Methods and devices for processing tissues and cells

Abstract

An apparatus and method for processing a sample is provided, in which one or more microfluidic chips mounted on a support plate are rotated. Each microfluidic chip can be rotated 180 degrees on the support plate, allowing sample to flow bidirectionally through each microfluidic chip. Each microfluidic chip may include a fluid path including multiple expansion and compression regions with increasing radii in three or more dimensions.

Description

[Technical Field]

[0001] [Incorporation by reference to priority application] All applications for which a foreign or domestic priority claim is identified in the Application Data Sheet filed with this application are hereby incorporated by reference pursuant to 37 CFR 1.57.

[0002] [Statement Regarding Federally Sponsored Research or Development] This invention was made with U.S. Government support under Contract No. 1R43DK116389-01 awarded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH). The U.S. Government may have certain rights in this invention.

[0003] [Field of Disclosure] This application generally relates to devices and methods for applying shear stress to living cells, tissues, reagents, particles, and fluids, and the use of the resulting cells in the treatment of damaged tissue. In particular, the technical field relates to utilizing rotational forces in conjunction with microfluidic-based devices to apply shear stress to living cells and / or tissues, reagents, particles, and fluids. In some embodiments, the methods and devices described herein can be used to treat wounds such as diabetic ulcers, venous stasis ulcers, arterial ulcers, and pressure ulcers (i.e., bed sores). The methods and devices described herein can be used in treatments associated with surgical specialties such as orthopedic surgery, arthroscopic surgery, neurosurgery, gastrointestinal and related organ surgery, urological surgery, general surgery, gynecological surgery, thoracic surgery, laparoscopic surgery, and plastic and reconstructive surgery when transplantation of processed and harvested adipose tissue is desired. [Background technology]

[0004] Diabetic foot ulcers (DFUs) are a serious complication of diabetes mellitus (DM) and the leading cause of non-traumatic lower limb amputations. Lower limb amputations in diabetes occur at a 2:1 ratio in men compared with women. The lifetime incidence of DFU formation in individuals with DM may be as high as 25%. In 2013, it was estimated that 384 million people suffered from DM, and this number is expected to increase to 592 million by 2035. The etiology of DFUs is multifactorial and primarily involves a combination of neuropathy, reduced vascularity, and isolated or repetitive trauma. Once DFUs form, the wound microenvironment is characterized by poor healing due to ongoing pressure and / or trauma, chronic infection, and displacement of the plantar fat pad. Current methods of DFU treatment are associated with cost and unpredictable outcomes. However, emerging evidence indicates that autologous stem cell therapy may be a safe and effective alternative to current treatment options.

[0005] Various techniques and procedures can be used to process tissue. In some applications, chemicals or enzymes are added to tissue to break down large chunks or aggregates of tissue into increasingly smaller fragments. For example, digestive enzymes such as collagenase, trypsin, or dispase are used to digest tissues such as adipose tissue. Such enzymatic treatments typically involve washing, followed by enzymatic digestion and centrifugation. This enzymatic approach can be variably characterized by different activity levels of the digestive enzymes. Furthermore, these methods require additional costs for reagents, including expensive enzymes derived from bacteria, and can take a significant amount of time to complete. Similarly, the methods may require additional processing and / or washing steps to minimize the effects of enzyme contamination.

[0006] Non-enzymatic approaches for processing tissues, including adipose tissue, have also been developed. For example, ultrasonic cavitation has been proposed to separate the stromal vascular fraction from adipose tissue. (See U.S. Patent No. 8,440,440, which is incorporated herein by reference in its entirety.) Yet another method involves using beads to homogenize adipose tissue, as disclosed, for example, in International Publication No. WO 2014 / 036094. (U.S. Patent No. 9,580,678, which is incorporated herein by reference in its entirety.) Patent Document 3 (U.S. Patent No. 9,580,678) (which is incorporated herein by reference in its entirety) discloses a microfluidic tumor isolation device used to destroy tumor tissue, which utilizes multiple sequentially arranged channels or stages with expansion and contraction regions. A syringe pump is used to move tumor tissue back and forth through the microfluidic device.

[0007] The processing of tissues such as adipose tissue is particularly important in the fields of plastic and reconstructive surgery, where adipose tissue is transferred from one location to another (i.e., fat grafting) to fill soft tissue defects. Cell-assisted fat grafting (CAL) is a technique that adds stromal vascular fraction (SVF) to fat grafts, significantly improving the retention of fat grafts. SVF is typically harvested from adipose tissue through a short digestion step using collagenase enzymes. Recently, a technique called "nanofat grafting" has been developed. This involves homogenizing standard lipoaspirate by manually passing it between two connected syringes, and then reinjecting the homogenized lipoaspirate into human patients for the correction of superficial wrinkles and pigmentation. Nanofat processing methods also serve as a means of mechanically isolating SVF while also stressing cells to generate multipotent or multipotent populations. For example, nanoadipose-derived SVF is known to contain a high percentage of mesenchymal stem cells (MSCs), adipose-derived stem cells (ADSCs), endothelial progenitor cells (EPCs), and Muse cells. It was hypothesized that the amount of stress applied to cells directly correlates with stem-like properties.

[0008] For example, MSCs can be used to treat diabetic ulcers. Current treatments for diabetic foot ulcers, such as allografts, are costly and may be ineffective due to potential rejection by the patient. If such ulcers are left untreated, patients must undergo limb amputation, which leads to further health complications. One innovative solution to treating these ulcers is to use MSCs for the direct treatment of these ulcers. However, current approaches to obtaining such cells are time-consuming, complicated, and produce variable results in terms of cell yield, quantity, and reproducibility. A rapid and cost-effective method for obtaining processed tissue is needed. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 8,440,440 [Patent Document 2] International Publication No. 2014 / 036094 [Patent Document 3] U.S. Patent No. 9,580,678 [Patent Document 4] International Publication No. 2017 / 214323 Summary of the Invention

[0010] In view of the need for devices, systems, and methods for efficiently, effectively, and reproducibly processing tissue, various embodiments of such devices, methods, systems, and their use in the development, production, or preparation of cells for treatment or administration to a subject are provided herein. Various details regarding the devices for generating activated stem cells disclosed herein can be found in International Publication No. WO 2017 / 214323, which is incorporated herein by reference in its entirety.

