Separation, dissociation, and / or disaggregation of cells using shock waves or mechanical impact

Abstract

To provide reproducible and robust manufacturing processes for the isolation and / or production of therapeutic cells for biopharmaceutical manufacturing.SOLUTION: Methods are based on external shockwaves, mechanical impacts and / or principles of lithotripsy to break up a tissue sample into smaller fragments, i.e., clusters of cells and / or single cells, after which a desired cellular fraction can be isolated from the sample. Devices of the present disclosure deploy focused and / or directed shockwaves, and / or focused and directed mechanical impacts, to break apart a tissue sample. The devices maintain the sample in a sterile, closed environment during exposure to the shockwaves or mechanical impacts. Therefore, the shockwaves and / or mechanical impacts are generated outside a closed device and are transmitted through one or more walls of the device into its interior, where the sample is located.SELECTED DRAWING: None

Description

[Technical field]

[0001] Cell bioprocessing is a form of biopharmaceutical manufacturing whose goal is to produce therapeutic cells. Establish a reproducible and robust manufacturing process for cell isolation and / or production. And so. [Background technology]

[0002] The current cell manufacturing process is highly user-dependent and involves human intervention at many points. Due to that dependency, the current process is cumbersome and extremely It is diverse, expensive and has low yields.

[0003] As an example, many current manufacturing processes proceed as follows: A tissue sample (adipose tissue) is A biological sample (e.g., aspirate, whole blood, bone marrow, cord blood, etc.) is obtained from the patient. The pellet is then transported to a laboratory for bioprocessing. Techniques are used to obtain the desired cell fraction. The cell fraction is then isolated and The cells are then transported back to the point-of-care facility for patient use or are subsequently expanded. either further processed by introduction into a bioreactor for Some known methods of cell separation involve the steps of: Ultrasonic cavitation requires contacting the sample with a foreign object (e.g., a bead). This includes the use of mechanical separation via centrifugation, enzymatic digestion, or other mechanical means. The use of such technologies will result in an increased regulatory compliance burden. The reason is that the enzyme needs to be in direct contact with the sample to produce the desired result. Also, enzymes can damage the integrity of the harvested cells. It has the potential to change its properties in some cases. The use of sonicators is similarly imperfect. The sonicator tip is fixed in frequency and The cells must be introduced into a cell or tissue suspension, which can adversely affect sterility. Additionally, the use of such high energy sound waves can result in large amounts of energy It introduces oxygen into the sample, which manifests as heat. This can damage cells. This reduces the yield of usable cells.

[0004] Once obtained, the expanded cell product is then transferred to a bioreactor where it is cleaned and concentrated. The test sample is washed, concentrated, and transported to the If the test results indicate an acceptable product, the artificial The cell products produced in are prepared for long-term storage or prepared for cryopreservation. or administered to the patient from whom the sample was derived, or any of the foregoing. It is any combination.

[0005] As can be appreciated, some of these steps involve transferring the sample from one vessel to another. Requires transfer to a container, requires significant user intervention, requires significant user intervention One or more samples were mislabeled or handled during processing. Not only does this increase the risk of error, but it can also compromise the sterility and identity of samples. Additionally, processing the sample in this manner may adversely affect the purity or potency of the sample. The time required to obtain the desired cell fraction may take many hours (even days) to complete. This means that the drug is not ready for delivery to patients (even if it is not This can have adverse effects on the patient's health. The potential for and the unreasonable amount of time required to process cell samples. Long periods of time can be hazardous for patients, especially those with weakened immune systems, or This is unacceptable for patients who require immediate care and is also a barrier to timely, high-quality care. This is unacceptable for providers who need to deliver treatment to many patients. Summary of the Invention

[0006] This disclosure includes the following [1] to

[33] . [1] A method for isolating a cellular fraction from a tissue sample, the method comprising: obtaining a tissue sample from a subject; contacting the tissue sample with a shock wave, a force from a mechanical impact, or both; isolating a cellular fraction from the tissue sample; Including, A method wherein the source of force from the shock wave and / or the mechanical impact does not make physical contact with the tissue sample. [2] The method according to [1] above, wherein the tissue is selected from adipose tissue, brain tissue, pharyngeal tissue, laryngeal tissue, heart tissue, arterial tissue, muscle tissue, liver tissue, gallbladder tissue, kidney tissue, small intestine tissue, large intestine tissue, lymph node tissue, lung tissue, spleen tissue, bone marrow tissue, stomach tissue, venous tissue, pancreatic tissue, bladder tissue, bone, tooth, dentin tissue, gingival tissue, skin tissue, pineal gland tissue, pituitary tissue, thyroid tissue, adrenal tissue, pancreatic tissue, ovarian tissue and testicular tissue. [3] The method according to [1] or [2] above, wherein the tissue is adipose tissue. [4] The method of any one of [1] to [3], wherein the tissue sample is contacted with shock waves and the source of the shock waves is a shock wave applicator powered by a shock wave generator. [5] The method of any one of [1] to [3], wherein the tissue sample is contacted with a force from a mechanical impact, and the source of the force from the mechanical impact is an impact arm powered by a motor. [6] The method of any one of [1] to [5], wherein the tissue sample is washed one or more times before contacting the tissue sample with the force from the shock wave and / or the mechanical impact. [7] The method of any one of [1] to [4] or [6] above, wherein the shock waves break down the tissue sample into a plurality of small cell groups, a plurality of individual cells, or both. [8] The method of any one of [1] to [3], [5] or [6] above, wherein the force from the mechanical impact breaks down the tissue sample into a plurality of small cell groups, a plurality of individual cells, or both. [9] The method according to [7] or [8], wherein the cell fraction comprises the plurality of small cell groups, a plurality of individual cells, or both, and isolation of the cell fraction is achieved by centrifugation.

[10] The method according to [9] above, wherein the centrifugation is carried out at a speed of 500g to 2,000g for 3 to 30 minutes.

[11] The method according to

[10] above, wherein the centrifugation is carried out at 1,200 g for 10 minutes.

[12] The method according to any one of [9] to

[11] above, wherein the isolated cell fraction is resuspended after centrifugation.

[13] The method according to any one of [1] to [3], [5], [6] or [8] to

[12] above, wherein the cell fraction is isolated within 30 minutes.

[14] A method for isolating stem cells from adipose tissue, the method comprising: obtaining an adipose tissue sample from a subject; placing the tissue sample into a container or cartridge; exposing the tissue sample to a force from a mechanical impact to release the stem cells; isolating a stem cell fraction from the adipose tissue; Centrifuging the stem cell fraction; Including,

[15] The method of

[14] , wherein the source of the force from the mechanical impact does not physically contact the adipose tissue.

[15] The method of

[14] , wherein the adipose tissue is washed one or more times before being exposed to the force from the mechanical impact.

[16] The method of

[14] or

[15] , wherein the source of the mechanical impact force is an impact arm powered by a motor, the motor including gearing, the gearing capable of reducing the speed of the impact arm when the motor is running at full speed.

[17] The method according to

[14] or

[15] , wherein the force from the mechanical impact is delivered to the adipose tissue through a wall of the container or cartridge.

[18] The method according to any one of

[15] to

[17] , wherein the gearing allows a reduction in speed of 1 / 10 within the impact arm.

[19] The method of any one of

[16] to

[18] , wherein the motor includes a gear diameter of 0.75 inches and at a motor speed of 3,000 rpm, the impact arm has a speed of 117.8 inches per second.

[20] The method of any one of

[14] to

[19] above, wherein the step of isolating the stem cell fraction comprises a step of allowing the adipose tissue to separate from an aqueous layer, the aqueous layer containing the stem cells.

[21] The method according to any one of

[14] to

[20] above, wherein the stem cells are isolated from the adipose tissue within 30 minutes.

[22] The method according to any one of

[14] to

[21] above, wherein centrifugation is carried out for 3 to 10 minutes at 500g to 1,600g.

[23] A method for isolating stem cells from adipose tissue, the method comprising: obtaining an adipose tissue sample from a subject; placing the tissue sample into a container or cartridge; exposing the tissue sample to shock waves to liberate the stem cells; isolating a stem cell fraction from the adipose tissue; Centrifuging the stem cell fraction; Including, A method wherein the source of the shock waves does not physically contact the adipose tissue.

[24] The method according to

[23] above, wherein the adipose tissue is washed one or more times before being exposed to the shock waves.

[25] The method of

[23] or

[24] , wherein the source of the shock waves is a shock wave applicator powered by a shock wave generator.

[26] The method according to any one of

[23] to

[25] , wherein the shock waves are delivered to the adipose tissue through a wall of the container or cartridge.

[27] A method according to any one of

[23] to

[26] , wherein the adipose tissue is exposed to shock waves having a power of 0.5 bar to 5.0 bar.

[28] A method according to any one of

[23] to

[27] , wherein the container is a plastic bag having a wall thickness of 0.25 mm and the adipose tissue is exposed to shock waves having a power of 2.0 bar to 2.5 bar.

[29] The total area of ​​the adipose tissue exposed to the shock waves at any one time is 1 cm 2 From 100cm 2 The method according to any one of

[23] to

[28] above, wherein the range is

[30] The container was a 19 ounce plastic bag with 0.25 mm thick walls, the total volume of the adipose tissue sample was 30 cc, and the total area of ​​the adipose tissue exposed to the shock waves at any one time was 5 cm 2 The method according to

[23] above.

[31] A method according to any one of

[23] to

[30] above, wherein the adipose tissue is exposed to a number of shock waves in the range of 5,000 to 100,000.

[32] The method of any one of

[23] to

[31] above, wherein the step of isolating the stem cell fraction comprises a step of allowing the adipose tissue to separate from an aqueous layer, the aqueous layer containing the stem cells.

[33] The method according to any one of

[23] to

[32] above, wherein centrifugation is carried out for 3 to 10 minutes at 500g to 1,600g. Therefore, there is a need for an optimized and scalable method of obtaining desired cell fractions directly in a short time period without the need for multiple user interventions at the point of care. There is also a need for a device capable of carrying out the method. Such a method and device are provided by the present disclosure. The disclosed method and device eliminate the need to transport the cell sample to a laboratory for processing, and also result in a processed sample that does not contact the cell sample with foreign objects during separation and provides the desired cell sample in a very short time period with little user contact, thereby allowing the optimization of expensive technical resources.

[0007] The devices and methods disclosed herein have several features, None of these is solely responsible for its desirable attributes. Without limiting the scope, certain features of the disclosed devices and methods will now be discussed briefly. After considering this argument, and in particular, After reading the chapter entitled "Forms of a Device and Method," one of ordinary skill in the art will recognize how the features of the device and method It is to be understood that the present invention provides several advantages over conventional systems and methods. This will be the case.

[0008] In one aspect, the present disclosure provides a method for the bioprocessing of a cell sample, e.g., the production of autologous stem cells. Suitable for bioprocessing of lipid tissue samples processed for cell therapy The system is not open to the atmosphere and therefore Sterile sample processing and sample storage throughout bioprocessing Allows transport.

[0009] In a first aspect, a method for isolating a cellular fraction from a tissue sample is provided, the method comprising: obtaining a tissue sample from the tissue sample; and applying force from a shock wave, a mechanical impact, or both. The method includes contacting a tissue sample with the cell fraction and isolating a cellular fraction from the tissue sample. and a source of force from the shock wave and / or mechanical shock is coupled to the tissue sample. No physical contact.

[0010] In some embodiments, the tissue is adipose tissue, brain tissue, pharyngeal tissue, laryngeal tissue, cardiac 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 tissue, thyroid tissue, adrenal tissue, pancreas The tissue is selected from pancreatic tissue, ovarian tissue and testicular tissue.

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

[0012] In some embodiments, the tissue sample is contacted with shock waves, the source of the shock waves being , a shock wave applicator powered by a shock wave generator.

[0013] In some embodiments, the tissue sample is contacted with a force from a mechanical shock, The source of force from the impact is an impact arm powered by a motor. .

