Methods and kits for preservation of adipose tissue grafts - Patents.com

Abstract

To provide methods, devices and kits for use in washing or cryopreserving live cells or live tissue, such as fat graft tissue.SOLUTION: A cell or tissue isolation, purification and storage device comprises: a vessel having first and second ends defining an internal chamber divided by a filter 30 that retains fat cells or fat tissue and having a volume between the second end and the filter of at least 3 mL; a first adaptor or connector 22, such as a Luer lock or slip connector, at the first end of the vessel defining a first opening; and a second adaptor or connector 24, such as a Luer lock or slip connector, at the second end of the vessel and defining a second opening.SELECTED DRAWING: Figure 1

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Provisional Patent Application No. 62 / 553,322, filed September 1, 2017. The benefit of this application is claimed and the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Soft tissue following craniofacial trauma, craniofacial deformities, congenital anomalies, and deformities related to cancer treatment Deformation and changes in volume / contour are difficult to correct. Injectable hyaluronic acid fillers are expensive. They can only be filled in small volumes and degrade over time, requiring repeated treatment. Polyethylene implants are permanent foreign bodies and carry the risk of long-term infection and migration. The soft tissue flap technique is complex, has high morbidity, and leads to significant donor site deformation and Autologous fat grafting is a promising approach to minimize donor-site morbidity while providing early recovery. Fat grafts have also been developed as an effective means of treating soft tissue deformities. It is also widely used in cosmetic surgery. In our clinical trials, we have demonstrated that it is particularly useful for craniofacial fat grafting. Autologous fat grafting is a procedure used in plastic and reconstructive surgery. It is widely used in the United States, with nearly 80,000 procedures performed by plastic surgeons in 2016. (American Society of Plastic Surgeons Data, https: / / www.plasticsurgery .org / news / plastic-surgery-statistics).

[0003] The major drawback of fat grafting is that only about 63% of the graft volume heals and survives for a long time. This means that optimal results are achieved with at least two treatments. Although fat harvesting is minimally invasive, the time, cost and time commitment inherent in this procedure are Additionally, this harvesting typically requires an operating room setting, which increases patient discomfort and recovery time. To minimize this, cost effective apparatus and methods are needed. Summary of the Invention

[0004] In accordance with one aspect of the present invention, a cell or tissue isolation, purification, and preservation device is provided. The device has a filtered inner chamber that holds the fat cells or tissue. The filter has a first end and a second end defining a gap, and a volume between the second end and the filter is small. a container having a volume of at least 3 mL; a luer rod at a first end of said container defining a first opening; a first adapter or connector, such as a slip connector or a plug; and a first adapter or connector, such as a a first connector, such as a luer lock or slip connector, at each end defining a second opening; and two adapters or connectors.

[0005] According to another aspect of the invention, the present invention is used in the isolation and preservation of cells such as fat grafts. The kit is provided with 2 to 25 cell or tissue preservation devices (each preservation device). The device has a filtered inner chamber that holds the fat cells or tissue. a first end and a second end defining a volume between said second end and said filter of at least a container having a volume of at least 3 mL; a luer lock or a cap at a first end of the container defining a first opening; a first adapter or connector, such as a casing or slip connector; and a second end of the container. a second connector, such as a luer lock or slip connector, defining a second opening. an adapter or connector; At least 3 mL of fat graft or other cells or tissue can be cryopreserved. and optionally an adapter such as a luer lock or slip connector. and at least one container having a feeder or connector.

[0006] In accordance with yet another aspect of the present invention, there is provided a method for preparing a fat graft. adipose tissue is passed through the second opening of the device according to any one of the embodiments described above or herein. introducing (e.g., injecting) the fat graft into the inner chamber of the device; and cryopreserving the fat graft. and delivering an effective amount of cryoprotectant to the interior chamber of the device through a first opening of the device. mixing said adipose tissue with said cryoprotectant; and and cooling to a temperature below 0°C.

[0007] In another aspect of the present invention, a method for preparing a fat graft is provided. The method comprises the steps of: The fabric is then passed through a second opening of the device according to any of the embodiments described above or herein into the interior of the device. introducing (e.g., injecting) into the chamber an amount effective to flush the fat graft; introducing the cleaning solution into the interior chamber of the device through a first opening of the device; mixing the adipose tissue with the washing solution; centrifugation with the opening facing down to separate the fat graft from the washing solution; and Discharging the cleaning solution from a first end of the device.

[0008] In another aspect of the invention, adipose tissue is treated with a device according to any of the above or herein aspects. introducing (e.g., injecting) the fat into an interior chamber of the device through a second opening; an effective amount of a washing solution for washing the graft or for cryopreserving said fat graft; introducing an effective amount of a cryoprotectant into an interior chamber of the device through a first opening of the device; and mixing said adipose tissue with said washing solution or said cryoprotectant; and and a washing solution is introduced, the device containing the fat graft is passed through the first opening. 4. Separate the fat graft from the washing solution by centrifuging the fat graft face down and removing the washing solution from the or if a cryoprotectant is introduced, the fat graft is discharged from the first end of the device. to a temperature below 0° C. . [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 illustrates a schematic of one embodiment of a fat collection and storage device described herein, with a partial cutaway showing the filter. [Diagram 2] FIG. 2 illustrates a schematic of one embodiment of a fat collection and storage device described herein with a partial cutaway showing the filter. [Figure 3A] FIG. 3A, together with FIG. 3C, partially illustrates a schematic diagram of one embodiment of a method for preserving a tissue graft, such as a fat graft, described herein. [Figure 3B] FIG. 3B illustrates a schematic of one embodiment of a storage device for use in the methods and kits described herein. [Figure 3C] FIG. 3C, together with FIG. 3A, partially illustrates a schematic diagram of one embodiment of a method for preserving a tissue graft, such as a fat graft, described herein. [Figure 4A] FIG. 4A shows the results of analysis of ASC (adipose-derived stem cells) for viability using trypan blue staining, as described in Example 3. [Figure 4B]FIG. 4B shows the results of analysis of adipocyte viability using trypan blue staining, as described in Example 3. [Figure 5A] FIG. 5A shows the results of an analysis of ASC viability using trypan blue staining, as described in Example 4. [Figure 5B] FIG. 5B shows the results of an analysis of adipocyte viability using trypan blue staining, as described in Example 4. Detailed Description of the Invention

[0010] The use of numerical values ​​in the various ranges specified in this application means that the values ​​fall within the stated ranges unless otherwise indicated. Both the minimum and maximum values ​​within are stated as approximations, as if preceded by the word "about." Thus, amounts above and below the stated ranges may be used to achieve substantially the same results as values ​​within the ranges. Some variation may be used, and unless otherwise noted, the disclosure of these ranges is intended to be minimum. A continuous range is intended, including all values ​​between the value and the maximum value.