[0011] In some embodiments, a system for processing biological samples is provided, including a support plate. The support plate includes a central portion configured to reversibly interact with a motor to provide a rotational force and at least one lateral arm, each of the at least one lateral arm including a first opening and at least one fixed opening. The system also includes at least one carriage assembly configured to reversibly interact with one of the at least one lateral arm. The at least one carriage assembly of the system includes a base portion, a spring platform, and a fixed element. The base portion of the at least one carriage assembly includes an upper surface including at least one circular groove, a bottom surface including a post configured to extend through the first opening in one of the at least one lateral arm, a first arm, and a second arm. The spring platform includes at least one circular groove disposed on the bottom surface of the spring platform, a first engagement portion, and a second engagement portion. The spring platform is disposed adjacent to the upper surface of the base portion. The securing element is configured to secure a tip assembly on the at least one carriage assembly.

[0012] The at least one carriage assembly may include at least one spring held between the base and the spring platform, the at least one spring having a first end secured within the at least one circular groove in the base and an upper end secured within the at least one circular groove in the spring platform, the at least one spring configured to provide an upward force to the spring platform. Each of the plurality of carriage assemblies may be rotatable at least approximately 180 degrees.

[0013] In another embodiment, the spring platform may be configured to move between a first position and a second position in response to insertion or removal of the chip assembly. In another embodiment, the spring platform may further include a tab, and the fixation element further includes a channel configured to receive the tab of the spring platform, the tab configured to move within the channel of the fixation element as the spring platform moves between the first position and the second position. In another embodiment, the fixation element includes at least one guide rail configured to guide and position the chip assembly. In another embodiment, the first engagement portion of the spring platform is configured to hold the first arm of the base, and the second engagement portion of the spring platform is configured to hold the second arm of the base. In another embodiment, the spring platform further includes a platform configured to guide the chip assembly to the at least one carriage assembly. In another embodiment, the spring platform includes a splash guard on the bottom surface of the spring platform, the splash guard configured to prevent fluid from being introduced into the at least one spring.

[0014] In another embodiment, the chip assembly includes a microfluidic chip, a first sample chamber, and a second sample chamber, the microfluidic chip having a fluid pathway extending from a first end to a second end of the microfluidic chip, the first sample chamber being fluidly connected to the first end of the microfluidic chip, and the second sample chamber being fluidly connected to the second end of the microfluidic chip. In another embodiment, the fluid pathway includes at least one expansion region and multiple compression regions. In another embodiment, the at least one expansion region increases in radius along first, second, and third axes, each perpendicular to a central axis of the fluid pathway, and at least one compression region has a diameter smaller than that of the at least one expansion region, and at least one compression region does not change in diameter. In another embodiment, the first, second, and third axes are perpendicular to each other. In another embodiment, at least one expansion region increases in three or more dimensions (increases in more than two dimensions). In other embodiments, the fluid pathway includes multiple teardrop-shaped expansion regions. In other embodiments, the fluid pathway includes spherical or elliptical expansion regions. In other embodiments, the fluid pathway includes multiple semi-teardrop-shaped expansion regions. In other embodiments, the fluid pathway includes hemispherical or semi-elliptical expansion regions. In other embodiments, the fluid pathway includes a D-shaped expansion region. In other embodiments, the fluid pathway includes an hourglass-shaped portion. In other embodiments, the fluid pathway includes at least two expansion regions and at least one compression region disposed between the at least two expansion regions. In other embodiments, the fluid pathway includes at least three expansion regions and at least two compression regions, each of the at least two compression regions disposed between adjacent expansion regions.

[0015] In other embodiments, a filter is disposed between the microfluidic chip and the first sample chamber and / or between the microfluidic chip and the second sample chamber. In other embodiments, the microfluidic chip of any of the systems disclosed herein comprises luer locks at a first end and a second end of the microfluidic chip.

[0016] In some embodiments, a system for processing biological samples is provided, comprising a support plate including a central portion configured to reversibly interact with a motor to provide a rotational force and at least one lateral arm, each of the at least one lateral arm having an interaction region. The system may also include at least one carriage assembly configured to reversibly interact with one of the at least one lateral arm. In some embodiments, each of the at least one carriage assembly comprises a base portion, a spring platform disposed on the base portion, at least one spring held between the base portion and the spring platform and configured to provide an upward force to the spring platform, and a fixation element. The system may also include at least one chip assembly having a microfluidic chip, the microfluidic chip having a fluid path extending from a first end to a second end of the microfluidic chip. The fluid path may include at least one expansion region and at least one compression region. The at least one expansion region may have an increasing diameter along a first axis, a second axis, and a third axis, each axis being perpendicular to a central axis of the fluid path. The at least one compression region may have a diameter smaller than that of the at least one expansion region, and the at least one compression region may have a constant diameter. The chip assembly may include a first sample chamber fluidly connected to a first end of the microfluidic chip and a second sample chamber fluidly connected to a second end of the microfluidic chip. The at least one chip assembly may be received within the at least one carriage assembly, each of the at least one carriage assembly being rotatable at least approximately 180 degrees.

[0017] In other embodiments, the first axis, the second axis, and the third axis are perpendicular to one another. In other embodiments, at least one expansion region increases in three or more dimensions (increases in more than two dimensions). In other embodiments, the fluid path includes multiple teardrop-shaped expansion regions. In other embodiments, the fluid path includes spherical or elliptical expansion regions. In other embodiments, the fluid path includes multiple semi-teardrop-shaped expansion regions. In other embodiments, the fluid path includes hemispherical or semi-elliptical expansion regions. In other embodiments, the fluid path includes a D-shaped expansion region. In other embodiments, the fluid path includes an hourglass shape. In other embodiments, the fluid path includes at least two expansion regions and at least one compression region disposed between the at least two expansion regions. In other embodiments, the fluid path includes at least three expansion regions and at least two compression regions, each of the at least two compression regions disposed between adjacent expansion regions. In other embodiments, a filter is disposed between the microfluidic chip and the first sample chamber and / or between the microfluidic chip and the second sample chamber. In other embodiments, the microfluidic chip comprises luer locks at a first end and a second end of the microfluidic chip.