[0014] In some embodiments, the tissue sample is subjected to force from shock waves and / or mechanical impact. The tissue is washed one or more times before contacting the tissue.

[0015] In some embodiments, the shock waves may be used to separate the tissue sample into small clusters of cells, individual cells, or a mixture of cells. The cells break down into individual cells, or both.

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

[0017] In some embodiments, a cell fraction may comprise a small group of cells, individual cells, or includes both, and isolation of the cellular fraction is performed by centrifugation.

[0018] In some embodiments, centrifugation is at 500 g to 2,000 g for 3 minutes to 30 minutes. This is done at a speed of

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

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

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

[0022] In a second aspect, a method for isolating stem cells from adipose tissue is provided, the method comprising: Obtaining a tissue sample and placing the tissue sample in a container or cartridge. and exposing the tissue sample to a force from a mechanical shock to release the stem cells. separating a stem cell fraction from the adipose tissue; and centrifuging the stem cell fraction. and wherein the source of force from the mechanical impact does not physically contact the adipose tissue.

[0023] In some embodiments, the adipose tissue is subjected to at least one thermal shock prior to exposure to the force from the mechanical shock. or multiple washes.

[0024] In some embodiments, the source of mechanical impact force is powered by a motor. the motor includes a gear ring, the gear ring being connected to the motor When the impact arm is operating at full speed, the speed of the impact arm can be reduced.

[0025] In some embodiments, the force from the mechanical shock is passed through the walls of the container or cartridge. The drug is delivered to the adipose tissue via the agonist.

[0026] In some embodiments, the gearing is 1 / 10 the speed of the impact arm. Allows for deceleration.

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

[0028] In some embodiments, isolating the stem cell fraction comprises isolating the adipose tissue from the aqueous layer. wherein the aqueous layer comprises stem cells.

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

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

[0031] In a third aspect, a method for isolating stem cells from adipose tissue is provided, the method comprising: Obtaining a tissue sample and placing the tissue sample in a container or cartridge. exposing the tissue sample to shock waves to liberate stem cells; and and centrifuging the stem cell fraction; The source of the shock waves does not physically contact the adipose tissue.

[0032] In some embodiments, the adipose tissue is washed one or more times prior to exposure to shock waves. Be purified.

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

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

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

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

[0037] In some embodiments, the total amount of adipose tissue exposed to shock waves at any one time is The total area is 1 cm 2 From 100cm 2 is in the range.

[0038] In some embodiments, the container is a 19 ounce vinyl container having walls 0.25 mm thick. The total volume of the adipose tissue sample was 30 cc. The total area of ​​adipose tissue exposed to the shock waves is 5 cm 2 It is.

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

[0040] In some embodiments, isolating the stem cell fraction comprises isolating the adipose tissue from the aqueous layer. wherein the aqueous layer comprises stem cells.

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

[0042] The features and advantages of the present disclosure will become more apparent from the following detailed description and accompanying drawings, which set forth illustrative embodiments. This may be further explained with reference to the accompanying drawings. [Brief description of the drawings]

[0043] [Figure 1] 1 is an overview of a method for isolating a cellular fraction from a tissue sample according to an embodiment provided by the present disclosure. [Diagram 2] FIG. 1 illustrates a representative example of an external shock wave device suitable for use in the systems and methods provided by the present disclosure. [Diagram 3] FIG. 1 illustrates a representative example of a mechanical impact device suitable for use in the systems and methods provided by the present disclosure. [Figure 4] 1 illustrates a first representative example of a rigid, self-contained cartridge suitable for use with an external shock wave and / or mechanical impact device provided by the present disclosure. In the embodiment shown, some of the internal components are flexible containers. [Diagram 5] FIG. 1 illustrates a representative example of a rigid, self-contained cartridge suitable for use with external shock wave and / or mechanical impact devices provided by the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Before embodiments of the present disclosure are described, such embodiments are provided by way of example only. and various alternatives to the embodiments of the present disclosure described herein. It should be understood that alternatives may be used in practicing this disclosure. Numerous variations, modifications, and substitutions will occur to those of ordinary skill in the art without departing from this disclosure. It will be attached.

[0045] Unless otherwise defined, all technical and scientific terms used herein are The terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Methods and materials similar or equivalent to those described herein may be used in the practice or manufacture of the present disclosure. Suitable methods and materials that may be used in the testing are described below. In cases where the materials, methods, The above and examples are for illustrative purposes only and are not intended to be limiting. Modifications, variations, and substitutions will occur to those skilled in the art without departing from the present disclosure. become.

[0046] As used in the specification and claims, the singular forms "a," "one," and "an" are used interchangeably. "an" and "the" are used unless the context clearly indicates otherwise. For example, the term "a cell" includes multiple references to multiple cells (including (including mixtures of

[0047] As used herein, "external shock wave," "multiple external shock waves," and / or or "ESW" stands for sudden, high amplitude pulses of mechanical energy, similar to sound waves. They are generated outside the sample of interest and then propagated to that sample. At that point, the ESW source does not make physical contact with the sample. The shock wave is in contact with the sample, but the source is not in contact with the sample. is generated by a source at some distance from the sample and then The transmission can be through air and / or through a tube in which the sample is located. The ESW can be carried out through the wall of a vessel that is sealed by a septic tank. Concentrate the ESW into an area of ​​interest covering the entire pull or just a portion of the sample. The image may be focused and / or directed to produce a desired effect.

[0048] As used herein, a "subject" or "patient" refers to a mammal, e.g., a human. It represents the space between the two.

[0049] The methods provided by the present disclosure include using external shock waves, mechanical impact, vibration and / or crushing. Using the principles of lithography, tissue samples are divided into smaller fragments, i.e., cell groups and / or The cells are then disrupted into single cells, after which a desired cell fraction can be isolated from the sample. In various embodiments, the method includes isolating a sample from a subject, the sample being Pull the sample containing the cell type of interest, and then separate it into a step to generate ESW. Instead of contacting the sample with a device used for ion exchange, the sample is exposed to an external shock wave, which causes the sample to shrink. disintegrating the sample into small cell clumps and / or individual cells; and then and isolating a cell type of interest from the cell.

[0050] The devices provided by the present disclosure are focused and / or oriented focused and directed shock waves and / or mechanical shocks In various embodiments, the present disclosure provides a method for fragmenting a sample of interest. The provided device is supported in a sterile, closed environment during exposure to shock waves or mechanical shock. Therefore, shock waves and / or mechanical shocks should be avoided in closed, sterile containers. is generated outside the vessel and transmitted to its interior through one or more walls of the vessel, Inside, the sample is located.

[0051] The ESW supply is optimized by maintaining the ESW source at a predetermined distance from the sample of interest. The source (the device that generates the ESW) does not come into physical contact with the sample. The sterility of the sample and any cellular fractions isolated from the sample is largely determined by the From the healthcare provider's perspective, this also greatly improves sterility requirements and This helps ensure that the cell fraction of interest derived from the pool is sterile for patient use. Additionally, keeping the ESW source away from the sample of interest allows the generator to - or adding heat into the sample of interest (which can damage or destroy cells) (which can kill cells, damage DNA, and dramatically reduce yields) In that respect, the device provided by the present disclosure is capable of detecting the presence of ultrasound waves, etc. This is an improvement over other known systems such as

[0052] The methods and devices provided by the present disclosure, in some embodiments, can be used in 30 minutes or less. In this manner, a desired cell fraction can be rapidly isolated from a tissue sample. The speed at which the disclosed methods and devices can be used depends on the desired cell fraction to be isolated from a sample. This confers a significant advantage in that minutes may be available for immediate use. .

[0053] method FIG. 1 illustrates a method for isolating a cellular fraction of interest from a tissue sample according to an embodiment provided by the present disclosure. A method for isolating it from

[0054] In the embodiment shown, the method begins by first extracting tissue from a subject. (100) Under the condition that tissue is destroyed when exposed to an external shock wave, The tissue can be of any type. In some embodiments, the tissue In some embodiments, the tissue is an adipose tissue or lipid. In some embodiments, the tissue is an internal organ, e.g. , brain, pharynx, larynx, heart, arteries, muscles, liver, gallbladder, kidneys, small intestine, large intestine, lymph nodes, lungs, From the spleen, bone marrow, stomach, veins, pancreas, bladder, bones, teeth, dentin, gums, or skin In some embodiments, the tissue is a component of the endocrine system, such as the pineal gland. glands, pituitary, thyroid, adrenal glands, pancreas, ovaries, or testes.

[0055] The extraction can be carried out via any number of known methods. In some embodiments, tissue extraction is performed via a syringe. , performed surgically, in which a tissue sample is removed from a subject and placed into a first container. It looks like this.

[0056] The tissue is then transferred into a processing vessel or cartridge 105. The device provided by the disclosure provides a fast and easy-to-use ESW cell bioprocessing Provides all of the components required for the disclosed method and / or Although some of the components of the device are separable from one another, , which are adapted to be connected to each other in a completely sterile manner, whereby Transfer of samples from one component to another during processing In that respect, the entire sample is processed within a single system. , thereby ensuring sterility.

[0057] A sterile transfer is performed without exposing the sample to the outside environment. Delivery can be via any number of methods that will maintain a closed, sterile environment. In some embodiments, the transfer is performed by transferring a first sample containing the sample extracted from the subject. Male and female connectors between the vessel and the processing vessel or cartridge. This is accomplished by attaching the earlock connectors to each other. In the embodiment, the transfer is performed by connecting the first container and the processing container using a sterile connecting device. This is accomplished by aseptic docking of the cartridge or sample. The first vessel is then transferred to a processing vessel or cartridge, either by gravity flow or by flow transfer. The patient is transferred to the facility.

[0058] In some embodiments, the first container is inserted into the cartridge and is sterile. The connecting device is used to aseptically dock the cartridge into the processing vessel. The first container, which serves as a processing chamber, already contains a sample. .

[0059] In that regard, in this disclosure, as being transferred from one container to another: Whenever tissue and / or cell samples are disclosed, such transfers must be performed in a closed system. This can be done in a way that maintains the integrity of the system, thereby preventing the sample from being exposed to the outside. Do not expose to harsh environments.

[0060] Once the sample is transferred to the processing vessel or cartridge, the tissue sample is The tissue is washed one or more times 110. The washing may be performed through the use of a sterile saline solution. Washing can be performed as described herein. This ensures that the product is free of as many impurities as possible before it is washed, and therefore This improves the purity level of the cell fraction that is isolated from the culture.

[0061] In some embodiments, washing comprises mechanically removing the processing vessel or cartridge. If multiple washing steps are desired and / or necessary, Sterile saline should be poured into the processing container or cart after a single wash is completed. The ridge is drained and fresh sterile saline is inserted for each subsequent flush. The sterile saline solution is then introduced into a container or cartridge, at which point it is drained. Each time new sterile saline is introduced into the container or cartridge, , as described herein, is performed in a manner that maintains a closed, sterile environment.

[0062] After washing, the tissue sample is processed. The sessing is performed by ESW, mechanical shock, vibration and shear or a combination of these. This can be done through use.

[0063] In some embodiments, the processing is performed via ESW, and the tissue sample is In some embodiments, the processing is carried out by disintegrating the ES cells into multiple small cell populations. Through W, tissue samples are broken down into small cell clusters and individual cells. In some embodiments, the processing is performed via ESW to obtain a tissue sample. The pull is completely disassembled into multiple individual cells.

[0064] In some embodiments, the processing is via mechanical impact and shear; The tissue sample is broken down into small clusters of cells. In some embodiments, the processing Singling is achieved through mechanical impact and shear, which breaks the tissue sample into multiple small cell clusters. and broken down into a plurality of individual cells. Through mechanical impact and shear, tissue samples are completely disaggregated into multiple individual cells. can be.