[0011] As used herein, the terms "right," "left," "upper," "lower," and derivatives thereof The terms should be related to the present invention in their orientation in the drawings in which they are depicted. However, the invention may assume various alternative orientations, and therefore such terms should not be considered limiting. It should be understood that such disclosure should not be made without the express written consent of the Except as otherwise described herein, it is understood that the present invention may assume various alternative variations and sequences of steps. Further, certain devices and methods are illustrated in the accompanying drawings and described in the following specification. It should be understood that the steps are examples. Specific dimensions and other physical characteristics are not to be considered as limiting.

[0012] The terms "distal" and "proximal" refer to directions with respect to the devices described herein, and "distal" The direction of the needle should be away from the user of the device, such as toward the sharp tip of a hypodermic syringe (the distal end of the needle). "Proximal" refers to the subcutaneous area away from the sharp needle tip at the proximal end of the syringe. Refers to the direction toward the user of the device, such as the direction approaching the end of a syringe plunger. The terms "distal" and "proximal" and other spatial relationships refer to the distal and proximal locations of the elements of the devices described herein. or other external reference point for the end use of the device in typical use as a skin device. These descriptors correspond to the relative position, orientation, and / or direction of the Relative positioning of the elements of the device described in the specification with respect to the device as a whole and with respect to the elements are provided merely to represent a general location, orientation, and / or direction, and unless otherwise specified As long as these elements are there, in that position, in that orientation at any given time, does not require or imply any physical relationship with the end user The drawings are, unless otherwise indicated, schematic in nature and are not drawn to scale. The drawings are not intended to be limiting, but are drawn to best illustrate the relationship between the various elements of the apparatus depicted in the drawings.

[0013] As used herein, the terms "comprising," "comprise," or "composition" are used interchangeably. The term "one or more combinations" and variations thereof means open ended. "A" and "an" are intended to refer to one or more.

[0014] As used herein, the term "treatment" or "treatment of" a condition, injury, or defect. "treating" refers to the desired clinical / therapeutic procedure, including tricuspid or mitral valve repair and / or replacement. Any suitable administration of the composition, device or structure for the purpose of achieving a medical endpoint. It means administration to a patient by a schedule, procedure and / or route of administration.

[0015] As used herein, the term "patient" or "subject" refers to, but is not limited to, "Mammal" refers to any member of the animal kingdom, including humans, and "mammal" includes, but is not limited to, humans. "Mammals" refers to all mammals, including humans.

[0016] Described herein is a simple and cost-effective method for preserving live adipocytes or live adipose tissue. Provided.

[0017] Syringes, cannulas, and devices and kits described herein The "fluid connector" for this purpose is, for example, a small-diameter fluid connector that is widely known in the medical field. The connector may be any suitable fluid connector known in the art (e.g., ISO 80369). Common connectors include those used with medical or laboratory instruments or devices such as hypodermic syringes. As is common for tubing, slip or threaded luer connectors are used. Connectors include plug-and-screw or threaded connectors. The connectors are designed to ensure proper orientation and use of the device. Can be matched by color or structure to ensure.

[0018] As used herein, a "syringe" is a medical device as it is commonly known in the medical field. or hypodermic syringe and may be made of glass, plastic, ceramic, and / or any approved The syringe may be manufactured from any suitable material. A "cannula" is a hollow tube with a blunt end, and a "needle" is a For the purposes of the devices and kits described herein, tissue, e.g., skin, blood vessels, or other tissue, Refers to a hollow tube with a sharp or pointed end to easily penetrate tissue. For this purpose, a cannula may be preferred to reduce the possibility of puncture wounds. When using a needle, the needle or other "sharp" device must first penetrate the patient's skin. Both needles and cannulas, as is commonly known, have an opening and In the case of a needle, a distal end (for insertion into a patient) comprising a sharp or pointed end; They have suitable fluid interfaces, such as a Luer connector for syringe attachment. The cannula also has a proximal end comprising a connector. The cannula for fat sampling has one or more openings. It may include a closed end adjacent its distal end and within the wall of the cannula in various patterns. Such cannulas are, inter alia, available as Tulip CellFriendly (trademark). Cannula and Tulip Medical P, San Diego, California roducts or MicroAire, Charlottesville, Virginia Various sampling cannulas and other suitable instruments are widely available.

[0019] A "cryoprotectant" is a substance that is added to living cells to protect them from freezing at a temperature such as -20°C or -80°C. This allows cells to be protected from exposure to temperatures below 0°C (e.g., cryopreservation), and as a result The result is a composition that provides substantial cell viability after the cells are subjected to freezing temperatures. Agents are generally non-toxic when used to preserve cells. Suitable cryoprotectants for use include dimethyl sulfoxide (DMSO), human serum albumin, trehalose, and any effective combination of the foregoing, optionally further comprising Also includes plasma or autologous serum. An effective amount of cryoprotectant, such as 10% v / v DMSO, may be used by those skilled in the art. The amount of cryoprotectant used is known in the art, and effective or optimal amounts are readily ascertained. Slow cooling and thawing of the explant may also be desirable to obtain good viability of the harvested tissue. For cryopreservation of cells, see Example 2 below.

[0020] Fat grafting is a surgical procedure that involves the aspiration of fat donor tissue from the subcutaneous tissue through a hollow cannula. The abdomen is a common donor site. Fat grafts are injectable, which allows This makes both the harvesting and implantation procedures minimally invasive. Fat grafts are made in the form of a viscous injectable slurry. The adipose tissue consists of small particles of fat tissue with a diameter of approximately 2 to 5 mm that form a sac. A saline solution containing epinephrine is injected into the subcutaneous tissue of the donor. This "wetting solution" Infusion not only minimizes bleeding but also provides aqueous flow to the harvested tissue, which must be separated before transplantation. The standard technique for harvesting and transplanting, called the "Coleman method," gives 10 c c Using a hollow sampling cannula attached to a Luer lock syringe, the surgeon makes a small incision The cannula is inserted through the tissue and the plunger is retracted to apply negative pressure. Upon passing through, small particles of fat are drawn into the opening of the harvesting cannula and are transported away by the movement of the cannula. The adipose tissue is then detached from the surrounding tissue and drawn into the syringe. The fat graft material is easily mixed with free lipids from the adipocytes and dissolved in water, making it a convenient benchtop blood vessel for blood grafting. The fat fraction is separated from the other fractions by gentle centrifugation in a liquid centrifuge. Fat grafts are suitable for transplantation and are present in most operating rooms for this purpose. It can also be injected into the recipient's tissue from a cc syringe, allowing for more precise injections. It can also be transferred to a smaller syringe.