[0018] In some embodiments, a system for processing biological samples is provided. The system may include a support plate having a central portion configured to reversibly interact with a motor to provide a rotational force and a plurality of lateral arms, each of the lateral arms including an interaction region. The support plate may include a plurality of holding arms, each of the plurality of holding arms disposed between each of the plurality of lateral arms, each of the plurality of holding arms configured to hold a syringe. The system may include at least one carriage assembly configured to reversibly interact with one of the at least one lateral arms, and the at least one tip assembly received within the carriage assembly. In some embodiments, each of the at least one carriage assembly is rotatable at least approximately 180 degrees.

[0019] In some embodiments, a method of treating damaged tissue is disclosed, comprising administering to a subject having damaged or diseased tissue a population of activated adipose stem cells at a concentration of about 1 x 10 per gram. 4 ~1×10 10 The activated adipose stem cells may result in one or more of the following: upregulation of a regenerative phenotype (e.g., CD34, CD13, CD73, or CD146), reduced inflammation, rapid tissue proliferation, rapid tissue remodeling, increased angiogenesis, or a combination thereof.

[0020] In another embodiment, the administering of the method is by subcutaneous injection. In another embodiment, the administering of the method is by intravenous injection. In another embodiment, the damaged tissue is the result of an ulcer. In another embodiment, the ulcer is selected from the group consisting of a diabetic foot ulcer, a bedsore (i.e., pressure sore), a venous stasis ulcer, and an arterial ulcer. In another embodiment, the damaged tissue is the result of a wound. In another embodiment, the wound is a burn-related wound, an abrasion (e.g., road rash), a laceration (e.g., a knife wound), a puncture wound, or an abrasion (e.g., a bullet wound or wound from another weapon). In another embodiment, the administering results in the damaged tissue healing time being reduced by at least 10% compared to damaged tissue not exposed to the activated adipose stem cells. In another embodiment, the administering results in the damaged tissue healing time being reduced by at least 50% compared to damaged tissue not exposed to the activated adipose stem cells.

[0021] In some embodiments, a method of treating damaged tissue is disclosed, which may comprise administering to a subject having damaged or diseased tissue a population of activated adipose stem cells in an amount sufficient to increase vascularization at the site of the injury.

[0022] In some embodiments, there is provided a use of activated adipose stem cells for treating damaged tissue, wherein the activated adipose stem cell population is about 1 x 10 per gram of a subject having damaged tissue. 4 ~1×10 10 The activated adipose stem cells are provided in an amount in the range of 100 mg / kg / day, and exposing the damaged tissue to the activated adipose stem cells reduces the time it takes for the damaged tissue to heal by at least 10% compared to damaged tissue not exposed to the activated adipose stem cells.

[0023] In another embodiment, the activated adipose stem cells result in one or more of the following: upregulation of a regenerative phenotype (e.g., CD34, CD13, CD73, or CD146), reduced inflammation, rapid tissue proliferation, rapid tissue remodeling, increased angiogenesis, or a combination thereof. In another embodiment, administration reduces the time it takes for the damaged tissue to heal by at least 50% compared to damaged tissue not exposed to the activated adipose stem cells.

[0024] In some embodiments, a method for activating adipose stem cells for use in tissue repair is disclosed. The method may include extracting a sample of adipose tissue from a patient in a volume sufficient to generate a sufficient amount of mechanically treated adipose-derived stem cells. The method may include inserting the adipose tissue sample into a first sample chamber located at one end of a microfluidic chip. The method may include rotating the microfluidic chip using a motor-driven rotating chuck to expose the adipose tissue sample to shear forces. While the microfluidic chip is rotating, the adipose tissue sample travels back and forth along multiple microfluidic channels from a first sample chamber to a second sample chamber located at the opposite end of the microfluidic chip. The method may include removing the mechanically treated adipose-derived stem cells from the microfluidic chip. The method may include administering the mechanically treated adipose-derived stem cells to a site of injury in a patient. [Brief explanation of the drawings]

[0025] These and other features, aspects, and advantages of the present disclosure will be described with reference to drawings of specific embodiments, which are intended to illustrate specific embodiments in a simplified manner and are not intended to limit the disclosure. [Figure 1] 1 illustrates one embodiment of a system for processing a biological sample. [Figure 1A]1 shows an exploded view of one embodiment of a system for processing a biological sample. [Figure 1B] 2 shows an exploded view of the same embodiment. [Figure 2A] 1 shows an exploded view of another embodiment of a system for processing a biological sample. [Figure 2B] 2 shows an exploded view of the same embodiment. [Figure 3A] A diagram of the carriage assembly is shown. [Figure 3B] 1 shows a diagram of the carriage assembly. [Figure 4A] FIG. 1 illustrates one embodiment of a chip assembly. [Figure 4B] FIG. [Figure 5A] 4A-4B illustrate an embodiment of a microfluidic chip configured to be secured to the chip assembly of FIGS. 4A-4B. [Figure 5B] This embodiment is shown. [Figure 5C] This embodiment is shown. [Figure 6A] 5A-5C are enlarged views of the microfluidic chip of FIGS. 5A-5C configured to be secured to the chip assembly of FIGS. 4A-4B. [Figure 6B] An enlarged view of the microfluidic chip is shown. [Figure 6C] An enlarged view of the microfluidic chip is shown. [Figure 6D] An enlarged view of the microfluidic chip is shown. [Figure 7A] 4A-4B illustrate another embodiment of a microfluidic chip configured to be secured to the chip assembly of FIGS. 4A-4B. [Figure 7B] This embodiment is shown. [Figure 7C] This embodiment is shown. [Figure 8A] 4A-4B illustrate another embodiment of a microfluidic chip configured to be secured to the chip assembly of FIGS. 4A-4B. [Figure 8B] This embodiment is shown. [Figure 9]4A-4B illustrate another embodiment of a microfluidic chip configured to be secured to the chip assembly of FIGS. 4A-4B. [Figure 10] 4A-4B illustrate another embodiment of a microfluidic chip configured to be secured to the chip assembly of FIGS. 4A-4B. [Figure 11A] 1 illustrates one embodiment of a three-dimensional channel in a microfluidic chip with expansion and compression regions. [Figure 11B] This embodiment is shown. [Figure 12A] 10 illustrates another embodiment of a three-dimensional channel in a microfluidic chip having expansion and compression regions. [Figure 12B] This embodiment is shown. [Figure 13A] 10 illustrates another embodiment of a three-dimensional channel in a microfluidic chip having expansion and compression regions. [Figure 13B] This embodiment is shown. [Figure 14A] FIG. 1 illustrates one embodiment of a sample chamber configured to be fixed to a microfluidic chip. [Figure 14B] FIG. [Figure 14C] FIG. [Figure 14D] FIG. [Figure 14E] FIG. [Figure 14F] FIG. [Figure 15A] FIG. 1 illustrates an embodiment of a filter. [Figure 15B] FIG. [Figure 16A] 15A-15B configured to be secured to a chip assembly. FIG. [Figure 16B] FIG. [Figure 17A] 3A-3B illustrate one embodiment of a base platform of the carriage assembly. [Figure 17B] This embodiment is shown. [Figure 18A] 3A-3B illustrate one embodiment of a spring platform for the carriage assembly of FIGS. 3A-3B. [Figure 18B] This embodiment is shown. [Figure 19A] 3C illustrates one embodiment of a locking mechanism for the carriage assembly of FIGS. 3A-3B. [Figure 19B] This embodiment is shown. [Figure 20A] 1 illustrates an embodiment of a screw cap. [Figure 20B] FIG. [Figure 21A] 1 illustrates one embodiment of a rotor foot insert. [Figure 21B] FIG. [Figure 22] FIG. 1 illustrates an embodiment of a base portion of a system for treating a biological system. [Figure 23A] FIG. 1 illustrates another embodiment of a system for treating a biological system. [Figure 23B] FIG. [Figure 24] FIG. 1 illustrates another embodiment of a system for treating a biological system. [Figure 25A] FIG. 1 illustrates an embodiment of a system for treating a biological system including an embodiment of a motor. [Figure 25B] FIG. [Figure 26A] FIG. 10 illustrates another embodiment of a system for treating a biological system including another embodiment of a motor. [Figure 26B] FIG. [Figure 27A] FIG. 1 illustrates one embodiment of a system for processing a biological system having a structure for holding multiple syringes. [Figure 27B] FIG. [Figure 27C] FIG. [Figure 27D]FIG. [Figure 27E] 27A-27D show the tip of a structure for holding multiple syringes in the embodiment of the system for treating a biological system of FIGS. 27A-27D. [Figure 27F] The tip of the same structure is shown. [Figure 28A] 1 shows a flow chart of a method for processing biological material. [Figure 29] FIG. 1 is a schematic diagram of one non-limiting embodiment of a treatment paradigm. [Figure 30A]