[0065] In some embodiments, the processing comprises a combination of ESW and mechanical impact and shear. This is done through a combination of techniques, which breaks down the tissue sample into small clusters of cells. In one embodiment, the processing comprises a combination of ESW and mechanical impact and shear. This is done via a procedure where the tissue sample is broken down into small cell clusters and individual cells. In some embodiments, the processing comprises a combination of ESW and mechanical impact and shear. Through combination, the tissue sample is completely broken down into a number of individual cells.

[0066] After processing, a cell suspension (which is made up of multiple small The cells that are separated from the culture medium (which may be a large group of cells, multiple individual cells, or both) are filtered and centrifuged. 120 Filtration is performed in a device separate from the processing vessel or cartridge. This may be done in the chair or in the processing vessel or cartridge itself. In some embodiments, the filtration is performed in a separate device, In that case, the cell suspension is prepared by sterile means as described herein. In some embodiments, the cell suspension is transferred to a container or cartridge. Filtration occurs as the sample is transferred from the tube into one or more centrifuge tubes. .

[0067] In some embodiments, the transfer to the filtration device is via a processing vessel or cart. Male and female Luer lock connectors were inserted between the ridge and the filtration device. In some embodiments, the transfer is accomplished by attaching the Using a sterile device, place the processing vessel or cartridge and the filtration device in a sterile This is accomplished by mixing the samples either mechanically or by gravity flow. from the processing vessel or cartridge to the filtration device.

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

[0069] In various embodiments, centrifugation is performed outside the processing vessel or cartridge. In some embodiments, the cell suspension is placed in a processing vessel or cartridge. The mixture is transferred to one or more centrifuge tubes located inside the centrifuge. Sometimes, centrifuge tubes are referred to as processing vessels or containers, as described herein. The cartridge is transferred via sterile means to a centrifuge.

[0070] In some embodiments, the cell suspension is processed as described herein. One or more centrifuge tubes positioned outside the suction vessel or cartridge. When filled, the centrifuge tubes are transferred via sterile means to the As described, sterile transfer from the processing vessel or cartridge to the centrifuge. It is moved via the means.

[0071] The duration and speed of centrifugation may vary depending on the tissue type being processed. In some embodiments, the cell suspension is allowed to thaw for a period of 3 to 30 minutes. In some embodiments, the cell suspension is centrifuged at a speed of 500 g to 2,000 g. The liquid is centrifuged at a speed of 1,000g to 1,800g for 10 to 25 minutes. In one embodiment, the cell suspension is centrifuged at 1,200 g for 10 minutes. In some embodiments, the cell suspension is centrifuged at 1,200 g for 7 minutes. In some embodiments, the cell suspension is centrifuged at 1,200 g for 5 minutes. The cell suspension is centrifuged at 1,200 g for 3 minutes. The suspension is centrifuged at 900 g for 10 minutes. In some embodiments, the cell suspension is centrifuged at 900 g for 5 minutes. In some embodiments, the cell suspension is centrifuged at 900 g for 3 minutes. In some embodiments, the cell suspension is centrifuged at 600 g 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 this embodiment, the cell suspension is centrifuged at 600 g for 3 minutes.

[0072] Multiple cell types present in a cell suspension can be separated from one another via centrifugation There are numerous means for separating cells. For example, a density gradient can be used to separate cells during centrifugation. Separates into bands according to its specific density. Additionally, a size exclusion protocol is used , can be used to separate cells via size. The tissue sample to be sampled is allowed to settle and Prior to cardioplegia, separation is performed by separating undesirable cell debris from the desired cell fraction. The type of separation technique used can be determined based on the type of isolated The amount of cellular fraction that is to be cultured can vary based on the desired cell fraction that is to be cultured.

[0073] Once the desired cell fraction is isolated, the cell fraction may be prepared for administration to the subject from which the cell fraction was derived. In some embodiments, the desired cell fraction can be resuspended in a centrifuge tube. In some embodiments, the desired The cell fraction is resuspended in sterile saline.

[0074] In one embodiment, the desired cell fraction is stem cells and the tissue from which the stem cells are derived is adipose tissue. It is.

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

[0076] Lipids are obtained from the patient via liposuction. Liposuction is performed by inserting a syringe into the The procedure can be performed via a liposuction machine, where the lipids are either directly drawn through a needle or through a liposuction machine. The amount of lipid removed during liposuction is determined by the amount of lipid removed from the patient via the liposuction tube. The number of stem cells to be cultured may vary depending on the number of stem cells to be cultured. The procedure is a mini-liposuction, and the volume is about 30cc to about 50cc. cc of lipids are removed from the patient. In some embodiments, liposuction is performed using a microsuction Liposuction is a method of removing lipid from the skin, and microliposuction is a method of removing lipid from the skin, from about 5cc to about 29cc. In some embodiments, liposuction is performed in a manner similar to that used in a typical clinical setting. Posuction, in which more than 300cc of lipids are removed from the patient.

[0077] Once the lipids are removed from the patient, they are placed into a sterile processing container or cartridge. The transfer is via sterile means as described herein. In some embodiments, the lipid is removed from the patient via a syringe, Male and female Luer locks between the The connectors can be attached to each other to allow for easy connection from the syringe to a processing vessel or container. In some embodiments, the lipid is delivered from the patient via a syringe to the cartridge. The syringe is removed and the male and female connectors are inserted between the syringe and the container or cartridge. By attaching the Luer lock connectors of the type to each other, processing vessels or In some embodiments, the lipid is a cysteine. The syringe is removed from the patient and transported to the processing vessel in the syringe and cartridge. Male and female Luer lock connectors are attached to the cartridge or By attaching the syringe to the processing vessel or cartridge, the In some embodiments, lipids are removed from a patient via a liposuction device. , using a sterile connecting device to connect the liposuction device and processing vessel or The cartridge is aseptically docked to remove the prosthesis from the liposuction device. The mixture is transferred to a processing vessel or cartridge.

[0078] In some embodiments, the transfer can occur via gravity flow. In some embodiments, the transfer can be accomplished mechanically, for example by the action of a syringe. By physically depressing the plunger and from inside the syringe, the process This can be done by forcing the lipids into the interior of a mixing vessel or cartridge. In some embodiments, the transfer is via a device such as a pump. It is possible.

[0079] The volume of the container or cartridge used to receive the lipid is obtained from the patient. In some embodiments, the amount of lipid added to the miniliposome may vary. 30cc to 50cc of lipids removed via filtration are 9 to 19 ounces of lipids. The liquid is delivered to a container or cartridge having an ounce capacity.

[0080] A volume of sterile saline equal to the amount of lipid removed from the patient is then added to wash the lipid out. For example, 30 cc of lipid is transferred from the patient to a container or cartridge. When the sample was removed from the tube, 30 mL of sterile saline was then added to wash the sample. In some embodiments, the amount of lipid removed from the patient is then greater than the amount of lipid removed. A large volume of sterile saline is transferred to the container or cartridge to wash the lipids. For example, if 30 cc of lipid is removed from the patient, then 35 mL of sterile saline is used to wash the sample. A volume of sterile saline less than the amount of lipid to be removed from the patient is then added to the lipid. For washing, the lipid is transferred to a container or cartridge. When the sample was removed from the subject, 25 mL of sterile saline was then used to wash the sample. In some embodiments, a combination of volumes of sterile saline is used. , which is added to the amount of lipid to wash the lipid. Water transfer is performed as described herein to maintain sterility of the system. In some embodiments, a container for holding sterile saline and a processing Male and female Luer lock connectors are inserted between the container or cartridge. By attaching the sterile saline solution to the container or cartridge, the sterile saline solution is transferred to the container or cartridge. In some embodiments, a sterile connection device is used to connect a sterile saline container and and processing container or cartridge by aseptic docking. The saline is transferred to a container or cartridge.

[0081] Washing is performed by gently shaking the container or cartridge containing the lipids and sterile saline. This is accomplished by rotating or twisting the

[0082] The container or cartridge is then filled with sterile saline to remove the (now washed) lipids. Since lipids are less dense than saline, they will dissolve in saline. They will rise to the top of the saltwater and float. The amount of time required for separation to occur is will vary depending on the particular composition of the lipid sample. Separation can occur in 1 to 30 minutes. In some embodiments, separation occurs in 1 to 15 minutes. In some embodiments, separation can occur in 1 to 10 minutes. In embodiments, separation can occur in 1 to 5 minutes. In some embodiments, separation can occur in 10 This can occur within seconds to a minute.

[0083] The saline is then drained from the container or cartridge as completely as possible. Thus, only the lipids remain. The drainage is performed by removing the lipids from the container or container utilized in the present method. Depending on the type of cartridge, this can be accomplished through a variety of means. In some embodiments, the saline is located at the bottom of the container or cartridge. The drain is discharged from the container or cartridge through a tube that is The flow can be actively advanced using either a syringe or pump, or by gravity flow. In some embodiments, the drainage can proceed passively through the This is done as described in the specification to maintain the integrity of the sterile system.

[0084] After washing is complete, the wash is repeated until the lipids are golden yellow and the saline is only slightly cloudy. In some embodiments, washing is performed 1 to 5 times, and in some embodiments, is performed 1 to 4 times, in some embodiments, 1 to 3 times, in some embodiments is performed 1 to 2 times, and in some embodiments, is performed once.

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

[0086] Optionally, but not required, sterile saline may be used as a buffer for processing. The addition of saline is necessary to allow the cells and / or small cell clusters to progress through the processing stage. This provides space for the players to move freely away from each other during play, When saline is added, it helps break down lipids into individual cells. As described herein, in a manner that maintains the integrity of the sterile system. , saline is added.

[0087] The volume of saline optionally added can vary. In this form, the volume of saline added is 0 to 2 times the volume of lipid removed from the patient. In some embodiments, an equal amount of saline is used (for example, For example, 30 mL of saline for 30 cc of lipid.

[0088] Excess air is removed from the container or cartridge using a pump or syringe. In some embodiments, excess air is purged from the drain by a sterile saline irrigation solution. The liquid is removed from the same tube located at the bottom of the container or cartridge from which it is discharged. In some embodiments, the air is stored in a sterile, closed container as described herein. Drains are discharged in a manner that maintains the integrity of the system.

[0089] The processing vessel or cartridge is then transferred to a processing device. An example of a suitable processing device 200 is shown in FIG. In one embodiment, the processing vessel or cartridge 205 is a reservoir for processing. The platform 210 is fixed to the ES It controls the movement of the container or cartridge 205 during W processing.

[0090] The platform 210 may be made of any suitable material, including wood, metal, plastic, acrylic, etc. It may be made of any suitable rigid material. The frame 210 is made from wood.

[0091] The processing device 200 delivers shock waves to the interior of the container or cartridge 205. The shock wave applicator 215 can be used to apply shock waves to the subject. 5 is placed on the processing device 200 via the applicator platform 220 The applicator platform 220 and the shock wave applicator 2 15 is spaced a predetermined distance from the lipids contained in the container or cartridge 205. The shock wave applicator 215 is never in direct contact with the lipids. This is because the shock waves delivered to the lipid are external or In some embodiments, the shock wave applicator 2 15 is placed in direct contact with a processing vessel or cartridge 205. In such an embodiment, the walls of the container or cartridge 205 may be impact resistant. It is ensured that the wave applicator 215 never comes into direct contact with the lipids.

[0092] The movement of the applicator platform 220, and thus the shock wave applicator 21 5 Movements can be made in three directional planes (X, Y, and Z), Each of them has its own motor: X-plane motor 230, Y-plane motor 235, and Z-plane motor 240. In some embodiments, these motors The motors are NEMA 17 stepper motors. Motors 230, 235, and Each of the motors 240 is controlled by a motor controller 225. The roller 225 moves the applicator over and around the container or cartridge 205. Allows movement of the platform 220 and shock wave applicator 215 in three dimensions In some embodiments, the motor is programmed by the Marlin firmware. It is controlled by a modular Arduino microprocessor v6 board. In this embodiment, the motor is powered by an Arduino v4 with Marlin firmware. 6 board. 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. Only the planar motor 230 is used. In some embodiments, the X-planar motor 230 and Y-plane motor 235 is used. In some embodiments, the X-plane motor In some embodiments, only the Y-plane motor 230 and the Z-plane motor 240 are used. Only the plane motor 235 and the Z plane motor 240 are used. are used by the X plane motor 230, the Y plane motor 235, and the Z plane motor 240. will be done.