[0021] For injections, a hollow cannula, similar to a collection cannula, is attached to the syringe. All common fat grafting devices are designed to be attached to a Luer connector. This is also taken into consideration in the design of one embodiment of the device described herein. The surgeon puts the fat directly into a 10cc syringe used for collection and performs the centrifugation process in the operating room. The syringe plunger is simply removed and a 10cc syringe is inserted. The centrifuge tubes fit into the sterile tubes of the centrifuge. In some embodiments, the described device is a standard For example, it has the same diameter and base length as a 10cc syringe, so it is directly related to device design. Therefore, the adipose tissue storage device can easily perform the post-thaw cleaning process without the need to change containers. The storage device may be adapted to a centrifuge to accommodate the fat transfer. The device is engineered to work seamlessly with current instrumentation and methods.

[0022] The main defect of autologous fat grafting is absorption of the fat graft during initial healing and persistence after healing. The volume of the graft that is filled is approximately 60%. The donor harvesting method for obtaining fat grafts requires a surgical setting and a fat Specialized equipment is required to process the tissue, so the fat tissue is stored at the time of surgery for later use. Preserving the data in a timely manner would significantly reduce costs, risks, and discomfort, and improve care. The results can be stored in small aliquots in a clinical setting for refinement. The tissue is preserved in situ, so the original fat harvest and the original Multiple treatments are possible with minimal additional cost to fat processing.

[0023] In some embodiments, the device includes a cryopreservation agent and a method for mixing the fat graft with the cryopreservation agent. It includes a special multi-purpose container used for storage and for the post-thaw cleaning process. In one example, each individual container holds approximately 10 cc of graft material. Hold the container, record the temperature constantly, and aliquot it while reserving other containers for continued storage. They are stored together in a special storage container that allows them to be removed individually. A large storage container may be included with each kit to hold the

[0024] In some embodiments, the individual disposable multi-function containers are configured to be used in conjunction with the system for fat grafting. It has a luer lock port for connection to a standard syringe. The built-in filter is Inject and eject both cryoprotectant and irrigation solutions without removing fat graft material. Add fat graft material, mix with cryoprotectant, and store in freezer. The steps of freezing, thawing, washing, and filtering are all carried out in the same vessel. This is a clear advantage of this system. In addition to the Luer lock connector, the device If desired, or if blockage of the Luer Lock port occurs, the fat graft can be transferred to another sterile One end may be open to allow pouring into a sterile container. Thus, in one embodiment, the multi-function container is the same diameter as a standard 10 cc syringe, approximately They are roughly the same length as those already used for fat grafting, and are commonly used in the operating room. Can be used in benchtop centrifuges.

[0025] Advantages of the devices, kits and methods described herein include: The tissue grafts can be prepared in the field very easily and stored at low cost; An aliquot of the fat graft is removed from storage and the remaining aliquot is stored for future use. To be left intact for use; Fat graft mixing, storage, and washing all occur in the same disposable multi-function container; Multi-function container works seamlessly with conventional fat graft instruments and standard syringes are designed to: The use of this instrument is highly intuitive for any surgeon performing fat grafting. That is.

[0026] Thus, herein, there are provided methods for use in harvesting and preserving adipocytes and adipose tissue. Apparatus, kits and methods for the preparation of a storage container are provided. Referring to FIG. 1, a cylindrical device 10 is depicted. The device 10 and its components are adapted to provide physiological temperature. (37°C) to storage under frozen storage conditions, e.g., -20°C, -80°C, or - Materials suitable for withstanding cell and tissue cryopreservation conditions up to 196°C (liquid nitrogen); As is known in the cryopreservation and centrifugation arts, suitable polycarbonate The filter is made of polyethylene or polypropylene and a suitable metal screen or filter. In some embodiments, the device 10 includes a device (e.g., a tube, a bottle, or a syringe) that is The dimensions are such that they fit into a typical clinical laboratory centrifuge capable of centrifuging the In this case, the device is typically 5 mL to 100 mL, e.g., 5 mL, 10 mL , 15mL, 20mL, 30mL, or 60mL medical syringes, typical 5mL to 50 mL, centrifuge tube (e.g., conical centrifuge tube) or typical blood collection tube dimensions, That is, an outer diameter of 5 mm to 25 mm, for example, 12 mm to 25 mm, and an outer diameter of 70 mm to 1 It has a length of 10 mm (body length excluding the connector). For example, it can be used with a 10 mL syringe. Storage containers for this purpose are available in sizes ranging from 16.5mm to 18mm in outer diameter and 80mm to 90mm in In one example, the ratio of the body length to the body outer diameter is at least 4:1, e.g. In some embodiments, the device 10 is custom sized, but the centrifuge Either custom or standard rotors or custom or standard rotor inserts The rotor is fitted with a volume between 3 mL and 100 mL or "Tubes, bottles, or syringes with volumes ranging from 1 mL to 4 L. "Centrifuges capable of centrifugation of These are centrifuges commonly found in factories or custom centrifuges, and can hold 1 mL to 4 mL tubes, such as 10 mL tubes. It is possible to centrifuge devices having volumes within the range of L, but not necessarily all of that range. For example, "the capacity to centrifuge volumes in the range of 1 mL to 4 L" A centrifuge that can centrifuge tubes, bottles, or syringes with a volume of 3 mL or more. Only tubes in the range of 30mL or 10mL to 60mL can be centrifuged. The outer tube does not have to be capable of being centrifuged.