[0023] Figure 1 relates to phenotypic data of cells obtained from adipose tissue using the systems and methods disclosed herein, showing the extent of CD26+ / CD55+ cells derived from adipose samples from healthy and diabetic patients. [Figure 30B]

[0023] Figure 1 relates to phenotypic data of cells obtained from adipose tissue using the systems and methods disclosed herein, showing the extent of CD34+ cells derived from adipose samples from healthy and diabetic patients. [Figure 31A] 10 relates to cell count and viability data for cells obtained from adipose samples using the systems and methods disclosed herein, showing cell densities obtained from processing adipose samples from healthy and diabetic patients. [Figure 31B] 10 relates to cell count and viability data for cells obtained from adipose samples using the systems and methods disclosed herein, showing cell viability obtained from processing adipose samples from healthy and diabetic patients. [Figure 32A] FIG. 10 shows the percentage of endothelial progenitor cells (EPCs) in cell populations derived from processing adipose samples from healthy and diabetic patients, with respect to the types of cells present after processing adipose samples using the systems and methods disclosed herein. [Figure 32B]FIG. 10 depicts the types of cells present after processing adipose samples using the systems and methods disclosed herein, showing the percentage of mesenchymal stem cells (MSCs) in cell populations derived from processing adipose samples from healthy and diabetic patients. [Figure 32C] This relates to the types of cells present after processing adipose samples using the systems and methods disclosed herein, and shows the percentage of Muse cells in cell populations derived from processing adipose samples from healthy and diabetic patients. [Figure 33A] The present invention relates to the characteristics of cells and cell types after processing adipose samples using the systems and methods disclosed herein, and shows the cell viability of cells derived from adipose samples under the conditions indicated. [Figure 33B] 10A-10C relate to the characteristics of cells and cell types after processing adipose samples using the systems and methods disclosed herein, showing the cell counts of cells derived from adipose samples under the indicated conditions. [Figure 33C] 10A-10C relate to the characterization of cells and cell types after processing adipose samples using the systems and methods disclosed herein, showing subpopulations of cell types derived from adipose samples under the indicated conditions. [Figure 34A] Data related to wound healing using cells obtained by processing adipose samples using the systems and methods disclosed herein is presented, particularly histological data regarding the presence of specific markers and tissue regeneration. [Figure 34B] 1 shows data related to wound healing using cells obtained by processing adipose samples using the systems and methods disclosed herein, and in particular, shows photographs of wound healing progression over time in a mouse injury model. [Figure 34C] Data related to wound healing using cells obtained by processing adipose samples using the systems and methods disclosed herein are presented, and in particular, summary data on wound healing for the indicated groups is provided. DETAILED DESCRIPTION OF THE INVENTION

[0026] Although some examples are described below, those skilled in the art will understand that the disclosure extends beyond the specifically disclosed examples and / or uses, as well as obvious modifications and equivalents thereof. Accordingly, it is not intended that the scope of the disclosure disclosed herein should be limited by the specific examples described below.

[0027] In some embodiments, a system for processing biological samples comprises a support plate and a plurality of carriages, the support plate having a central portion including a receiving element and side portions including a plurality of interaction areas, each interaction area configured to reversibly interact with a plurality of carriages, each of the plurality of carriages configured to be operably coupled to a side portion of the support plate.

[0028] In some embodiments, the receiving element is configured to reversibly interact with a drive shaft of a motor configured to impart centrifugal motion to a support plate, hi one embodiment, the central portion lies in a plane perpendicular to an axis of rotation of the drive shaft of the motor, and the lateral portions extend radially from the central portion and lie in planes at least partially parallel to the plane of the central portion.