[0093] The third embodiment of the applicator platform 220 and the shock wave applicator 215 The original transfer was determined by the fact that all of the lipids in the extracted sample were not exposed to external shock during processing. Allows exposure to waves.

[0094] Shock waves are then applied from a shock wave applicator 215 to break up the lipids. Through the transmission gel or a membrane containing the transmission gel, or through the walls of the cartridge 205 into the lipid sample, where small cell populations and and detaching individual cells from the lipid. In some embodiments, the transmission gel ,DJO,LLC,1430 Decision Street,Vista,CA 9 2081, REF 4248 manufactured for Chattanooga by USA Conductor Transmission Gel. Before processing In those embodiments in which sterile saline is added to the lipid, small particles detached from the lipid Clusters of cells and / or individual cells will migrate into the saline solution.

[0095] The shock wave applicator 215 is powered by a shock wave generator 245 and generates shock waves. The generator 245 produces the ESW, which is then applied to the fat layer via the shock wave applicator 215. In some embodiments, the shock wave generator is a Storz Medica Master manufactured by AG, 8274 Tagerwilen, Switzerland In some embodiments, the shock wave generator is a Lumsai l Industrial Inc.,4 / F,No. 9Yi,Lane 2,Sui BS-SWT2X manufactured at de Road, Shanghai, 200331, China be.

[0096] The power of the ESW delivered to the lipid can be varied. For example, in thick-walled Increasing the power of the ESW to penetrate the walls of the enclosed vessel or cartridge 205 In general, the power of ESW delivered to lipids is The wall thickness of the container or cartridge 205 and / or the Depending on the material from which the edge 205 is made, this can vary. In this configuration, the ESW power delivered to the lipid ranged from 0.5 bar to 5.0 bar. In some embodiments, the power of the ESW delivered to the lipid is between 1.0 bar and In some embodiments, the power of the ESW delivered to the lipid is in the range of 4.5 bar. In some embodiments, the delivery to lipids is in the range of 1.5 bar to 4.0 bar. The power of the ESW used ranges from 2.0 bar to 3.5 bar. In form, the container 205 is a vinyl bag having a wall thickness of 0.25 mm, and the ESW The power levels range from 2.0bar to 2.5bar.

[0097] The area covered by ESW delivered by the shock wave applicator 215 is , can vary. The area covered by the shock wave applicator 215 The larger the diameter, the greater the surface area of ​​the lipid exposed to the ESW. In an embodiment, the total area covered by the shock wave applicator 215 is less than 1 cm 2 mosquito 100cm 2 In some embodiments, the range is 1 cm 2 From 90cm 2 Range and in some embodiments, 1 cm 2 From 80cm 2 In some cases, In this embodiment, 1 cm 2 From 70cm 2 In some embodiments, the range is 1 cm 2 From 60cm 2 In some embodiments, the range is 1 cm 2 From 50cm 2 Range and in some embodiments, 1 cm 2 From 40cm 2 In some cases, In this embodiment, 1 cm 2 30cm from 2 In some embodiments, the range is 1 cm 2 20cm from 2 In some embodiments, the range is 1 cm 2 From 10cm 2 Range and in some embodiments, 1 cm 2 From 5cm 2 There are several implementations. In this embodiment, the total area covered by the shock wave applicator 215 is 5 cm 2 Yes do.

[0098] In one embodiment, the container 205 is a 19 ounce vinyl container having 0.25 mm thick walls. The total amount of lipid obtained from the patient is 30 cc, and An additional 30 mL of saline (a volume equal to the lipid sample) was added prior to processing. In this embodiment, the total surface area of ​​the bag exposed to the ESW is The volume, and therefore the total volume of the lipid sample, is approximately 5 cm 2 It is.

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

[0100] In this embodiment, stem cells are isolated from a lipid sample obtained from a patient. 5cm above wave applicator 215 2 Use the applicator tip of 2.0 to The shock wave number range of 10,000 to 50,000 at 2.5 bar range is suitable for stem cells and It is sufficient to separate both the lipid and stromal vascular cell fractions from lipid. As used herein, a "stromal vascular cell fraction" refers to a cell fraction (which in some embodiments is adipose tissue). Processed cells include cells obtained from isolating and dissociating cells from a culture medium (which is a tissue-forming medium). It refers to a cell fraction obtained from a tissue sample. The stromal vascular cell fraction is not limited to These include, among others, stem cells, growth factors, and progenitor cells.

[0101] After the ESW is applied to the lipid sample, the container or cartridge is In some embodiments, the mechanical agitation is performed by shaking. In some embodiments, the mechanical agitation is performed one or more times by stirring the container or In some embodiments, the cartridge 205 may be turned upside down for a short period of time. The time period may range from 1 second to 30 seconds, and in some embodiments may range from 1 second to 15 seconds. In some embodiments, the time is in the range of 1 second to 10 seconds. The short period of time is approximately 10 seconds. The stirring is performed to dissolve the stem cells and lipids released from the lipids. The stromal vascular cell fraction is then removed from the adipose tissue that may still be present in the container or cartridge 205. This allows the cells to be separated from the cellular debris and any persistent lipid tissue.

[0102] The (now processed) lipids were then separated from the lipids by ESW. As the stem cell and stromal vascular cell fractions are now isolated from the sterile saline containing The container or cartridge 205 is placed in the vessel. The lipid is dissolved in saline containing the stem cells. Since lipids are less dense than lipids, they rise to the top of the saline solution and float. The amount of time required for the lipid sample to react will vary depending on the particular composition of the lipid sample. In some embodiments, separation can occur in 1 minute to 30 minutes. In some embodiments, separation can occur in 1 to 15 minutes. In some embodiments, separation can occur in 1 to 10 minutes. In some embodiments, separation can occur in 1 to 5 minutes. In some embodiments, separation can occur in 10 seconds to 1 minute. The separation can be accomplished in less than a minute.

[0103] The stem cells are now separated from the lipid sample, where the stem cells are then separated from the lipid sample by a sterile saline layer. It is suspended in.

[0104] The saline layer containing the stem cells is then removed as completely as possible from the container or cartridge. 205, so that only the lipids remain. The removal is utilized in the present method. This can be accomplished through a variety of means depending on the type of container or cartridge 205. In some embodiments, the saline solution is provided in a container or cartridge 205. out of the container or cartridge 205 via a tube located at the bottom of the Drainage is actively performed using either a syringe or a pump. It can move in a circular manner or passively via gravity current. In embodiments, the stem cell-containing layer is placed in a closed, sterile container as described herein. It is removed in a manner that maintains the integrity of the environment.

[0105] In some embodiments, the saline layer containing the stem cells is a container or cartridge. During removal from 205, the material is passed through filters, the size of which varies. In some embodiments, the filter size is between 40 μm and 100 μm. In some embodiments, the filter size is in the range of 70 μm. In various embodiments, the filter is nylon.

[0106] Either after or during removal from the container or cartridge 205, the stem cells The containing saline is transferred into one or more centrifuge tubes. In some embodiments, the transfer may involve maintaining the integrity of a closed, sterile environment, as described herein. The stem cells are then pelleted at the bottom of the centrifuge tube in a manner that maintains the integrity of the stem cells. Centrifugation follows to concentrate the cells.

[0107] Stem cells are concentrated by centrifugation at 500g to 1,600g for 3 to 10 minutes. In some embodiments, the stem cells are amplified by centrifugation at 1,200 g for 10 minutes. It is concentrated by separation.

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

[0109] A small amount of fluid is added to the centrifuge tube to resuspend the stem cells. In one embodiment, the amount of fluid added to the centrifuge tube is in the range of 1 mL to 5 mL. In some embodiments, the fluid is saline, platelet rich plasma, hyaluronic acid, fixative Fixing gel, hydrogel, scaffold, fibrin, glue, or In some embodiments, the cells are centrifuged. The mixture is resuspended in the extract.

[0110] With the stem cells floating in suspension, the stem cells are ready for use. Such uses include, but are not limited to, reintroduction into the patient from which the stem cells were derived, freezing, In some embodiments, the method may include concentrating, storing, and expanding the The suspended stem cells are transferred through a hypodermic needle into a syringe for patient use. In some embodiments, the concentrated resuspended stem cells are used to treat cardiac, muscle, bone, cartilage, and other conditions. Tissue scaffolding for growth into bone, liver, kidney, or other tissue and organ structures In some embodiments, the concentrated resuspended stem cells are seeded into a medium. In fact, stem cells express pluripotency transcription factors. By inducing the cells to undergo cell division, they are converted into induced pluripotent stem cells.

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

[0112] Lipids are obtained from the patient via liposuction. Liposuction is performed by inserting a syringe into the The procedure can be performed via a liposuction machine, where the lipids are either directly drawn through a needle or through a liposuction machine. The amount of lipid removed during liposuction is determined by the amount of lipid removed from the patient via the liposuction tube. The amount of lipid in the stem cells can vary depending on the number of stem cells being cultured. The syringe is removed from the patient via the syringe, which is then inserted between the syringe and the cartridge. The cartridge is fitted with male and female Luer lock connectors. In some embodiments, the lipid is delivered via a syringe. and removed from the patient and placed in a processing container or cart in the syringe and cartridge. Attach male and female Luer lock connectors to each other between the ridges. The syringe is then transferred to a processing vessel or cartridge by In one embodiment, the liposuction is a miniliposuction. In some embodiments, about 30 cc to about 50 cc of lipid is removed from the patient. Liposuction is microliposuction, and in microliposuction, the rate is about 5 cc to about 29 cc of lipid is removed from the patient. The procedure is a typical clinical liposuction procedure, in which more than 300cc of lipid is removed. is removed from the patient.

[0113] Once the lipids are removed from the patient, they are placed into a sterile processing container or cartridge. The transfer is via sterile means as described herein. In some embodiments, the lipid is removed from the patient via a syringe, Male and female Luer locks between the The connectors can be attached to each other to allow for easy connection from the syringe to a processing vessel or container. In some embodiments, the lipid is transferred to the cartridge by using a liposuction device. The catheter is then removed from the patient via the liposuction device and the catheter is then attached to the patient using a sterile connecting device. The liposac is then inserted into the processing vessel or cartridge by sterile docking. The sample is transferred from the processing device to a processing vessel or cartridge.

[0114] In some embodiments, the transfer can occur via gravity flow. In some embodiments, the transfer can be accomplished mechanically, for example by the action of a syringe. By physically depressing the plunger and from inside the syringe, the process This can be done by forcing the lipids into the interior of a mixing vessel or cartridge. In some embodiments, the transfer is via a device such as a pump. It is possible.

[0115] The volume of the container or cartridge used to receive the lipid is obtained from the patient. In some embodiments, the amount of lipid added to the miniliposome may vary. 30cc to 50cc of lipids removed via filtration are 9 to 19 ounces of lipids. The liquid is delivered to a container or cartridge having an ounce capacity.

[0116] A volume of sterile saline equal to the amount of lipid removed from the patient is then added to wash the lipid out. For example, 30 cc of lipid is transferred from the patient to a container or cartridge. When the sample was removed from the tube, 30 mL of sterile saline was then added to wash the sample. In some embodiments, the lipid is removed from the patient via a syringe. The syringe has male and female Luer lock connectors between the syringe and cartridge. The connectors are attached to each other in a sterile dock in the cartridge. In some embodiments, the lipid is removed from the patient via a syringe and Male and female connectors between the processing vessels in the cartridge or between the cartridge The luer lock connectors of the syringes are attached to each other to allow easy connection from the syringe to the processing vessel. The saline solution is transferred to a container or cartridge. The transfer is performed in a manner that maintains the sterility of the system, as described herein. In some embodiments, the container holding the sterile saline solution and the processing container are Male and female Luer lock connectors are attached to the cartridge or By attaching the sterile saline solution, the sterile saline solution is transferred to the container or cartridge. In some embodiments, a sterile connection device is used to connect a sterile saline container and a processor. Sterile saline is delivered by aseptically docking the dispensing container or cartridge. The saltwater is transferred to a container or cartridge.