[0027] Referring again to FIG. 1, the device 10 has a wall and an interior chamber (i.e., an interior cavity, or The vessel comprises a vessel depicted as a cylindrical barrel 20 having an internal bore. Although depicted as a cylindrical barrel shape, any suitable shape is acceptable. The device 10 also includes an opening extending through a first fluid connector 22 into the lumen of the barrel 20. a first end having a first fluid connector 22 comprising a mouth 23; and a second fluid connector a second fluid connector comprising an opening 25 extending through the nozzle 24 into the lumen of the barrel 20; and a second end having a first fluid connector 22, a barrel 20, and The second fluid connector 24 together with the opening of the first fluid connector 22 and the second fluid connector The filter 30 is disposed in the barrel 20 and forms a closed flow path between the filter 30 and the opening of the nectar 24. The first fluid connector 22 is disposed adjacent to the first fluid connector 22, and the opening of the first fluid connector 22 and the first 2. The apparatus is configured to filter liquid passing through a closed flow path between the opening of the fluid connector 24 of the first fluid connector 22 and the opening of the second fluid connector 24 of the second fluid connector 22. That is, the filter 30 is disposed between the opening of the first fluid connector 22 and the opening of the second fluid connector 23. The fluid passing through the closed flow path between the opening of the connector 24 passes through the filter 30. The filter 30 is adapted to operate at a temperature between -196°C and 37°C. Any suitable material capable of withstanding cryopreservation temperatures in the range of °C, e.g., metal mesh, e.g. The filter can be made from stainless steel mesh. Any suitable pore size or mesh may be used so that the adipose tissue is retained by the filter. The size may be, for example, a filter in the range of 50 microns (μ) to 250 μ, or or other porous barrier that serves a filtering purpose. The internal volume between the second end and the filter 30 is in the range of 3 mL to 100 mL, for example, approximately 5 mL, 10mL, 15mL, 20mL, 25mL, 30mL, 50mL, 60mL, or The volume is 100 mL, and the intervals between them are, for example, 10 cc cylinders, in one non-limiting example. The device 10 is 9 mL to 15 mL in order to accommodate the tissue volume obtained using the This allows the internal chamber of the barrel 20 to receive live cells or tissue, e.g., live fat cells or Raw adipose tissue, for example, comprises a fat graft, for example, a human fat graft.

[0028] The device is designed to withstand the centrifugal conditions typical for washing and pelleting viable fat grafts. , at least 500g, for example at least 1000g, 1200g, or 1500g They are designed and manufactured to withstand G-forces (multiples of the force of gravity at the Earth's surface, 1 g). There are a variety of common centrifugation conditions and protocols used to prepare fat grafts that result in G-forces are generally in the range of 400g to 1500g for a typical rotation duration of 1 to 5 minutes, e.g. For example, 1200g for 3 minutes as shown below.

[0029] FIG. 2 shows a second embodiment of the device 11, including a first barrel portion 26 and a slip fitting. 1, except that the barrel has a separable barrel that splits into a second barrel portion 28 that includes a nozzle 29. 2. The device is essentially the same as device 10 shown in FIG. 1. The purpose is to separate barrel portions 26 and 28. It is possible to open the barrel of device 11 and release the contents in the event of a blockage of the device. The device 11 has a first fluid connector 22 extending into the lumen of the barrel 20. a first end having a first fluid connector 22 including an opening 23 extending therethrough; A barrel formed by a first barrel portion 26 and a second barrel portion 28 from the nectar 24. and a second end having a second fluid connector 24 including an opening 25 extending into the lumen of the The slip fitting 29 effectively seals the barrel during use and prevents the barrel from configured to engage an inner surface of the first barrel portion to prevent separation of portions 26 and 28 Alternatively, to prevent separation of barrel portions 26 and 28 during use, a fitting 29 is fitted to the fitting's mating ridges and the first barrel so that the barrel portions 26 and 28 mate. Alternatively, the fitting may include suitable structural features such as valleys on the inner surface of fitting portion 26. The barrel 29 is threaded and the barrel portions are threaded to connect the first barrel portion 26 to the second barrel portion. The first barrel portion 26 is secured to the barrel 28 such that the first barrel portion 26 and the barrel 28 can be twisted relative to each other to separate the first barrel portion 26 and the barrel 28 from each other. The inner surface of section 26 is threaded or tapped. A fitting 29 is attached to the second barrel section. It is shown as one with the part 26, or it can be reversed, i.e. it is the first bar It is integral with the rail portion 29.

[0030] A storage container as described herein and exemplified by device 10 in FIG. 1 may be used to store live adipocytes or Methods are provided for processing and preserving, e.g., cryopreserving, raw adipose tissue (fat grafts). 3A-3C show methods and kits useful for preserving, e.g., cryopreserving, fat grafts. Referring to FIG. 3a, a fat graft 113, i.e., freshly aspirated fat tissue, is shown. The tissue and fat cells are drawn into a syringe 112. The fat graft 113 is then drawn from the syringe. Apparatus 110, for example apparatus 10 essentially as shown in FIG. 1 (with elements of apparatus 10 of FIG. 1) 2. Inject the solution into the second end (opposite the filter) of the tube (see reference 2.1.1 for details). The container is removed from the syringe 112 and cryoprotectant 114 is poured into the first end (filler) of the device 110. The second end of the device 110 is then capped 144, and then the second end of the device 110 is capped 145. The first end of the device 110 is capped 146. The device 110 is then shaken to separate the fat graft and the frozen tissue. This process is repeated as many times as desired, for example, to obtain a mixture of protective agents 115 from the patient. ~25 tubes of fat (fat grafts) were harvested and the fat grafts were mixed with cryoprotectant. The fat graft is then stored at a temperature below 0°C, e.g., −20°C, −80°C, or in a liquid Store under nitrogen. 150 As used herein, "mixing" includes shaking, inversion, shaking water bath, etc. Place in a vortex mixer, gently mix on a vortex mixer, or place in a container that is not designed to substantially affect cell viability. This may be by any suitable method, including any suitable method that does not adversely affect the

[0031] FIG. 3B illustrates an exemplary fat preservation kit that can be distributed as part of a kit for preserving fat grafts. Container 152 is shown. Container 152 contains the cryoprotectant mixed in as shown. The container 152 contains five pre-filled storage devices 110 containing fat grafts. A storage container is shown having an individual patient label with indicia regarding Also includes an outer label containing a safety seal and indications including patient information and storage dates for the fat graft. In addition, a temperature recorder (e.g., a widely known The container may also include a suitable number of storage devices 1 10 and may be of any shape and size sufficient to hold the fat graft in place. The substrate may be fabricated from any suitable material capable of withstanding the temperature changes associated with storage.