[0029] In some embodiments, each of the plurality of carriages comprises a first end, a second end, a base extending between the first and second ends, and a receiving area configured to reversibly interact with the microfluidic chip. The microfluidic chip is fluidly coupled to at least one sample chamber configured to receive a sample for processing. In some embodiments, each of the plurality of carriages comprises a post, rod, shaft, or other extension extending substantially perpendicularly from the base and configured to interact with (e.g., connect, attach, or otherwise) one of the plurality of interaction areas of the side portion. In some embodiments, each of the plurality of carriages is coaxially disposed about one of a plurality of axes, each axis extending substantially parallel to the axis of rotation of the motor drive shaft during operation, and each of the plurality of carriages is at least intermittently rotatable about one of the plurality of axes. Depending on the embodiment, the carriages can rotate to various angles. For example, in some embodiments, the carriages are induced to rotate through an arc of approximately 180 degrees.

[0030] In some embodiments, the system further includes at least one microfluidic chip responsible for holding and processing samples according to the system. In some embodiments, each microfluidic chip includes a central body portion located between a first end and a second end and at least one microfluidic channel extending between the first end and the second end, the at least one channel having various dimensions and configured to allow passage of a sample from the first end to the second end. In some embodiments, each of the first and second ends is configured to fluidly interact with the sample chamber. For ease of use, each microfluidic chip is sized to fit within a corresponding receiving area of ​​a corresponding carriage. In some embodiments, each microfluidic chip is reversibly fluidically coupled to a sample chamber at each of the first and second ends.

[0031] Optionally, in some embodiments, the sample chamber comprises a vent and a vent channel fluidically connected to the interior of the sample chamber, hi some embodiments, each sample chamber is reversibly fluidically coupled to the microfluidic chip via an adapter.

[0032] In some embodiments, each carriage includes a capture element at its first and second ends, configured to communicate with a release element on a side of the support plate, allowing the release element to intermittently rotate each of the carriages. In other words, the capture element serves to hold the carriage in a desired position until there is a signal (or force, or lack thereof) that allows the capture element to disengage or otherwise stop the release element. The carriage is then allowed to rotate, followed by re-engagement of the capture element to stop the carriage's movement (which, in some embodiments, allows the carriage to rotate through a 180-degree arc at a desired time in a tissue processing protocol). In some embodiments, the capture element includes a magnet of one polarity, and the release element includes a magnet of the opposite polarity.

[0033] In some embodiments, the side portions of the support plate include a disk and a plurality of interaction areas are circumferentially spaced about the disk. In such embodiments, the side portions and the central portion are of unitary construction, although in other embodiments, the support plate may be comprised of multiple pieces that are connected or joined together prior to use.

[0034] In some embodiments, the side portions of the support plate include multiple arms, each arm including a corresponding interaction area. In one embodiment, the arms and the central portion are a unitary structure. In another embodiment, the arms and the central portion are separate structures that are coupled to each other. In some embodiments, the arms are hinged to the central portion. In such embodiments, the hinge allows the arms to move in a plane of an axis substantially parallel to the axis of rotation of the motor's drive shaft during operation. In some embodiments, this allows for a gentle start and stop process so that the sudden application or removal of centrifugal force does not disrupt the cell / tissue sample.

[0035] In some embodiments, the interaction area of ​​the side portion comprises a through-hole that receives a post (or other structure) from the corresponding carriage. In some embodiments, the receiving area is located on the top surface of the base portion of the carriage. In some embodiments, the post extends from the bottom surface of the base portion of the carriage. In such embodiments, the post (or other structure) extends from the bottom of the carriage, passes through a hole (receiving area) in the side portion (e.g., arm), and is fixed (to allow rotation relative to the side portion) by, for example, a nut, pin, clamp, or other mechanism. In some embodiments, the intermittent rotation of each carriage is achieved by interaction of a gear located on the side portion with a fixed tooth that induces rotation of each carriage.

[0036] In some embodiments, the side portion includes at least three arms, each of the three arms including an interaction area configured to interact with one of at least three carriages including first and second ends, each of the carriages configured to reversibly interact with one of at least three microfluidic chips, each microfluidic chip including a first end, a second end, and a body therebetween, each end of the microfluidic chip being fluidly coupled to a sample chamber, the body of the microfluidic chip including a plurality of microfluidic paths extending between the first end and the second end, the carriage configured to intermittently rotate between a first position and a second position, the first position being a position where the first end is positioned at a first location a first distance from a receiving element in the central portion, and the second position being a position where the first end is positioned at a second location a second distance from a receiving area in the central portion, the first distance being greater than the second distance.

[0037] Depending on the embodiment, the system may optionally include a housing, which isolates the system from the external environment.

[0038] Depending on the embodiment, the system may further comprise a motor operatively connected to the drive shaft, hi some embodiments, the motor is controlled by a controller unit that allows control of the rotational speed of the motor, the controller unit comprising an interface that allows a user to program (or select from pre-programmed) protocols for processing tissue.

[0039] Also provided herein are methods for processing biological samples. For example, in some embodiments, the method includes loading a biological sample into a first sample chamber configured to be fluidly coupled to a microfluidic chip. The microfluidic chip has a central body portion located between a first end and a second end. The first end is configured to be fluidly coupled to the first sample chamber, and the second end is configured to be fluidly coupled to the second sample chamber, with at least one microfluidic channel extending between the first end and the second end. The at least one channel includes various dimensions and is configured to allow passage of a sample from the first end to the second end. The method includes reversibly coupling the microfluidic chip to a receiving area of ​​one of a plurality of carriages that are part of a centrifuge device. The centrifuge device includes a support plate having a central portion and side portions. The side portions of the centrifuge device extend radially from the central portion and lie in planes parallel to the plane of the central portion. Each carriage is operably coupled to a side portion of the support plate and includes a first end, a second end, and a base extending between the first and second ends. The carriage bases include receiving areas, and each carriage is configured to be rotatable about an axis substantially perpendicular to the plane of the central portion. The carriages are rotatable from a first position to a second position. In the first position, the first end is located a first distance from the receiving area, and in the second position, the second end is located the first distance from the central portion of the support plate. The method includes applying a rotational force to the centrifuge device. By applying the rotational force to the centrifuge device, the sample passes from a first sample chamber coupled to the first end of the microfluidic chip, through at least one microfluidic channel extending between the first and second ends, and into a second sample chamber. The method includes allowing the carriage to rotate between the first and second positions.