[0117] Washing is performed by gently shaking the container or cartridge containing the lipids and sterile saline. This is accomplished by rotating or twisting the

[0118] The container or cartridge is then filled with sterile saline to remove the (now washed) lipids. Since lipids are less dense than saline, they will dissolve in saline. They will rise to the top of the saltwater and float. The amount of time required for separation to occur is will vary depending on the particular composition of the lipid sample. Separation can occur in 1 to 30 minutes. In some embodiments, separation occurs in 1 to 15 minutes. In some embodiments, separation can occur in 1 to 10 minutes. In embodiments, separation may occur in 1 to 5 minutes.

[0119] The saline is then drained from the container or cartridge as completely as possible. Thus, only the lipids remain. The drainage is performed by removing the lipids from the container or container utilized in the present method. Depending on the type of cartridge, this can be accomplished through a variety of means. In some embodiments, the saline is located at the bottom of the container or cartridge. The drain is discharged from the container or cartridge through a tube that is The flow can be actively advanced using either a syringe or pump, or by gravity flow. In some embodiments, the drainage can proceed passively through the This is done as described in the specification to maintain the integrity of the sterile system.

[0120] After washing is complete, the wash is repeated until the lipids are golden yellow and the saline is only slightly cloudy. In some embodiments, washing is performed 1 to 5 times, and in some embodiments, is performed 1 to 4 times, in some embodiments, 1 to 3 times, in some embodiments is performed 1 to 2 times, and in some embodiments, is performed once.

[0121] Once washing is complete, the lipids are ready for processing.

[0122] Optionally, but not required, sterile saline may be used as a buffer for processing. The addition of saline is necessary to allow the cells and / or small cell clusters to progress through the processing stage. This provides space for the players to move freely away from each other during play, When saline is added, it helps break down lipids into individual cells. As described herein, in a manner that maintains the integrity of the sterile system. , saline is added.

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

[0124] Excess air is removed from the container or cartridge using a pump or syringe. In some embodiments, excess air is purged from the drain by a sterile saline irrigation solution. The liquid is removed from the same tube located at the bottom of the container or cartridge from which it is discharged. In some embodiments, the air is stored in a sterile, closed container as described herein. Drains are discharged in a manner that maintains the integrity of the system.

[0125] The processing vessel or cartridge is then transferred to a processing device. An example of a suitable processing device 300 is shown in FIG. In one embodiment, the processing vessel or cartridge 205 is a reservoir for processing. The platform 315 is fixed to the top of the platform 315. The device controls the movement of the container or cartridge 205 during processing.

[0126] The platform 315 may be made of any suitable rigid material, including wood, plastic, and the like. In some embodiments, the platform 315 may be made from wood. It has been produced by

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

[0128] The mechanical shock is generated by a motor 305, which generates an impact The impact arm 310 drives the fat to be processed. In this embodiment, the ion exchange agent is placed in physical contact with a reservoir or cartridge 205 that contains the substance. The motor 305 drives the impact arm 310, which moves up and down. It is adapted to articulate and at the bottom of its range of motion, In some embodiments, the motor is a Black and cker,1000 Stanley Drive,New Britain,CT 0 6053, 4.5Amp motor from USA made model BDEJS300C.

[0129] In some embodiments, the motor 305 is a variable speed motor. The rotor rotates at a rate ranging from 0 rpm to 30,000 rpm, in some embodiments. at a rate ranging from 0 rpm to 20,000 rpm, and in some embodiments At rates ranging from 0 rpm to 10,000 rpm, in some embodiments, Adjustable to drive the impact arm at rates ranging from 1000 to 5,000 rpm It is possible to save.

[0130] In some embodiments, the motor 305 includes gearing, the gearing being more In order to generate a lot of torque, the motor 305 is at full speed (in some embodiments, 30,000 rpm), the speed of the impact arm 310 is reduced. When this occurs, the force generated by the impact arm 310 The momentum is transferred to the tissue in the container or cartridge 205. is broken down and stem cells are released from fatty tissue, greatly increasing the rate at which they are released.

[0131] In one embodiment, the motor 305 is configured to rotate at a speed between 30,000 rpm and 3,000 rpm. It is geared to allow a 1 / 10 reduction in speed. In the configuration, the motor 305 includes a gear diameter of 0.75 inches, which is 1 / 10 of this. At 3,000 rpm and a gear diameter of 0.75 inches, The impact arm 310 reaches a speed of 117.8 inches per second, at which speed: The impact arm 310 articulates back and forth, thus impacting the container or cartridge 2. 05. When the impact arm 310 comes into contact with the container or cartridge 205 The speed at which this can be done allows for greatly reduced processing times. In contrast to known or existing tissue processing techniques that require long processing times, and has significant advantages.

[0132] The tissue is then incubated until the lipids have been degraded to a desired level or until a desired amount of stem cells have been extracted from the lipids. In some embodiments, the lipid sample is exposed to mechanical shock until released. In some embodiments, the lipid sample is processed within 30 minutes. and in some embodiments, the mixture is processed for between 30 seconds and 20 minutes. In some embodiments, the processing is performed for between 30 seconds and 10 minutes. The mixture is filtered and, in some embodiments, processed for between 30 seconds and 5 minutes.

[0133] The movement of the platform 315 occurs in three directional planes (X, Y, and Z). Each of them has its own motor: X-plane motor 230, Y-plane motor 23 5, and Z-plane motor 240. In some embodiments, the X-plane Only motor 230 is used. In some embodiments, only Y-plane motor 235 is used. In some embodiments, only the Z plane motor 240 is used. In some embodiments, only the X-plane motor 230 is used. , only the X-plane motor 230 and the Y-plane motor 235 are used. In this embodiment, only the X plane motor 230 and the Z plane motor 240 are used. In some embodiments, only the Y plane motor 235 and the Z plane motor 240 are used. In some embodiments, the X-plane motor 230, the Y-plane motor 235, and the Z-plane motor A surface motor 240 is used. Each of the motors 230, 235, and 240 has a The motor controller 225 controls the motor Allows movement of platform 315 in dimensions.

[0134] This maximum three-dimensional movement of the platform 315 allows the lipids in the extracted sample to be analyzed. All of these allow the samples and This allows optimal positioning of the imaging and processing equipment.

[0135] The length, area of ​​the impact surface, number of impact surfaces, and impact arm 310 The shape of the impact surface may vary. Increasing the area and / or total number of impact surfaces allows for easier preparation of lipid samples. This reduces the amount of time required for processing.

[0136] The (now processed) lipids are then separated from the lipids by mechanical impact. The container or cart is then placed in a container or cart so that the stem cells are separated from the sterile saline that now contains the stem cells. The ridges 205 are placed in the cavity 202. Since lipids are less dense than saline, the lipids are more dense than saline. It will rise to the top of the water and float. The amount of time required for separation to occur is This will vary depending on the particular composition of the lipid sample. Separation can occur in 1 to 30 minutes. In some embodiments, separation occurs in 1 to 15 minutes. In some embodiments, separation can occur in 1 to 10 minutes. In some embodiments, separation can occur in 1 to 5 minutes. In some embodiments, separation occurs in less than 1 minute. In some embodiments, separation can occur in 10 seconds to 1 minute. In some embodiments, the separation can be performed in less than a minute.

[0137] wherein the stem cell and stromal vascular cell fractions are separated from the lipid sample, , suspended in saline.

[0138] The saline layer containing the stem cells is then removed as completely as possible from the container or cartridge. 205, so that only the lipids remain. The removal is utilized in the present method. This can be accomplished through a variety of means depending on the type of container or cartridge 205. In some embodiments, the saline solution is provided in a container or cartridge 205. out of the container or cartridge 205 via a tube located at the bottom of the Drainage is actively performed using either a syringe or a pump. It can move in a circular manner or passively via gravity current. In embodiments, the stem cell-containing layer is placed in a closed, sterile container as described herein. It is removed in a manner that maintains the integrity of the environment.

[0139] In some embodiments, the saline containing the stem cells is placed in a container or cartridge 2. During removal from 05, the material is passed through a filter. The size of the filter can vary. In some embodiments, the filter size is between 40 μm and 100 μm. In some embodiments, the filter size is 70 μm. In various embodiments, the filter is nylon.

[0140] Either after or during removal from the container or cartridge 205, the stem cells The containing saline is transferred into one or more centrifuge tubes. In some embodiments, the transfer may involve maintaining the integrity of a closed, sterile environment, as described herein. The stem cells are then pelleted at the bottom of the centrifuge tube in a manner that maintains the integrity of the stem cells. Centrifugation follows to concentrate the cells.

[0141] Stem cells are concentrated by centrifugation at 500g to 1,600g for 3 to 10 minutes. In some embodiments, the stem cells are amplified by centrifugation at 1,200 g for 10 minutes. It is concentrated by separation.

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

[0143] A small amount of fluid is added to the centrifuge tube to resuspend the stem cells. In one embodiment, the amount of fluid added to the centrifuge tube is in the range of 1 mL to 5 mL. In some embodiments, the fluid is saline, platelet rich plasma, hyaluronic acid, fixative Gel, hydrogel, scaffold, fibrin glue, glue, or any of the foregoing In some embodiments, the cells are isolated from the centrifugation aspirate. The mixture is resuspended in 100 mL of PBS.

[0144] With the stem cells in suspension, they are ready for use. Such uses include, but are not limited to, reintroduction into the patient from whom the stem cells were derived, cryopreservation, and In some embodiments, the concentrated and resuspended The stem cells are then transferred through a hypodermic needle into a syringe for patient use. In some embodiments, the concentrated resuspended stem cells are used to generate stem cells for cardiac, muscle, bone, cartilage, liver, of tissue scaffolds for growth into kidneys or other tissue and organ structures. In some embodiments, the concentrated resuspended stem cells are seeded into a stem cell culture medium. are converted into induced pluripotent stem cells by inducing them to express pluripotency transcription factors. do.

[0145] In the first and second methods described above, a container or cartridge and The mechanical energy source is mobile. In some embodiments, the lipid is ES The air is pumped through a chamber located under the impact surface. In these embodiments, both the bag and the mechanical energy source are fixed in place. The tissue is passed through a chamber and exposed to ESW and / or mechanical energy. exposed to energy sources.

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

[0147] Lipids are obtained from the patient via liposuction. Liposuction is performed by inserting a syringe into the The procedure can be performed via a liposuction machine, where the lipids are either directly drawn through a needle or through a liposuction machine. The amount of lipid removed during liposuction is determined by the amount of lipid removed from the patient via the liposuction tube. The number of stem cells to be cultured may vary depending on the number of stem cells to be cultured. The procedure is a mini-liposuction, and the volume is about 30cc to about 50cc. cc of lipids are removed from the patient. In some embodiments, liposuction is performed using a microsuction Liposuction is a method of removing lipid from the skin, and microliposuction is a method of removing lipid from the skin, from about 5cc to about 29cc. In some embodiments, liposuction is performed in a manner similar to that used in a typical clinical setting. Posuction, in which more than 300cc of lipids are removed from the patient.

[0148] Once the lipids are removed from the patient, they are transferred into a sterile processing cartridge. Transfer is via sterile means as described herein. In some embodiments, the lipid is removed from the patient via a syringe and the syringe and the processing Male and female Luer lock connectors are attached to the single cartridge. By attaching the syringe to the processing cartridge, the sample is transferred from the syringe to the processing cartridge. In some embodiments, the lipid is removed from the patient via a syringe, the syringe comprising: Male and female Luer lock connectors are attached between the cartridge and the In some embodiments, the cartridge is sterile docked by inserting the In this method, lipids are removed from the patient via a syringe and then transported through the syringe and into the cartridge. Male and female Luer lock connectors for connection to processing vessels or cartridges The syringe is then attached to the processing vessel or cartridge. In some embodiments, the lipids are transferred to the liposome via a liposuction device. Removed from the patient, the liposuction device and process are connected using a sterile connection device. The surgical cartridge is then aseptically docked to the liposuction device. The sample is then transferred to the processing cartridge.