[0032] Referring now to FIG. 3C, for example, with respect to the use of fat grafts as tissue fillers, The storage container containing the graft and cryoprotectant mixture 115 is then placed, for example, on ice or in a refrigerator. The fat graft 113' is thawed under appropriate conditions, e.g., 4° C., and the fat graft 113' is then cooled to room temperature with a cryoprotectant 114'. The container is centrifuged 154 to separate the cryoprotectant from the cryoprotectant 114′, and then the cryoprotectant 114′ is poured into the first end of the container. 156 For purposes herein, fat grafts are defined as fat grafts that preserve and optimize graft viability. or maximizing the liquid in which the fat graft is dispersed (e.g., cryoprotectant or washing agent). Generate sufficient G-force for a sufficient period of time to effectively separate the fat graft from most of the fluid Centrifuge at the appropriate speed (RPM) for the appropriate time to freeze the adipose tissue. Since the containers are suspended in water, the containers are centrifuged with the first end down.

[0033] A wash solution 115, such as saline, water, phosphate buffered saline, serum-free medium, or Any suitable cleaning solution is introduced 158 into the first end of the container, capped and shaken 160, and the cleaning solution is The fluid and fat graft mixture 116 is produced. The container is centrifuged as described above (not shown). ), separating the wash solution 115' from the cells and withdrawing 162 the wash solution 115' from the container. Washing steps 158, 160, and 162 are performed to remove cryoprotectant, free lipids, and cell debris. Repeat the centrifugation as many times as necessary to obtain the desired results, for example, 2 to 6 times, for example, 3 times. If present, the lipids will be in the upper layer of the fat graft. Free lipids can be removed by decantation. The container is inverted and the washed fat graft 113' is withdrawn 164 from the container into a syringe; The solution is then ready to be injected into the patient. Optionally, the free lipids are then irrigated to the patient after removal of the washing solution 115′. The fat graft 113'' is transferred into a syringe 164 and 166 for delivery to the is removed from the explant.

[0034] Centrifugation is particularly effective and rapid, but cells or tissues may be lysed using any convenient method. For example, but not limited to, cryoprotectants or washing agents may be used to separate the liquid. The cells or tissue material mixed with the solution may be, for example, but not limited to, cells or 2. Place and hold the device in a suitable rack for a period of time sufficient to separate the tissue layer from the liquid layer. (depending on where the layer or layers of liquid are separated, the liquid is then The separation can be performed without the use of a centrifuge. When the cells or tissue is a fat graft, the fat graft is generally cryoprotectant or or the cleaning solution, in which case the denser liquid will be absorbed by the equipment. The acellular lipids, which have a lower density than the fat graft, are extracted through the second opening of the device. It can be removed from the opening by tilting.

[0035] After injecting the fat graft into the device 110 and before mixing 142 the fat graft with the cryoprotectant. The fat grafts were then incubated with PBS or saline as previously described (156, 158, 160, 162). The lipids can be washed with a suitable washing solution, such as saline, and the free lipids can be removed after centrifugation, e.g., by decanting. After thawing and washing, the fat graft can be removed by the coagulation method before use. The cells are treated with lagenase, filtered to remove structural fragments, and optionally, progenitor cells, e.g., adipose tissue. The adipose tissue is then isolated from other cellular components, including the stromal vascular fraction (SVF), which comprises adipose derived stem cells (ASCs). For example, by centrifuging to separate cells from SVF cells adhered to plastic The compound can be further processed, such as by subjecting the compound to treatment with a solvent.

[0036] In one embodiment, a kit for use in preserving a graft, such as a fat graft, is provided. The kit may be used in any of the methods described above and elsewhere herein. , boxes, molded containers or inserts, identifying contents and instructions for use of kit elements The kit comprises suitable packaging for the components thereof, including suitable labeling outlining the At the very least, the kit may comprise any of the components described herein, e.g., with respect to FIG. 1 or FIG. 2. The kit also optionally comprises two or more storage containers as described herein. The cryoprotectant described herein is added to the container of the kit in a volume that can be drawn into the container of the kit, such as adipose tissue. The method comprises the steps of: (a) providing a cryoprotectant for a graft comprising: providing a cryoprotectant for a graft; Two 10mL storage volumes for use with fat grafts drawn into 0mL syringes The device provides an amount of cryoprotectant that would be effective for cryoprotecting 20 mL of fat graft material. As described above, DMSO, human serum albumin, or trehalose, or Any suitable cryoprotectant may be included in the container, such as any effective combination of: The protectants may be contained in one multi-use container or may be individually packaged in a single container. The cryoprotectant may also be contained in a single-use container that contains only enough cryoprotectant to process the tissue. The kit also contains a suitable washing solution in an amount appropriate to the number and size of the kit's containers. These may optionally contain a cryoprotectant or a washing solution. The container may include a suitable connector, such as a Luer connector, for use with a syringe. The kit also optionally includes a method for obtaining a fat sample from a patient or a method for detecting a patient using the method described herein. and one or more sterile syringes for administering the graft described in the document. A cannula may also be included in the kit. In addition, the cannula may be used in combination with any of the devices shown and described with respect to FIG. A storage container may be included in the kit so as to optionally contain one or more of the following: : Label for storage container, label for storage container, temperature logger, or container contents In one example, the kit may be packaged in a suitable packaging. 5, 10, 15, 20, or 25 storage containers, one or more of which contain a cryoprotectant. a storage vessel comprising a container, one or more containers comprising a cleaning solution, and optionally a temperature logger The present invention relates to a method for producing a semiconductor device comprising the steps of: EXAMPLES

[0037] Example 1 To determine whether the devices described herein can withstand processing and effectively separate fat from liquid. 1, 3A, and 3C, and a 10 mL syringe was used. A 100μ stainless steel screen was placed at the first end of the device as shown. The syringe was essentially made from the distal ends of two 10 mL syringes that were then solvent welded together. Dispose of fresh human fat graft tissue retrieved from surgery using a storage container as shown in 1. Instead of cryoprotectant and washing solution, saline (0.9% saline) was used. Saline) was used. Throughout this protocol, fat grafts were centrifuged at 1200 g for 3 min. After the final wash, excess fat was removed before drawing the fat graft into the syringe for transfer to the patient. The mass was removed by decantation.

[0038] Example 2 Tissue samples Abdominal liposuction was performed on a 51-year-old female donor at the Department of Plastic Surgery, University of Pittsburgh. The study was approved by the Institutional Review Board.

