[0317] obtaining an adipose tissue sample from the subject;

[0318] placing the tissue sample into a container or cartridge;

[0319] subjecting the tissue sample to shock waves to release the stem cells;

[0320] Isolating a stem cell fraction from adipose tissue; and

[0321] Centrifuge the stem cell fraction;

[0322] The source of the shock waves does not physically contact the adipose tissue.

[0323] Item 24. The method of Item 23, wherein the adipose tissue is washed one or more times before being subjected to shock waves.

[0324] Item 25. The method of Item 23 or Item 24, wherein the shock wave source is a shock wave applicator driven by a shock wave generator.

[0325] Item 26. The method of any one of Items 23-25, wherein the shock waves are delivered to the adipose tissue through the wall of the container or cartridge.

[0326] Item 27. The method of any one of Items 23-26, wherein the adipose tissue is subjected to a shock wave having a force of 0.5-5.0 bar.

[0327] Item 28. The method of any one of Items 23-27, wherein the container is a vinyl bag having a wall 0.25 mm thick, and the adipose tissue is subjected to a shock wave having a force of 2.0-2.5 bar.

[0328] Item 29. The method of any one of Items 23-28, wherein the total area of ​​adipose tissue subjected to shock waves at any one time is 1 cm 2 -100cm 2 .

[0329] Item 30. The method of Item 23, wherein the container is a 19 ounce vinyl bag with a wall thickness of 0.25 mm, the total amount of the adipose tissue sample is 30 cc, and the total area of ​​adipose tissue subjected to the shock wave at any one time is 5 cm 2 .

[0330] Item 31. The method of any one of Items 23-30, wherein the adipose tissue is subjected to 5,000-100,000 shock waves.

[0331] Item 32. The method of any one of Items 23-31, wherein isolating the stem cell fraction comprises allowing the adipose tissue to separate from an aqueous layer, wherein the aqueous layer contains the stem cells.

[0332] Item 33. The method of any one of Items 23-32, wherein the centrifugation is performed at 500 g-1,600 g for 3-10 minutes.

Claims

1. A method for isolating a stromal vascular fraction from adipose tissue, comprising: placing the tissue sample into a processing container of a tissue processing device; subjecting the tissue sample to forces from mechanical impact to disintegrate the tissue; and Isolation of stromal vascular fraction from adipose tissue; in: The mechanical impact is generated by an impact arm that physically contacts the container by articulating up and down at a speed of up to 30,000 rpm; and The impact arm does not physically contact the adipose tissue.

2. The method according to claim 1, wherein the processing device comprises: a platform to which the processing vessel is connected; and A variable speed motor drives the impact arm to be hinged up and down.

3. The method of claim 2, wherein the motor is a variable speed motor, the variable speed motor being adjustable to drive the upper and lower articulations of the impact arm at a speed of 3,000 rpm to 30,000 rpm.

4. The method of any one of claims 1-3, wherein the separating occurs by separating the tissue from an aqueous layer, the aqueous layer comprising a stromal vascular fraction.

5. The method according to any one of claims 1 to 3, wherein the separation occurs by: Filtering; and / or Centrifuge.

6. The method of claim 4, wherein the stromal vascular fraction is isolated from the tissue in 30 minutes or less.

7. The method according to any one of claims 1 to 3, wherein: The processing container is a bag; and The bag comprises one or more outlets connected to the interior fluid of the bag; and / or The bag includes an inlet fluidly connected to the interior of the bag.

8. The method of any one of claims 1-3, wherein the stromal vascular fraction comprises stem cells, growth factors, and progenitor cells.

9. The method of claim 1, wherein the stromal vascular fraction comprises adipose stem cells and the separation occurs in less than 1 minute.

10. The method of any one of claims 1-3, further comprising subjecting the tissue to shear forces and mechanical impact to disintegrate the tissue.

11. The method according to any one of claims 1 to 3, wherein: After separation, the stromal vascular fraction is filtered and / or centrifuged.

12. The method according to any one of claims 2-3, wherein the platform is movable in three directional planes.

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