[0149] In some embodiments, the transfer can occur via gravity flow. In some embodiments, the transfer can be accomplished mechanically, for example by the action of a syringe. By physically depressing the plunger and from inside the syringe, the process This is done by forcing the lipids into the inner chamber of the cartridge. In some embodiments, the transfer is via a device such as a pump. It is possible to do so.

[0150] The volume of the internal chamber of the cartridge 400, 500 that receives the lipid (e.g., The cartridge embodiment (see FIG. 4 and FIG. 5 ) is a cartridge for collecting fat from a patient. In some embodiments, mini-liposuction can be used. 30cc to 50cc of lipids are removed through the cartridge 400, 500. The mixture is transferred to the fat chamber 405, 505.

[0151] In this embodiment, the lipids are a specific component of the self-contained cartridge 400, 500. The sample is injected into the component 405, 505, and the cartridge 400, 500 extracts the sample. The cartridge 400, 500V, and 600V are used for processing lipid samples without exposing them to the outside environment. In some embodiments, the lipid-receiving The fat chambers 405, 500 are positioned in the cartridges 400, 500. The 05 is a flexible bag with a volume ranging from 9 fluid ounces to 19 fluid ounces.

[0152] In various embodiments, the cartridge 400, 500 is an ESW or mechanical shock device. and designed for use with any of the methods disclosed herein. In some embodiments, the processing is carried out according to the processing described below. Some of the processing steps are performed automatically by a machine. In this embodiment, the processing is performed by a user, who may then The steps described above are performed manually. In both embodiments, the processing is The internal processing of the cartridge 400, 500 is controlled externally, while the sample is The procedure is performed in a sterile manner without exposure to harsh environments.

[0153] In the embodiment shown, the lipid sample is passed through the first one-way valve 410, 510. The fat is introduced into the fat chamber 405, 505 by passing the first sample through the fat chamber. The directional flow valves 410, 510 are oriented as shown to direct the flow of the fat chambers 405, 5 05. In some embodiments, after removal from the patient, A container for holding the lipids and a first one-way valve of the processing cartridge 400, 500 Male and female Luer lock connectors are attached to the 410 and 510. By this, lipids are introduced into the inner chamber of the fat chamber 405, 505. In some embodiments, lipids are removed from the patient via a liposuction device. The liposuction device and processing device are then removed using a sterile connection device. The cartridge is sterile docked to remove the process from the liposuction device. The sample is transferred to the insert cartridge.

[0154] Once inside the fat chamber 405, 505, the lipids are washed prior to processing. The cartridge 400, 500 contains a fluid reservoir 415, 515, The fluid reservoirs 415, 515 are filled with sterile saline and, in some implementations, The form is filled with sterile 1x phosphate buffered saline, which is used to wash the tissue. Inject sterile saline into the fat chamber 405, 505. To do this, saline is introduced into the fat chamber 405 through the second one-way valve 420, 520, 505. The flow direction is as shown. Water is introduced into the fat chamber 405, 5 through the use of external actuators 425, 525. 05, and the external actuator 425, 525 operates the first one-way valve 4 A first plunger 430, 530 is used to push saline through the catheter 20, 520. In some embodiments, the external actuator 425, 525 moves the The device is manually operated by a user, and the user allows the desired amount of saline to flow through the second one-way valve. 420, 520 into the fat chamber 405, 505. In some embodiments, the external actuator 425, 525 The movement of 425, 525 is automated, and an external machine controls the external actuator 425 , causing 525 to move in the direction shown, thereby A desired amount of saline enters the interior of the fat chamber 405, 505 through the valve 420, 520. Measure out the salt water.

[0155] Wash the cartridges 400 and 500 gently with a solution containing lipids and sterile saline. The measurement is performed by shaking or swirling the sample.

[0156] The cartridges 400, 500 are then refilled with sterile saline to remove the (now cleaned) lipids. In this embodiment, the cartridges 400, 500 are In some embodiments, the cartridge 400, 500 has two variations: It has a fixed vertical orientation and is contained within a fat chamber 405, 505. The lipid and saline washes will always naturally separate from each other via gravity. and the lipids float to the top of the fat chamber 405, 505. In some embodiments The cartridges 400, 500 have a horizontal orientation and are rotated vertically. This allows the lipids to float and separate from the saline wash. Since lipids are less dense than lipids, they will rise to the top of the saline solution and float. The amount of time required for this to occur will vary depending on the particular composition of the lipid sample. In some embodiments, separation can occur in 1 to 30 minutes. In embodiments, separation can occur in 1 to 15 minutes. In some embodiments, separation can occur in 1 to 5 minutes. In some embodiments, separation can occur in 10 seconds to 1 minute. , separation can be accomplished in less than a minute.

[0157] The saline is then drained as completely as possible from the fat chambers 405, 505. The drain is discharged from the cart utilized in the present method, so that only the lipids remain. Depending on the type of Ridge 400 or 500, this can be done through a variety of means. In some embodiments, the saline is applied to the outside of the cartridge 400, 500. The fat is fed through a tube located at the bottom of the fat chamber 405, 505. Drains are drawn out of the fat chambers 405, 505 and away from the cartridges 400, 500. Drainage is actively accomplished using either a syringe or a pump. The flow can be conducted by a suction pump or can proceed passively via gravity flow. In the configuration, drainage is used to maintain the integrity of the sterile system as described herein. This is done to maintain.

[0158] After washing is complete, the wash is repeated until the lipids are golden yellow and the saline is only slightly cloudy. In some embodiments, washing is performed 1 to 5 times, and in some embodiments, is performed 1 to 4 times, in some embodiments, 1 to 3 times, in some embodiments is performed 1 to 2 times, and in some embodiments, is performed once.

[0159] In some embodiments, sterile saline is dispensed from the fluid reservoir 415, 515. The washing is repeated until the fat chamber 405 is exhausted. , 505 saline wash should be clear enough that after washing, 50% to 100% The washing is repeated until the light passes through the saline solution and shows a high degree of transparency. In some embodiments, cleaning is repeated until a set number of cleaning cycles are completed, which may be In some embodiments, the washing is 1 to 5 times, and in some embodiments, the washing is 1 time. in some embodiments, 1 to 4 washes, in some embodiments, 1 to 3 washes, In some embodiments, one to two washes, and in some embodiments, one wash. In some embodiments, two washes, and in some embodiments, three washes. It is.

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

[0161] The lipids in the lipid chambers 405, 505 are added to the lipids in order to release the stem cells from the lipids. The above-mentioned methods 1 and 2 are carried out by subjecting the material to ESW and / or mechanical shock. The resulting product is processed according to one of the methods described above. In this embodiment, the cartridge 400, 500 has an opening above the fat chamber 405, 505. The nozzle allows for optimal processing and is protected from ESW and / or mechanical shocks. These forces are prevented from passing through the rigid plastic exterior of the Cartridge 400 and 500. In such an embodiment, the flexible fat chamber 405, 505 walls are , exposed for processing.

[0162] After tissue processing is complete, the lipids 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 and the fat chambers 405, 505 The processed lipids and saline contained in the The lipids will then float to the top of the fat chamber 405, 505. In some embodiments, the cartridges 400, 500 have a horizontal orientation. The plate is then rotated vertically, allowing the lipids to float and separate from the saline. Because lipids are less dense than saline, they rise to the top of the saline and float. Here, the stem cells released from the lipids are contained in the physiological saline layer. .

[0163] The saline containing the stem cells is then removed from the fat chamber 405, 505 and dispensed into the cart. The lipid is introduced into the cell reservoir 435, 535 located within the ridge 400, 500. The saline is moved through the third one-way valve 440, 540 away from the through the use of a second external actuator 445, 545, the cell reservoir 435 , 535, and the second external actuator 445, 545 is moved to the inside of the third A second plunger 45 is inserted to push the saline through the one-way valve 440, 540. 0, 550. In the cell reservoir 435, 535, a second plunger 450, 550 are oriented in a fully closed configuration and the cell reservoirs 435, 535 In this manner, the second actuator 445 is fully closed. , 545 moves the second one-way valve 440, 540 away from the third one-way valve 440, 540. The plunger 450, 550 is moved to apply a slight pressure to the inside of the cell reservoir 435, 535. A vacuum is created, which removes the saline layer containing the stem cells from the fat chamber 405. , 505 through a third one-way valve 440, 540 to a cell reservoir 435, 535 In some embodiments, the second external actuator 445, 54 5 is operated manually by a user, and the user injects a physiological saline solution containing stem cells into the The entirety of the ion exchange membrane is introduced into the cell reservoir 435, 535 through the third one-way valve 440, 540. The second actuator 445, 545 is pulled until it is retracted. In this embodiment, the movement of the second external actuator 445, 545 is automated and the external The machine pulls the second external actuator 445, 545, causing the stem cell-containing bioreactor to move. Saline is moved into the cell reservoir 435,535.

[0164] In some embodiments, the saline containing the stem cells removes any remaining unwanted cell debris. The pieces are pulled through a mesh to filter them out of the processed fatty tissue. In some embodiments, the mesh is placed in the fat chamber 405, 505. In the fat chamber 405, 505, the mesh is positioned The mesh covers the opening of the one-way valve 440, 540. In some embodiments, the mesh is located inside the cell reservoir 435, 535, and the cell reservoir 435 Inside 535, a mesh covers the outlet of the third one-way valve 440, 540. In some embodiments, the mesh is positioned within the cell reservoir. Inside the cell reservoir, the mesh is connected to the opening of the fourth one-way valve 455, 555. In some embodiments, the pores in the mesh are 40 In some embodiments, the pores range in size from 70 μm to 100 μm. In some embodiments, the mesh is nylon.

[0165] In the embodiment shown in FIG. 4, the stem cell-containing fraction is From the inside, through the fourth one-way valve 455, the remote control contained within the cartridge 400 The stem cell fraction is transferred into the heart tube 460 by a second external actuator 445. through the use of a second external actuator, 445 pushes saline through the third one-way valve 440 into the centrifuge tube 460. To do this, the second plunger 450 is moved. This transfer can be performed manually or it can be automated. The tube 460 is removed from the cartridge 400 for centrifugation. proceeding as described in either of the two embodiments described above, The pellet remains at the bottom of the centrifuge tube 460 .

[0166] In the embodiment illustrated in FIG. 5, the fraction containing stem cells is stored in cell reservoir 535. from the inside of the cartridge 500 through the fourth one-way valve 555. The mixture is transferred into the centrifuge component 560. The centrifuge component 560 The square chamber and the triangular chamber act as centrifuge components. This spins the cells out of the saline solution so that they settle on the outer edge of the chamber. The access port 565 allows the user to Allows access to the stem cell pellet, resuspends the pellet, and / or Alternatively, the pellet may be removed from the centrifugation component 560. In some embodiments, the operation of the centrifuge component 560 may be automated and controlled by an external device. and controlling the external device, wherein the external device is located in the center of the chamber and in the cartridge. The spin chamber is engaged through a motor and gears that insert into the The centrifuge component 560 may be direct drive or geared. The layer / supernatant containing the stem cells can be separated by centrifugation as described above. The tube is loaded into the isolation component and the other side is balanced prior to centrifugation. Once introduced into the centrifuge component, the stem cells are centrifuged at 500g for 3 to 10 minutes. In some embodiments, the centrifugation is at 120° C. for 10 minutes. It is performed at 0g.

[0167] A small amount of fluid is pumped into the tube to resuspend the cells. Typically, the volume is 1 mL to 5 mL. Fluids can be saline, platelet-rich plasma, hyaluronic acid, or or fixative gel, hydrogel, scaffold, or fibrin or glue, or or other compounds.