[0039] Cryopreservation solution Dimethyl sulfoxide (DMSO) in PBS at concentrations of 5% and 10% (V / V) a) was used as the intracellular cryoprotectant. To achieve extracellular cryopreservation, 2% human alfa was used. Albumin (Invitrogen) alone or in the presence of 0.25 M trehalose (Sigma) The following four combinations of cryoprotectant solutions were used in combination with fresh, non-frozen fat. Comparison was made using fat aspirate. 1-10% DMSO + 2% albumin 2-10% DMSO + 2% albumin + 0.25M trehalose 3-5% DMSO + albumin 4-5% DMSO + albumin + 0.25M trehalose

[0040] Freezing and Thawing Procedure Two different freezing methods were used to freeze and store lipoaspirates to avoid intracellular ice crystal formation. Conditions were used: (1) controlled freezing to -80°C at a rate of 1°C per minute; and (2) -80 The lipoaspirate was frozen in a polystyrene foam box in a 30°C freezer. The lipoaspirate was centrifuged at 500 g for 5 minutes. The cells were washed twice with phosphate-buffered saline (PBS) (Dulbecco) by After washing, 10 mL of the lipoaspirate was placed in a 50 mL Falcon tube. The control samples were transferred to a -80°C freezer. After 48 hours, the samples were thawed in a water bath at 37°C. The thawed tissue samples were then resuspended in PBS. The cryopreservative was removed by washing twice with 50 mL of 1000 mM sodium chloride.

[0041] Digestion of lipoaspirate Fresh and thawed lipoaspirates were digested with collagenase enzyme. In this study, 10 mL of lipoaspirate was treated with 0.8 mg / mL type II collagenase (Wort hington, NJ) / 3% BSA / HBSS (Sigma) in a shaking water bath at 37°C. The digestion solution was passed through sterile gauze to remove structural fragments, and the digested tissue was then incubated at 4°C for 75 min. The upper adipocyte layer was collected in a separate tube, and the stromal vascular fraction pellet was centrifuged at 0 g for 5 min. The cells were treated with ACK red blood cell lysis buffer, passed through a 70 μm cell strainer, and centrifuged (2 00g, 5 min) and resuspended in DMEM, 10% FBS medium.

[0042] Cell viability analysis The viability of adipocytes and SVF cells was determined using the Countess cell counter (Invitrogen). rogen) according to the manufacturer's protocol.

[0043] cell culture Stromal vascular fraction cells were collected from 25 cm 2 Flasks of DMEM / 10% FCS / gentamicin medium In the ground, 10,000 cells / cm 2 The cells were incubated at 37°C in the presence of 5% CO2. The medium was changed every 2 days and the cells were counted after 8 days.

[0044] histological study Lipoaspirates were then washed with 10% formalin-buffered saline (Fisher Scientific The tissues were then fixed in 5% CO 2 for 48 h. The fixed tissues were embedded in paraffin and cut into 3–5 μm sections. Hematoxylin and eosin staining was performed to visualize the structures.

[0045] Example 3 - Cryopreservation test Methods: The lipoaspirate was washed with PBS, and the lower liquid layer and the upper fat layer were removed. The extract (10 mL) was frozen in a controlled temperature drop at −80 °C in a Styrofoam box. The frozen aspirates were thawed at 37°C after 48 hours and washed with PBS. After digestion with trypan blue staining, ASCs (Figure 4A) and adipocytes (Figure 4B) were isolated. The percentage of viable cells isolated from fresh fat was set as 100%. The percentage of viable cells after cryopreservation was calculated and plotted. Al = albumin , Th = trehalose

[0046] Example 4 - Cryopreservation test Methods: The lipoaspirate was washed with PBS, and the lower liquid layer and the upper fat layer were removed. The extract (7 mL) was flash frozen in liquid nitrogen or cooled to reduce the temperature. Slow freezing was performed in a lyobox at -20°C and then at -80°C for 4 hours. Regarding freezing conditions, the following cryopreservation agent (CPA) settings were used: 1) no CPA; 2) 2 % albumin; and 3) 2% albumin + 5% DMSO. Frozen aspirates were cultured at 3°C ​​for 48 h. The cells were thawed at 7°C and washed with PBS. After digestion with collagenase, ASC (Figure 5A) and Adipocytes (FIG. 5B) were analyzed for viability using trypan blue staining.

[0047] The following numbered paragraphs describe various non-limiting aspects of the present invention.

[0048] 1. Define a filtered inner chamber that holds the fat cells or adipose tissue. a first end and a second end, the volume between the second end and the filter being at least 3 a container having a volume of 100 mL; a luer lock or a first adapter or connector, such as a slip connector, at a second end of the container; and a second adapter, such as a luer lock or slip connector, defining a second opening. with adapter or connector; 1. A cell or tissue isolation, purification, and preservation device comprising:

[0049] 2. Centrifuging a container, such as a tube, bottle, syringe, or device The dimensions of the centrifuge must be such that it can be fitted into a centrifuge capable of carrying out the above steps, or it must have a volume in the range of 1 mL to 4 L. 13. The apparatus according to claim 1.

[0050] 3. At least 500g, e.g. at least 1000g, 1200g, or 1500 3. The device of claim 1 or 2, constructed to withstand centrifugation at G-forces of g.

[0051] 4. The internal chamber of the container has a volume in the range of 5 mL to 100 mL, for example, 5 mL, 10 mL Any one of items 1 to 3 having a volume of 1 L, 15 mL, 30 mL, or 50 mL Item 1. The device described in item 1.

[0052] 5. A container, such as a tube, bottle, or syringe, having a volume in the range of 1 mL to 4 L. The dimensions are suitable for a centrifuge capable of centrifuging containers such as cereals, etc. 13. The device according to any one of claims 1 to 12.

[0053] 6. The filter is closer to the second end of the container than to the first end of the container. Item 10. The device according to any one of items 1 to 5.

[0054] 7. The container comprises two separable members forming a continuous sealed container, These members may be positioned closer to the first end of the container than to the second end of the container, and optionally a slip or screw connector adjacent the first end of the container; The device according to any one of claims 1 to 6, wherein the device is connected to a

[0055] 8. Any of items 1 to 7, wherein the filter is a 100 micron (μ) filter. 13. The device according to claim 12 .

[0056] 9. The first adapter and / or the second adapter are Luer lock adapters. The device according to any one of items 1 to 8, which is a photodetector.

[0057] 10. The first adapter and the second adapter are Luer lock adapters. The device according to any one of items 1 to 9.

[0058] 11. The device according to any one of claims 1 to 10, having a cylindrical cross-sectional shape.

[0059] 12. A syringe having a diameter of 10 mL, as described in any one of paragraphs 1 to 11. Equipment.

[0060] 13. The container has an outer diameter in the range of 16.5 to 18 mm and a length in the range of 80 mm to 90 mm. 13. The apparatus of claim 12, comprising:

[0061] 14. The container has an internal volume between the filter and the first end of the container of 5 mL or more, e.g. 12. The device according to any one of claims 1 to 11, having a volume in the range of 8 mL to 10 mL.