[0040] The method includes applying a further rotational force to return the sample from the second sample chamber to the first sample chamber through at least one microfluidic channel extending between the first and second ends. In some embodiments, the biological sample includes adipose tissue, although other tissue types may be processed using the systems and methods disclosed herein. For example, adipose tissue, tumor tissue, cell preparations, lipoaspirates, cultured cells, etc. may be readily processed.

[0041] Further, in some embodiments, a system for processing a sample is provided, the system comprising: a support plate having a plurality of rotatable carriages radially arranged around the support plate; and at least one microfluidic chip disposed on one of the rotatable carriages, the at least one microfluidic chip defining a fluid pathway formed by one or more microfluidic channels disposed therein, the fluid pathway extending from a first port of the microfluidic chip to a second port located at an opposite end of the microfluidic chip.

[0042] In some embodiments, the support plate comprises a plurality of arms, each of which carries a rotatable carriage. In such embodiments, the arms are fixed to a separate central hub. In some embodiments, the support plate includes a first magnetic element disposed in or on the support plate and adjacent an end of the rotatable carriage, and the rotatable carriage further includes a second magnetic element disposed in or on the carriage.

[0043] Alternatively, in some embodiments, the rotatable carriage is coupled to a gear set disposed on a gear assembly attached to the support plate, the gear set including gears exposed on a radially outer portion of the gear assembly, hi some embodiments, the gear assembly or the support plate further includes a stationary magnet disposed therein, and the rotatable carriage includes a pair of magnetic elements disposed on opposite ends thereof.

[0044] In some embodiments, the plurality of rotatable carriages are rotatable in a plane substantially parallel to the plane of rotation of the support plate. In some embodiments, the plurality of rotatable carriages are rotatable in a plane substantially perpendicular to the plane of rotation of the support plate.

[0045] In some embodiments, the system further comprises an electromagnet disposed on a support plate beneath each of the plurality of rotatable carriages, the rotatable carriages comprising a magnetic post element extending through an opening formed in the support plate.

[0046] In some embodiments, at least one of the first sample retention chamber and the second sample retention chamber comprises a syringe barrel, e.g., a standard 2 mL, 5 mL, 10 mL, 20 mL, or 60 mL syringe barrel. Some embodiments further include a filter interposed between at least one microfluidic chip and one of the first sample retention chamber or the second sample retention chamber. In some embodiments, the filter is positioned upstream or before the microfluidic chip and configured to filter the sample to prevent clogging of the microfluidic chip. In some examples, the upstream filter may include a mesh configured to cut or pulverize tissue or tissue fragments so that the sample can pass through the microfluidic chip without clogging. The cutting or pulverization of the sample is configured to generate macroscopic aggregates for the purpose of microfluidic shearing in the microfluidic chip. In some embodiments, the filter is positioned downstream or after the microfluidic chip and allows only samples of a specific size to pass out of the device for collection.

[0047] In some embodiments, the system also includes a sample-holding chamber disposed within the rotatable carriage and coupled to a first port of the microfluidic chip, and a syringe coupled to a second port of the microfluidic chip, the syringe mounted approximately perpendicular to the plane of rotation of the microfluidic chip. In some embodiments, the systems disclosed herein may optionally include a vertically movable plate or ring coupled to the plunger of the syringe. In some embodiments, the vertically movable plate or ring comprises an internally threaded bearing attached to a rotatable threaded rod. Optionally, certain embodiments further include a second motor coupled to the threaded rod.

[0048] In some embodiments, the system has at least one of a first sample holding chamber and a second sample holding chamber including an inlet having a one-way valve disposed therein.

[0049] In some embodiments, a method of using the systems disclosed herein is provided, comprising rotating a support plate to move a sample through a first port into one or more microfluidic channels of at least one microfluidic chip and out a second port; rotating a rotatable carriage including at least one microfluidic chip by approximately 180 degrees; rotating the support plate to move the sample through the second port into one or more microfluidic channels and out the first port; and repeating the above steps multiple times until the sample has been processed to a desired extent.

[0050] In such methods, the sample moves between a first sample holding chamber fluidly coupled to a first port and a second sample holding chamber fluidly coupled to a second port, hi some embodiments, at least one of the first sample holding chamber and the second sample holding chamber comprises a syringe barrel.

[0051] In some embodiments, the sample comprises tumor tissue. In some embodiments, the sample comprises adipose tissue. In some embodiments, the sample comprises a fluid containing one or more reagents. In some embodiments, the sample comprises particles (e.g., nanoparticles, magnetic particles, particles coated with reagents or antibodies, etc.). In some embodiments, the sample comprises a cell-containing fluid.

[0052] In some embodiments, after processing the tissue, the methods disclosed herein further include injecting the processed tissue (e.g., adipose tissue) into a subject.

[0053] Complementing the above systems, devices, and methods, the following system for processing samples is also provided herein: The system includes a support plate, a plurality of arms extending radially from the support plate, a motor coupled to the support plate and configured to rotate the support plate, and a plurality of carriages, each of the plurality of carriages disposed on one of the plurality of arms on the support plate, each of the plurality of carriages disposed coaxially around one of a plurality of axes, each axis extending perpendicularly from the arm on which the carriage is disposed, each of the plurality of carriages configured to receive a microfluidic chip and at least one sample chamber for receiving a sample for processing, the at least one sample chamber having an opening fluidly connected to the microfluidic chip, and each of the plurality of carriages rotatable about one of the plurality of axes.

[0054] In some embodiments, the system further comprises a controller configured to drive the motor, the controller configured to adjust the rotational speed or revolutions per minute (RPM) of the motor. In some embodiments, the controller is adjustable or programmable with a predetermined spin program or sequence of operations. In some embodiments, the controller is configured (or configurable) to increase the spin speed of the motor to an RPM speed such that the sample is configured to flow from the first end of the microfluidic chip to the second end of the microfluidic chip. In some embodiments, the controller is configured to accelerate or decelerate the RPM of the motor such that each of the multiple carriages is configured to rotate about one of the multiple axes.

[0055] In some embodiments, each of the plurality of carriages is configured to rotate 180 degrees about one of the plurality of axes. Further, in some embodiments, each of the plurality of carriages is configured to receive a first sample chamber and a second sample chamber, the first sample chamber being disposed at a first end of the microfluidic chamber and the second sample chamber being disposed at a second end of the microfluidic chamber.