[0168] With the cells in suspension, the cells are then injected through a hypodermic needle for further use. It is drawn into a syringe. This is done automatically by the processing machine. or manually by an operator.

[0169] A small amount of fluid is used to resuspend the stem cells. The amount of fluid added to resuspend the cells ranges from 1 mL to 5 mL. In some embodiments, the fluid may be saline, platelet rich plasma, hyaluronic acid, fixative gel, hyaluronic acid, or the like. A gel, scaffold, fibrin, glue, or any combination of the above. In some embodiments, the cells are resuspended in the centrifugation aspirate. .

[0170] With the stem cells floating in suspension, the stem cells are ready for use. Such uses include, but are not limited to, reintroduction into the patient from which the stem cells were derived, freezing, In some embodiments, the method may include concentrating, storing, and expanding the The suspended stem cells are transferred through a hypodermic needle into a syringe for patient use. In some embodiments, the concentrated resuspended stem cells are used to treat cardiac, muscle, bone, cartilage, and other conditions. Tissue scaffolding for growth into bone, liver, kidney, or other tissue and organ structures In some embodiments, the concentrated resuspended stem cells are seeded into a medium. By inducing stem cells to express pluripotency transcription factors, will be converted.

[0171] device As discussed above, the present disclosure provides a method for bioprocessing a tissue sample and In various aspects, the present disclosure also provides a method and a system for To provide an easy-to-use device suitable for use with the Such devices are closed to the environment and designed for single use. , including single-use cartridges, which are completely contained within a single device. This enables

[0172] container The embodiments described above include withered tissue samples, lipid samples, A fraction containing the desired cell fraction, a fraction containing stem cells, or any of the foregoing. Such a container may be one or more containers as shown in FIG. Containers utilized in step 105 of 1; container 205; fat chamber 405, 50 5; fluid reservoirs 415, 515; and cell reservoirs 435, 535. These containers are suitable for use with the systems and methods provided by the present disclosure. and can be flexible, rigid, or semi-rigid. In some embodiments, the container is a syringe.

[0173] The vessel utilized in step 105 of FIG. 1; vessel 205; fat chamber 405; 505; fluid reservoirs 415, 515; and cell reservoirs 435, 535, respectively. It is protected from the outside environment by itself or as part of a sealed cartridge. They are closed and disposable.

[0174] In some embodiments, the container utilized in step 105 of FIG. ; fat chambers 405, 505; fluid reservoirs 415, 515; and cell reservoirs Each of the containers 435 and 535 is a flexible container, for example a flexible bag. In such an embodiment, each container is made of ethylene vinyl acetate (EVA), poly( Polyvinyl chloride (PVC), ethylene vinyl acetate (EVA), nylon, etc. The plastic may be of the type described above.

[0175] Each of the flexible containers may be blow molded. In some embodiments, the flexible Each vessel is radio frequency, high frequency, or induction welded. It may also be blow molded.

[0176] In some embodiments, each of the flexible containers comprises an inner chamber of the flexible container. A three-dimensional bag having at least one outlet port in fluid communication with the In embodiments, a single container may include two or more outlets in fluid communication with the interior chamber of the container. Such an outlet may, for example, be provided for the passage of air from the interior chamber of the container. To drain fluid from a container or to transfer fluid from inside a container to another container. It can be used for:

[0177] In some embodiments, the container includes one or more lids positioned on the exterior of the container along the edge. or a plurality of holes, one or more of which may be adapted to suspend the container in space. It can be used to lower

[0178] The total volume of the container can be from 5 fluid ounces to 50 fluid ounces, and several In some embodiments, it can be from 9 flow ounces to 19 flow ounces.

[0179] In some embodiments, the tissue sample, sterile saline, or other form of To receive the input, the vessel receives inlet lines or connections at separate points. and the inlet line or connection is in fluid communication with the interior of the vessel. To maintain the integrity of a closed, sterile environment, the inlet line or connection must be The connection includes a female Luer connection, the female Luer connection allowing the container to be connected to an external device. The container is connected to a syringe containing a tissue sample, from which the substance is dispensed. Another container to be transferred into the container in question, or the like. The inlet line or connection should be connected to an external device using a sterile connection device. Includes a sterile dock for

[0180] In some embodiments, the container can be a flat bag, and the flat bag The gag has a top edge, a bottom edge, and two substantially similar side edges. The edge is in fluid communication with the interior of the bag for draining fluid out of the bag. The flat bag also allows for the aseptic introduction of materials into the interior of the bag. It has an inlet portion through which

[0181] In some embodiments, the container is a three-dimensional bag that is rectangular in shape; The three-dimensional bag has a top edge, a bottom edge, and two substantially similar side edges. The bottom edge is in fluid communication with the interior of the vessel for draining fluid out of the vessel. The upper edge includes an outlet portion that is in communication with the interior of the vessel for introduction of material into the vessel. The inlet includes an inlet portion that is connected to the

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

[0183] In some embodiments, the container can be semi-rigid. In an embodiment, the container includes a rigid plastic housing. The housing maintains its shape and does not move easily inside the housing during tissue processing. The housing can hold a flexible container. is configured to contain a point of attachment to 315.

[0184] Rigid plastic housings are made of e.g. high density polyethylene (HDPE) or may be made from any suitable rigid plastic material, including polypropylene (PP). Rigid plastic materials do not exhibit elastic deformation and The material exhibits any of the elastic behavior typically exhibited by flexible plastics. The rigid plastic housing can be injection molded or die cut.

[0185] In some embodiments, the flexible container is contained within a rigid plastic housing. The rigid plastic housing may completely enclose the flexible container. In some embodiments, the flexible container may or may not be a rigid plastic. The device is not completely enclosed in a plastic housing. Rather, the housing is The flexible container is partially sealed to hold it in place during tissue processing. do.

[0186] cartridge In various aspects, cartridges suitable for use with the present disclosure include, among others: It is a three-dimensional container that can house one or more containers within its interior. The inner container is , may be connected by one or more lines and / or valves. Examples are the cartridges 400, 500 shown in FIGS.

[0187] In some embodiments, the cartridge is made from a rigid plastic material. The rigid plastic material maintains its shape and remains in place during tissue processing. holding at least one, and preferably a plurality, of flexible containers inside the cartridge The containers can be mounted on the platform 210 or 315. The method is configured to contain one or more points of:

[0188] Rigid plastics are, for example, high density polyethylene (HDPE) or polypropylene. It may be made from any suitable rigid plastic material, including polypropylene (PP). A flexible plastic material does not exhibit elastic deformation, and a rigid plastic material It does not exhibit any of the elastic behavior typically exhibited by flexible plastics. The rigid plastic cartridge may be injection molded or die cut.

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

[0190] Centrifuge tube Centrifuge tubes suitable for use with the methods and devices of the present disclosure are suitable for use in a centrifuge rotor. Precision finished glass or plastic designed to fit your device exactly The capacity of the centrifuge tube is 100% of the volume of the sample to be processed. Depending on the total volume of the tissue sample being sampled, the amount of tissue sample may vary. In some embodiments, the centrifuge tube has a capacity ranging from 1 mL to 50 mL. Volumes ranging from 0.5 mL to 20 mL, and in some embodiments, 250 μL The volumes range from 0.1 to 2.0 mL.

[0191] In some embodiments, the centrifuge tube is an Eppendorf® tube. and in some embodiments a microfuge tube. In one embodiment, the tube is a microcentrifuge tube.

[0192] The material of the centrifuge tube can vary. In some embodiments, the centrifuge tube In some embodiments, the centrifuge tube is made of glass. In each embodiment, the centrifuge tube is designed for single use. In some embodiments, the centrifuge tube is made of polyethylene. It is made from a flexible, transparent plastic such as a plastic stencil and is shaped like a half cone. It includes an integral hinged sealing cap.

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

[0194] In various aspects, one-way sensors suitable for use with the devices and methods of the present disclosure The valve includes a two-port valve having two openings, one through which the fluid can flow and one through which the fluid can flow. One way valves allow fluid to enter and the other allows fluid to leave. It works to provide unidirectional flow automatically, without any user intervention or control. In some embodiments, one-way valves useful in the methods and devices of the present disclosure are not required. is made from a rigid plastic such as poly(propylene).

[0195] In some embodiments, a suitable The directional valve is a ball check valve, and the component that prevents the backflow of the fluid in the ball check valve is The component is a spherical ball. In some embodiments, the ball closes a one-way valve. It is spring loaded to help keep it in place.

[0196] In some embodiments, a suitable The directional valve is a diaphragm check valve, which is adapted to produce a valve closure. In such an embodiment, the flexing rubber diaphragm is positioned in the The upstream pressure can be generated by user intervention or via an automated process. This causes the diaphragm to open and fluid to flow through the valve. When pressure on the glass ceases, the diaphragm closes, terminating fluid flow.

[0197] In some embodiments, a suitable The directional valve is a swing check valve or a tilting disc check valve. In this state, the disks are spaced apart to allow fluid flow in one direction and in the opposite direction. The valve is the moving part of the valve that is used to prevent backflow of fluid.

[0198] example The materials, methods, and embodiments described herein are further defined in the following examples. Specific embodiments are also defined herein in the examples. These examples, while showing specific embodiments, are provided for illustrative purposes only. It should be understood that, from the disclosure herein and these examples, one of ordinary skill in the art will be able to It is possible to ascertain the essential features of the disclosure, and it is not necessary to deviate from the spirit and scope of the disclosure. Various modifications of the present invention may be made without departing from the spirit and scope of the present invention, to adapt it to various uses and conditions. Various variations and modifications are possible.

[0199] Example 1 General Protocol for ESW Tissue Processing Lipoaspirate is obtained from the subject, the lipoaspirate being approximately 30 cc in volume. Prior to processing, the lipoaspirate was cut into an endothelial cavity having a sidewall approximately 0.25 inches thick. Ethylene vinyl acetate (EVA), poly(vinyl) chloride (PVC), ethylene vinyl acetate The catheter is then transferred into a sterile flexible bag of EVA or nylon. There is enough

[0200] Place the male Luer lock connector from the saline bag onto the flexible bag. By connecting to the female Luer lock connector that is installed in the By manually dispensing saline into the flexible bag, the lipoaspirate is Washing is performed using an equal volume of sterile saline solution introduced into the flexible bag. The lipoaspirate and saline irrigation solution were then removed by gently shaking the flexible bag. It is mechanically stirred.

[0201] The tissue sample / saline wash mixture was allowed to sit for at least 10 min and then aspirated. Allow the object to float to the top of the saline solution.

[0202] The supernatant or saline wash is drained into a drain located at the bottom of the flexible bag. The syringe is connected to the line and removed from the flexible bag. by drawing the bottom liquid out of the bag or by gravity flow. , removal can be performed.

[0203] The wash was repeated 3-4 times, with the final wash being filled with sterile saline wash solution in the bag. leave.

[0204] The next step is to use the Masterpuls MP100 external shock wave generator to The objective is to apply external shock waves to the lipoaspirate.

[0205] The generator may contact the walls of the flexible bag, but the shockwave generator may not penetrate the lipoaspirate itself. Care should be taken to ensure that no contact is made with the

[0206] If necessary, ultrasound gel may be applied to the flexible bag prior to application of shock waves to the lipoaspirate. It may also be applied to the sides.

[0207] ESW is applied to the lipoaspirate for 1 to 30 minutes.

[0208] The flexible bag is then gently washed by shaking or gently vortexing. Stir thoroughly.

[0209] The now processed lipoaspirate is left to rest and the remaining fat tissue is washed with saline. It is designed to float on the top of the water.

[0210] By attaching a sterile syringe to the drain tube as described above. Remove the supernatant from the flexible bag by pulling as much of the supernatant as possible. Remove the clear fluid. Do not remove any remaining adipose tissue.

[0211] During removal, three nibs with pore sizes of 40 μm, 70 μm, or 100 μm were used. Pass the supernatant through one of the Ronmesh filters.