[0062] 15. The container has an outer diameter in the range of 5 mm to 25 mm and a length in the range of 70 mm to 110 mm. The device according to any one of claims 1 to 11, comprising:

[0063] 16. The ratio of the barrel length to the barrel outer diameter of the container is at least 4:1, or 4:1 to 6: 16. The device according to any one of claims 1 to 15, wherein:

[0064] 17. The live cells, such as live adipocytes, and optionally a cryoprotectant are placed in the inner chamber of the container. Any of items 1 to 16, comprising: 13. The apparatus described in claim 1.

[0065] 18.2 to 25 cell or tissue storage devices (each storage device is for fat cells or fat tissue) a first end and a second end defining an interior chamber separated by a filter for holding a container having a volume between said second end and said filter of at least 3 mL; and a first opening. a first connector, such as a luer lock or slip connector, at a first end of the container defining a portion; an adapter or connector at a second end of the container defining a second opening. Includes a second adapter or connector, such as a Luer lock or slip connector. And then, At least 3 mL of fat graft or other cells or tissue can be cryopreserved. and optionally an adapter such as a luer lock or slip connector. at least one container having a feeder or connector; 13. A kit for use in isolating and preserving cells, such as fat grafts, comprising:

[0066] 19. The device has a volume ranging from 1 mL to 4 L, for example, a tube, a bottle, The dimensions are such that the container can be spun in a centrifuge, such as a syringe or device. 19. The kit according to item 18,

[0067] 20. The device weighs at least 500 g, for example at least 1000 g, 1200 g, or or 1500 g of G force, Kit included.

[0068] 21. The internal chamber of the container has a volume in the range of 5 mL to 100 mL, for example, 5 mL, 10 Any of paragraphs 18 to 20 having a volume of 1 mL, 15 mL, 30 mL, or 50 mL The kit according to any one of claims 1 to 4.

[0069] 22. A container, e.g., a tube, bottle, or syringe, having a volume ranging from 1 mL to 4 L. The dimensions of the centrifuge container, such as a centrifuge, are as described in any one of paragraphs 18 to 21. Equipment.

[0070] 23. Labels for displaying the storage container and / or storage vessel may be included. a storage container configured to store the 2 to 25 cell or tissue storage devices, 23. The kit according to any one of items 18 to 22, further comprising:

[0071] 24. A temperature logger that may be optionally attached to the storage container. 24. The kit according to any one of items 18 to 23, further comprising:

[0072] 25. The filter is disposed in front of the first end of one or more or all of the containers 2 to 25. 25. The kit according to any one of items 18 to 24, wherein the second end of the container is adjacent to the second end of the container.

[0073] 26. One or more or all of the containers 2 to 25 form a continuously sealed container. the second end of the container comprises two separable members, the members being closer to the container than the second end of the container. a slip connector proximate the first end, and optionally adjacent the first end of the container; Any one of items 18 to 25 connected by a wire or screw connector Item 3. The kit according to item 2.

[0074] 27. One or more or all of the filters in said containers 2 to 25 have a resolution of 100 microns (μ 27. The kit according to any one of items 18 to 26, wherein the kit is a filter.

[0075] 28. One or more or all of the first adapters and / or Any one of paragraphs 18 to 27, wherein the second adapter is a Luer lock adapter. The kit according to claim 1,

[0076] 29. One or more or all of the first adapters and second adapters of the containers 2 to 25. 28. The method according to any one of claims 18 to 27, wherein the adapter is a luer lock adapter. kit.

[0077] 30. A first method, wherein one or more or all of the containers 2 to 25 have a cylindrical transverse shape. 30. The kit according to any one of items 8 to 29.

[0078] 31. One or more or all of the containers 2 to 25 have a diameter of a 10 mL medical syringe. The kit according to any one of items 18 to 30,

[0079] 32. Between the filter and the first end of one or more or all of the containers 2 to 25. The internal volume of the container is 5 mL or more, for example, in the range of 8 mL to 10 mL. The kit according to any one of the preceding claims.

[0080] 33. One or more of the containers 2 to 25 have an outer diameter in the range of 5 mm to 25 mm and 7 32. The method according to any one of claims 18 to 31, having a length in the range of 0 mm to 110 mm. kit.

[0081] 34. One or more or all of the containers 2 to 25 have an outer diameter in the range of 16.5 to 18 mm. and a length in the range of 80 mm to 90 mm, Kit.

[0082] 35. One or more of the above 2 to 25, or the ratio of the total barrel length to the barrel outer diameter is at least 4 32. The kit according to any one of claims 18 to 31, wherein the ratio of the β-amino acid to the β-amino acid is 4:1 to 6:1, or 4:1 to 6:1. .

[0083] 36. The cryoprotectant is dimethyl sulfoxide (DMSO), human serum albumin, Rehalose, or a combination of two or more of the above, such as a combination of DMSO and hSA For example, a combination of 5%-10% (v / v) DMSO and 2% (w / v) hSA may be used. The kit according to any one of items 18 to 35,

[0084] 37. The adipose tissue is fed from the second opening of the device according to any one of items 1 to 16. introducing (e.g., injecting) the fat graft into the interior chamber of the device; and cryopreserving the fat graft. and injecting a cryoprotectant into the inner chamber of the device through a first opening of the device in an amount effective to mixing said adipose tissue with said cryoprotectant; and to a temperature below 0°C.

[0085] 38. The device has a volume ranging from 1 mL to 4 L, e.g., a tube, bottle, syringe The container is sized to fit into a centrifuge capable of centrifuging a container such as a syringe or device. 38. The method according to claim 37.

[0086] 39. The device weighs at least 500 g, for example at least 1000 g, 1200 g, or or 38, which is designed to withstand centrifugation at a G force of 1,500 g. Method of posting.

[0087] 40. After cooling the fat graft, increase the temperature of the fat graft to 38. The method of claim 37, further comprising thawing.

[0088] 41. The temperature change of the fat graft during freezing or thawing is less than 2°C per minute, or 1°C per minute 41. The method according to any one of items 37 to 40, wherein:

[0089] 42. Separating the fat graft from the cryoprotectant; from the first end of the fat graft; and into a first end of the device, mixing the washing solution with the fat graft, and from the cleaning solution and withdrawing the cleaning solution from a first end of the device, 42. Any one of items 37 to 41, further comprising washing the fat graft. The method described above.