[0056] Some embodiments include at least one sample chamber that is attached to the end of the microfluidic chip using an adapter. The adapter may include any of a luer slip, slip-tip connector, luer lock, and rotation collar. The adapter may be constructed of a metal or polymeric material, depending on the embodiment and whether the microfluidic chip is disposable or reusable (e.g., sterilizable).

[0057] In some embodiments, the support plate is disposed within a housing configured to protect the user from the sample processing system. The housing may be constructed of any material, such as plastic or metal, and may be provided with a thickness sufficient to prevent penetration or rupture of the housing by debris generated by a drop in vacuum pressure, low temperatures, thermal changes, or centrifugal rotation. In some embodiments, the housing may be configured to be opened and closed as needed to place or remove samples or manually rotate one or more carriages. In some embodiments, the housing may be optically transparent and configured to allow monitoring of the operation of the sample processing system.

[0058] In embodiments including multiple arms, each of the multiple arms further includes a first engagement structure and a second engagement structure positioned a distance from the first engagement structure, the first engagement structure and the second engagement structure each configured to engage one of the first structure located at a first end of the carriage and the second structure located at a second end of the carriage. In some such embodiments, the first and second engagement structures are configured to interchangeably disengage and engage the first structure and the second structure such that the carriage moves in multiple orientations about one of multiple axes.

[0059] In some embodiments, the first engagement structure and the second engagement structure are magnets, and the first structure and the second structure comprise a magnetically responsive material. In some embodiments, the first engagement structure and the second engagement structure comprise a magnetically responsive material, and the first structure and the second structure are magnets. In some embodiments, the first engagement structure is disposed distally from the second engagement structure along the length of each of the multiple arms. To move the carriage, in some embodiments, an acceleration or deceleration force is configured to move the carriage between multiple orientations. In some embodiments, the carriage includes a gear assembly, and the gear assembly is configured to move the carriage between multiple orientations. In some embodiments, the carriage includes a centripetal ratchet, and the centripetal ratchet is configured to move the carriage between multiple orientations.

[0060] To enable proper flow of the sample through the microfluidic chip, in some embodiments, at least one sample chamber includes a vent and a vent channel fluidly connected to the interior of the sample channel, the vent configured to provide laminar flow through the sample chamber (e.g., by preventing a vacuum). In some embodiments, the vent is located at an end of the sample chamber opposite the opening. Depending on the embodiment, the sample chamber can be any desired shape, including rectangular, square, ellipsoidal, cylindrical, oval, or other polygonal. In one embodiment, the sample chamber is rectangular. In some alternative embodiments, the sample chamber is a syringe. In some such embodiments, the syringe comprises a chamber having an adapter end, the adapter end having an opening configured to fluidly connect to the microfluidic chip, and a plunger comprising a seal disposed within the chamber. An indenter is attached to the distal end of the plunger and configured to advance and retract the plunger. In some embodiments, the syringe comprises a vent and a vent channel fluidly connected to the interior of the chamber, the vent configured to provide laminar flow through the syringe. Further, in some embodiments, the syringe can optionally include a secondary syringe plunger disposed within the vent channel and configured to selectively open and close the vent channel. In such embodiments, the secondary syringe is optionally coupled to the indenter such that movement of the indenter is configured to advance and retract both the plunger and the secondary plunger. In some embodiments, the adapter end of the syringe is configured to accept a needle. In some embodiments, the syringe is configured to be detachable from the microfluidic chip, and the sample is configured to be injected directly into the injection site.

[0061] In some embodiments, each of the plurality of chambers (e.g., chambers for holding microfluidic chips) is held in a respective opening of the plurality of arms, and each of the plurality of chambers extends through a respective opening of the plurality of arms. In some embodiments, each of the plurality of chambers is held along a respective plane of the plurality of arms. In some embodiments, each of the plurality of chambers is held in a respective opening of the plurality of arms with at least one pin configured to enable out-of-plane rotation of each of the plurality of chambers. In some embodiments, the out-of-plane rotation of each of the plurality of chambers is configured to move each of the plurality of chambers between a plurality of orientations. In some embodiments, each of the plurality of chambers moves between 180-degree rotations (in-plane or out-of-plane). In some embodiments, each of the plurality of chambers moves between orientations in which each of the plurality of chambers lies along a respective plane of the plurality of arms.

[0062] In some embodiments, the systems disclosed herein further comprise at least one filter configured to prevent larger-sized sample components from entering and clogging the microfluidic chip. In some embodiments, the filter is attached to the sample chamber to filter the sample before passing it through the microfluidic pathway of the microfluidic chip. In some embodiments, the filter is positioned upstream or before the microfluidic chip and configured to filter the sample to prevent clogging of the microfluidic chip. In some examples, the upstream filter may include a mesh configured to cut or pulverize tissue or tissue fragments so that the sample can pass through the microfluidic chip without clogging. The cutting or pulverization of the sample is configured to generate macroscopic aggregates for the purpose of microfluidic shearing in the microfluidic chip. In some embodiments, the filter is positioned downstream or after the microfluidic chip to allow only samples of a specific size to exit the device for collection.

[0063] Additional systems are also provided herein, such as a system for processing a sample, comprising: a support plate; a motor coupled to the support plate and configured to rotate the support plate; and at least one carriage disposed on the support plate, the at least one carriage configured to receive a microfluidic chip and at least one sample chamber for receiving a sample for processing, the at least one carriage configured to rotate in a plane parallel to the plane of the support plate.

[0064] Further provided is a system for processing a sample, the system comprising: a support plate; a motor coupled to the support plate and configured to rotate the support plate; at least one carriage disposed on the support plate and configured to rotate in a plane parallel to the plane of the support plate; and a microfluidic chip received in the at least one carriage, the microfluidic chip comprising a port, at least one microfluidic channel extending along a length of the microfluidic chip, and at least one sample chamber for receiving a sample for processing, the at least one sample chamber being fluidly connected to a first port of the microfluidic chip and configured to allow the sample to flow from the at least one sample chamber along the length of the microfluidic chip.