[0212] Distribute the supernatant into one or more sterile centrifuge tubes. If used, a balance tube containing sterile saline is used for balancing. Ensure that it is installed inside the rotor.

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

[0214] Optional: Resuspend pellet in 1 mL RBC lysis buffer and incubate at 37 °C for 10 min. Incubate in a water bath at 37°C. Neutralize the RBC buffer.

[0215] If RBC lysis buffer is not desired, dissolve the cell pellet in 1 mL of sterile saline. Resuspend the pellet.

[0216] Count the cells.

[0217] Example 2 Comparison of lipoaspirate processing methods Summary: The purpose of the tests performed in this example is to isolate adipose tissue using collagenase digestion. The yield of nucleated cells from mesenchymal stem cells extracted from tissue and the effect of extracorporeal shock wave (ESW) disruption The purpose of the study was to compare the

[0218] material: 1,500 mL 1x Phosphate Buffered Saline (PBS) 35mL of 1x Red Blood Cell (RBC) Lysis Buffer 200mL of 0.1% collagenase type I 15 mL of 10% fetal bovine serum (FBSA) in Dulbecco's modified Eagle's medium (DMEM) S)

[0219] method: control Approximately 250 mL of lipoaspirate was removed using a sterile 60 mL catheter for 100 m. The excess fluid was transferred into the L beaker. Little or no excess fluid was introduced into the beaker. Care was taken to ensure that this was not introduced.

[0220] The lipoaspirates were washed four times with approximately equal volumes of sterile 1xPBS. For each wash, sterile 1xPBS was added to the beaker and swirled twice to mix. The mixture was allowed to settle. The supernatant was removed from the beaker by aspiration. The pulls were washed a total of four times.

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

[0222] A sterile 10 mL serological pipette is used to transfer 45 mL of the bottom supernatant into the fourth and final From the washings, 40 μm, 70 μm, and 100 μm were placed into three sterile 50 mL conical tubes. and transferred through a 100 μm tube sterile filter. A total of 135 mL of supernatant was were transferred for use as controls.

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

[0224] The bottles were then placed in a water bath at 37° C. Every 10 minutes, the bottles were removed from the water bath. The bottle was removed and inverted several times to redistribute the lipoaspirate and allow the bottle to settle. 30 min After incubation for 1 h, the bottles were agitated one final time and removed from the aquaria.

[0225] The exterior of the bottle was sprayed with 70% ETOH and placed in a fume hood. The lipoaspirate and collagenase mixture was allowed to sit for 10 minutes.

[0226] A sterile 10 mL serological pipette was used, and three sterile 50 mL conical-shaped tubes were inserted. into the tube through 40 μm, 70 μm, and 100 μm tube sterile filters The supernatant was transferred. A total of 135 mL of supernatant was transferred. 45 mL was added for balance. Three tubes contained collagenase digested cells, including the fourth tube containing water. To obtain a pellet, the mixture was centrifuged at 1,200 g for 10 min at 37°C.

[0227] The exterior of the three tubes containing the collagenase suspension was cleaned with 70% ETOH. The tubes were then washed and placed in a fume hood, leaving the balance tube. The supernatant was aspirated. and leaving the cell pellet in each tube. 25mL serological pipette Using the 5 mL of 10% FBS dispensed into each tube, Each tube was vortexed for 5 seconds to resuspend the pellet. S was used to neutralize any remaining collagenase activity.

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

[0229] ESW Processing 200 mL of lipoaspirate was then dispensed into the remaining lower extremities using a sterile 60 mL catheter syringe. The entire effluent was transferred into a sterile 500 mL leg catheter bag. 1xPBS is added to achieve a total fluid volume of 200 mL. Approximately 2 mL of ultrasound gel was placed in the catheter bag. Using the large tip, at 2 bar and 21 Hz, approximately A 10,000 ESW pulse was administered to the sample inside the bag. This process An additional 10,000 ESW pulses were repeated on the other side of the bag. The exterior of the catheter bag was cleaned with 70% ETOH, which was then He was placed in the Muhud.

[0230] The bag is stirred and emptied into a 500 mL beaker and the suspension is stirred for 10 min. Using a sterile 10 mL serological pipette, the supernatant was pipetted into three sterile into 50mL conical tubes, 40μm, 70μm, and 100μm tubes The supernatant was transferred through a sterile filter. A total of 135 mL of supernatant was transferred.

[0231] Concentration and dissolution Three tubes containing a control suspension and a processed suspension The three tubes containing were centrifuged for 10 min at 1,200 g and 37°C. The supernatant was aspirated from all tubes, leaving behind the pellet in each, and 5 mL of 1xR BC Lysis Buffer was added to each tube. Each tube was pelleted. The pellet is then vortexed for 5 seconds to resuspend the pellet, and the tube is placed in 37°C water for 10 minutes. was placed in the tank.

[0232] The exterior of each tube was disinfected with 70% ETOH, and the tubes were then The plates were placed in a hood. 25 mL of sterile 1xPBS was added to dilute the lysis buffer and was added to each tube to neutralize.

[0233] Cell counting Using a sterile micropipette tip, 300 μL of sterile 1xPBS was pipetted into the six 75 μL of each was transferred into a sterile 0.5 mL microcentrifuge tube. A 50 mL sample tube was placed in each microcentrifuge tube for cell counting. was transferred into the tube.

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

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

[0236] [Table 1-1]

[0237] [Table 1-2]

[0238] [Table 1-3]

[0239] Hemocytometer Table 2 details the counts obtained from the visual hemocytometer. Cell counts from each quadrant, quadrant totals, dilution factor, and total counts. The cell numbers refer to the cell counts in the control group. The ratio of processed cells to control cells should be considered. It is an important indicator of the rate.

[0240] [Table 2]

[0241] overview Valid counts using the MoxiZ automated cell counter included controls, The overall averages for cell counts for lagenase and ESW are shown in Table 3. This is true.

[0242] [Table 3]

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

[0244] When looking at the cell ratios in the hemocytometer counts, the ESW process showed the highest ratio The ratios ranged from 1.78 to 9.0 times higher than the control. Only collagenase was in the range of 1.29- to 2.84-fold higher than the control. Created a ratio.

[0245] Discussion These data indicate that ESW is effective in generating high numbers of mesenchymal stem cells from adipose tissue. Based on these data, it has been shown that the method for obtaining mesenchymal stem cells is To achieve this, ESW is used in combination with ultrasonic processing of adipose tissue or collagenase processing. It was concluded that this is a viable alternative to either the In comparison, ESW has a collagenase peak at 7.08e+0 compared to 4.32e+05. This produced a cell yield of 5, which was a 164% increase in yield.

[0246] Given the data presented above, a sample of 50cc to 60cc of lipoaspirate The pull resulted in the production of approximately 5 million cells from ESW processing, and collagenase It is reasonable to conclude that the enzyme digestion would yield approximately 3 million cells. It is.

[0247] Therefore, using ESW to liberate stem cells from adipose tissue means: It is clear that this has significant advantages over known techniques. [Explanation of symbols]

[0248] 200 Processing Equipment 205 Processing vessel or cartridge 210 Platform 215 Shock Wave Applicator 220 Applicator Platform 225 Motor Controller 230X Planar Motor 235 Y Planar Motor 240 Z-plane motor 245 Shock Wave Generator 300 Processing Equipment 305 Motor 310 Impact Arm 315 Platform 400 Cartridges 405 Fat Chamber 410 First one-way valve 415 Fluid Reservoir 420 Second one-way valve 425 External Actuator 430 First Plunger 435 Cell Reservoir 440 Third one-way valve 445 Second External Actuator 450 Second Plunger 455 Fourth One-Way Valve 460 Centrifuge Tube 500 cartridges 505 Fat Chamber 510 First one-way valve 515 Fluid Reservoir 520 Second one-way valve 525 External Actuator 530 First Plunger 535 Cell Reservoir 540 Third one-way valve 545 Second External Actuator 550 Second Plunger 555 Fourth One-Way Valve 560 Centrifugation Components 565 Access Port

Claims

1. 1. A method for disintegrating tissue, comprising: placing the tissue sample in a processing container of a tissue processing device; and The tissue samples are subjected to forces from mechanical impact and shear forces to cause the tissue to: Multiple cell populations; A plurality of groups of cells and a plurality of individual cells; or A plurality of individual cells; Decomposing the the mechanical impact is generated by an impact arm that makes physical contact with the container by articulating up and down at speeds of up to 30,000 rpm; The method, wherein the impact arm does not physically contact the tissue.

2. The processing device comprises: a platform to which the processing vessel is attached; and The method of claim 1 comprising a variable speed motor driving the up and down articulation of the impact arm.

3. 3. The method of claim 2, wherein the motor is a variable speed motor adjustable to drive the up and down articulation of the impact arm at speeds from 3,000 rpm to 30,000 rpm.

4. The mechanical impact and shear forces disintegrate the tissue into a plurality of cell groups and a plurality of individual cells; and 4. The method of claim 1, wherein the method further comprises isolating the plurality of individual cells from the plurality of cells.

5. 5. The method of claim 4, wherein the isolating step is performed by filtration and / or centrifugation.

6. 6. The method of claim 4, wherein the isolating step is performed in 30 minutes or less.

7. 7. The method of any one of claims 1 to 6, 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, vein tissue, pancreatic tissue, bladder tissue, bone, tooth, dentin tissue, gum tissue, skin tissue, pineal tissue, pituitary tissue, thyroid tissue, adrenal tissue, pancreatic tissue, ovarian tissue, and testicular tissue.

8. 8. The method of claim 7, wherein the tissue is adipose tissue, the plurality of cell populations comprises adipose tissue and cells, and the plurality of individual cells comprises adipose-derived cells.

9. The method of claim 1 , wherein the platform is movable in three planes.

10. the processing vessel is a bag; the bag includes one or more outlets in fluid communication with the interior of the bag; and / or The method of claim 1 , wherein the bag comprises an inlet in fluid communication with an interior of the bag.

11. 1. A method for isolating cells from a first tissue, comprising: placing said first tissue sample in a processing receptacle of a tissue processing device; and subjecting the first tissue sample to forces from mechanical impact and shear forces; The organization is: Multiple cell populations; A plurality of groups of cells and a plurality of individual cells; or A plurality of individual cells; Decomposing the the mechanical impact is generated by an impact arm that makes physical contact with the container by articulating up and down at speeds of up to 30,000 rpm; The method, wherein the impact arm does not physically contact the first tissue.

12. The processing device comprises: a platform to which the processing vessel is mounted; and The method of claim 11 comprising a variable speed motor driving the up and down articulation of the impact arm.

13. 13. The method of claim 12, wherein the motor is a variable speed motor adjustable to drive the up and down articulation of the impact arm at speeds between 3,000 rpm and 30,000 rpm.

14. filtration, Centrifugation, Separating the tissue from an aqueous layer, the aqueous layer containing the cells; or Any combination of the above; 14. The method of any one of claims 11 to 13, further comprising isolating the cells by:

15. 15. The method of claim 14, wherein said isolating is performed in 30 minutes or less.

16. 16. The method of any one of claims 11 to 15, wherein the first 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 tissue, thyroid tissue, adrenal tissue, pancreatic tissue, ovarian tissue, and testicular tissue.

17. 16. The method of claim 14 or claim 15, wherein the cells are stem cells, the method further comprising the step of seeding the stem cells onto a tissue scaffold for growth into a second tissue structure and / or organ structure.

18. 18. The method of claim 17, wherein the second tissue structure 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 tissue, thyroid tissue, adrenal tissue, pancreatic tissue, ovarian tissue, and testicular tissue, and combinations thereof.

19. 18. The method of claim 17, wherein the organ structure is selected from heart, muscle, bone, cartilage, liver, and kidney.

20. 20. The method of any one of claims 11 to 19, wherein the cells are stem cells, and the method further comprises transforming the stem cells into induced pluripotent stem cells.

21. 21. The method of claim 20, wherein transformation occurs by causing the stem cell to express one or more pluripotency transcription factors.

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