[0090] 43. The device containing the thawed fat graft is centrifuged with the first opening facing down. and separating the fat graft from the cryoprotectant; and removing the cryoprotectant from the apparatus. from the first end; and providing an effective amount of cleaning solution to cleanse the fat graft. a first end of the device, mixing the washing solution with the fat graft, and The device containing the fat graft is centrifuged with the first opening facing down to remove the fat graft from the washing solution. and withdrawing the washing solution from the first end of the device. 42. The method according to any one of items 37 to 41, further comprising washing the

[0091] 44. The method of paragraph 42 or paragraph 43, wherein the fat graft is washed 2 to 6 times.

[0092] 45. The wash solution is phosphate buffered saline or saline (e.g., 0.9% w / v physiological saline).

[0093] 46. ​​The cryoprotectant is dimethyl sulfoxide (DMSO), human serum albumin (hSA), SA), trehalose, or a combination of any of the above, such as a combination of DMSO and hSA. For example, the combination of 5% to 10% (v / v) DMSO and 2% (w / v) hSA is not included. The method according to any one of items 37 to 45,

[0094] 47. The adipose tissue is passed through the second opening of the device according to any one of items 1 to 15. introducing (e.g., injecting) the fat graft into the interior chamber of the device; introducing a cleaning solution into the interior chamber of the device through a first opening of the device in an amount effective to mixing said adipose tissue with said washing solution; and and centrifuging the container with the first opening facing down to separate the fat graft from the washing solution. and withdrawing the cleaning solution from the first end of the device. Method for preparing the compound.

[0095] 48. The adipose tissue is fed from the second opening of the device according to any one of items 1 to 15. introducing (e.g., injecting) the fat graft into the interior chamber of the device; or an effective amount of a cryoprotectant to cryopreserve said fat graft. introducing an agent into an interior chamber of the device through a first opening of the device; and mixing the adipose tissue with said washing solution or said cryoprotectant; and If the fat graft is to be removed, the device containing the fat graft is centrifuged with the first opening facing down. Separating the fat graft from the washing solution and draining the washing solution from the first end of the device. or, if a cryoprotectant is introduced, the fat graft is cooled to a temperature below 0° C. cooling said fat graft.

[0096] 49. A wash solution is introduced into the device after the fat graft is introduced into the device; The washing solution and the fat graft are mixed; and the device containing the fat graft is The fat graft is separated from the washing solution by centrifugation, opening down; 49. The method of claim 48, wherein liquid is withdrawn from a first end of the device.

[0097] 50. The cryoprotectant is introduced into the device after the fat graft is introduced into the device. the cryoprotectant and the fat graft are mixed and the fat graft is cooled to a temperature below 0° C. 49. The method according to claim 48,

[0098] These embodiments are described with reference to various examples. Upon reading and understanding the above examples, Modifications and variations will readily occur to others. Therefore, the above examples should not be construed as limiting the present disclosure. should not be interpreted as such.

Claims

1. 1. A method for the preservation and isolation of adipose tissue, comprising: introducing adipose tissue into an interior chamber of the device; wherein the device comprises: a container having a first end and a second end defining an interior chamber configured to hold fat cells or adipose tissue, the container having a continuous annular outer surface formed by the two separable members sized to fit into a centrifuge configured to centrifuge the container, the two separable members being connected by a fitting portion that is a slip connector or a screw connector located closer to the second end of the container than to the first end of the container; a first adapter or connector defining a first opening; a second adapter or connector defining a second opening; and Including, The two separable members of the container are a second member that connects with or is integral with the second adapter or connector, the second member including a second barrel portion having a closed end, an opposing opening, a sidewall having an interior surface extending between the closed end and the opening, and the fitting portion extending from the opening of the second barrel portion; a first member that interfaces with or is integral with a first adapter or connector, the first member including a first barrel portion including a closed end, an opposing opening, and a sidewall having an interior surface extending between the closed end and the opening; Including, said fitting portion of said second member is inserted into the first barrel portion of said first member and engages an inner surface of the first barrel portion, thereby forming a continuous, sealed container having an interior chamber defined by the inner surfaces of the first barrel portion and the second barrel portion; introducing a cryoprotectant into an interior chamber of the container through a first adapter or connector of the device in an amount effective to cryopreserve the adipose tissue; mixing the adipose tissue with the cryoprotectant; and cooling said adipose tissue to a temperature below 0° C. The method comprising:

2. The method of claim 1 , wherein the adipose tissue is introduced into the internal chamber of the device through the second adaptor or connector.

3. cooling the adipose tissue and then increasing the temperature of the adipose tissue to thaw the adipose tissue; and Centrifuging the device containing the thawed adipose tissue to separate the adipose tissue from the cryoprotectant. The method of claim 1 or 2, further comprising:

4. 4. The method of claim 3, wherein centrifuging the device comprises vertically inserting the device into a centrifuge with the second member of the container adjacent to an upper portion of the centrifuge and the first member of the container adjacent to a lower portion of the centrifuge.

5. 5. The method of claim 4, wherein the device further comprises a filter disposed in the first barrel portion of the first member and configured to retain fat cells or adipose tissue adjacent to a bottom of the centrifuge when the device is inserted into the centrifuge, the filter having a diameter that matches an inner diameter of the first barrel portion.

6. After centrifugation, withdrawing a cryoprotectant from an interior chamber of the container through a filter disposed in the first barrel portion and through a first adapter or connector of the device; and The adipose tissue held in the inner chamber introducing a washing solution into a first adapter or connector of the device in an amount effective to wash the adipose tissue; mixing said washing solution with said adipose tissue within an internal chamber of said device; Centrifuging the device containing the adipose tissue to separate the adipose tissue from the washing solution; and drawing said cleaning fluid through a filter disposed in said first barrel portion and out of a first adapter or connector of said device. To be washed by The method of claim 3 further comprising:

7. The method of claim 6, wherein the adipose tissue is washed 2 to 6 times.

8. The method of claim 6, wherein the washing solution is phosphate buffered saline or saline (e.g., 0.9% w / v saline).

9. 3. The method of claim 1 or 2, wherein the cryoprotectant comprises 5%-10% (v / v) dimethylsulfoxide (DMSO) and 2% (w / v) human serum albumin (hSA).

10. 3. The method of claim 1 or 2, wherein the cryoprotectant comprises 5% (v / v) dimethylsulfoxide (DMSO) and 2% (w / v) human serum albumin (hSA).

11. 11. The method of claim 9 or 10, wherein cooling the adipose tissue comprises changing the temperature of the adipose tissue at 2°C or less per minute.

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