[0065] In some embodiments, the microfluidic chip has a length of about 10 mm to 100 mm. In some embodiments, the length may be about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, about 90 mm, about 100 mm, or about 10-20 mm, about 30-40 mm, about 40-50 mm, about 50-60 mm, about 60-70 mm, about 70-80 mm, about 80-90 mm, about 90-100 mm, or any value within these ranges, inclusive. In some embodiments, the length of at least one microfluidic channel is less than (or equal to) the length of the microfluidic chip. In some embodiments, the width and depth of the microfluidic channel are within the range of 5 μm to 8 mm. In some embodiments, the microfluidic channels may be about 5-200 μm, about 200-400 μm, about 400-600 μm, about 600-800 μm, about 800-1000 μm, about 1-2 mm, about 2-4 mm, about 4-6 mm, about 6-8 mm, or any value within these ranges, inclusive. It should be understood that in some embodiments, the microfluidic chip is removable.

[0066] The microfluidic channels can have various configurations depending on the embodiment and the tissue being processed. For example, in one embodiment, the microfluidic channel has an hourglass-shaped configuration. In some embodiments, at least one microfluidic channel has a first region including a stepwise taper that gradually decreases in width along the length of the at least one microfluidic channel, a constricted region, and a second region including a stepwise taper that gradually increases in width along the length of the at least one microfluidic channel. In another embodiment, at least one microfluidic channel has a series of regions of increasing width and a region of decreasing width. In some embodiments, at least one microfluidic channel has a diamond pattern. In some embodiments, at least one microfluidic channel includes multiple pockets, which may optionally be fin-shaped. In some embodiments, at least one microfluidic channel includes a first region including a series of branches and a second region where pairs of branching channels recombine. In another embodiment, at least one microfluidic channel includes multiple wells, which are configured to sort portions of a predetermined size of a sample.

[0067] Further methods are provided herein, such as, for example, methods for processing a sample. The method includes providing a sample in a sample chamber, inserting the sample chamber into at least one of a plurality of carriages, and rotating a support plate about a first axis. In the method, the sample chamber is fluidly connected to a microfluidic chip including at least one microfluidic channel, one of the plurality of carriages is attached to the support plate, the support plate configured to rotate about the first axis, and at least one of the plurality of carriages configured to rotate about a second axis, the second axis being parallel to the first axis, and rotation of the support plate about the first axis is configured to drive the sample from the sample chamber, through the at least one microfluidic channel, and in a first direction away from the sample chamber. In some embodiments, the method further includes rotating at least one of the plurality of carriages in a second orientation about the second axis. In some embodiments, the method further includes rotating the support plate about a first axis, the rotation configured to drive the sample through the at least one microfluidic channel in a second direction toward the sample chamber. Additionally, the method optionally further includes removing the sample chamber from at least one of the plurality of carriages.

[0068] Further provided is a system comprising: a support plate including a plurality of arms; a motor; a plurality of carriages; and a case (housing). In the system, the plurality of arms extend radially from the support plate; a motor is coupled to the support plate and configured to rotate the support plate; each of the plurality of carriages is disposed on one of the plurality of arms on the support plate; each of the plurality of carriages is disposed coaxially around one of a plurality of axes, each axis extending perpendicularly from the arm on which the carriage is disposed; each of the plurality of carriages is configured to receive a microfluidic chip and at least one sample chamber for receiving a sample for processing; each of the plurality of carriages is rotatable about one of the plurality of axes; and a case configured to receive the support plate, the case configured to protect a user from the system for processing the sample, the case including an opening configured to provide access to the sample chamber. In some embodiments, the at least one sample chamber includes a vent and a vent channel fluidly connected to an interior of the sample channel, the vent configured to provide laminar flow through the sample chamber. In some embodiments, at least one sample chamber includes an opening fluidly connected to the microfluidic chip. In some embodiments, at least one sample chamber includes an inlet configured to allow a sample to be processed to be inserted or removed from the at least one sample chamber. In one embodiment, the inlet is located opposite the opening, although other locations may optionally be used. In some embodiments, at least one sample chamber includes a one-way valve configured to allow the sample to remain within the chamber during processing. In some embodiments, the interior of the sample chamber has a beveled, chamfered, or otherwise shaped surface adjacent the inlet, which is configured to concentrate the sample adjacent the inlet for easy sample removal after processing. In one embodiment, a vent is located at the end of the sample chamber opposite the opening.In some embodiments, the injection port is configured to engage a syringe, which is configured to withdraw a sample and inject the sample directly into a target site.

[0069] In some embodiments, each of the plurality of chambers is held in a respective opening of a respective one of the plurality of arms, and each of the plurality of chambers extends through the respective opening of the respective one of the plurality of arms. In such embodiments, each of the plurality of chambers is optionally held along a plane of a respective one of the plurality of arms. In some embodiments, each of the plurality of chambers is optionally held in a respective opening of a respective one of the plurality of arms using at least one pin configured to enable out-of-plane rotation of each of the plurality of chambers. In some such embodiments, the out-of-plane rotation of each of the plurality of chambers is configured to move each of the plurality of chambers between a plurality of orientations. In some embodiments, each of the plurality of chambers moves between 180-degree rotations (e.g., 0-45 degrees, 45-90 degrees, 90-135 degrees, 135-180 degrees, etc.). In some embodiments, each of the plurality of chambers moves between orientations in which each of the plurality of chambers is positioned along a plane of a respective one of the plurality of arms. In some embodiments, the system further comprises a filter configured to prevent larger-sized sample components from entering and clogging the microfluidic chip. In one embodiment, the filter is attached to the sample chamber. In some embodiments, a filter is placed upstream or before the microfluidic chip and configured to filter the sample to prevent clogging of the microfluidic chip. In some examples, the upstream filter may include a mesh configured to cut or pulverize tissue or tissue fragments so that the sample can pass through the microfluidic chip without clogging. The cutting or pulverization of the sample is configured to generate macroscopic aggregates for the purpose of microfluidic shearing in the microfluidic chip. In some embodiments, a filter is placed downstream or after the microfluidic chip to allow only samples of a certain size to exit the device for collection.

[0070] In some embodiments, a system for processing samples is provided, the system comprising: a support plate including a plurality of arms; a motor; a plurality of carriages; a case (housing); a spin stand; an externally threaded rod; and a plate, the plurality of arms extending radially from the support plate; a motor coupled to the support plate and configured to rotate the support plate; each of the plurality of carriages being disposed on one of the plurality of arms on the support plate; each of the plurality of carriages being disposed coaxially around one of a plurality of axes, each axis extending perpendicularly from the arm on which the carriage is disposed; each of the plurality of carriages being configured to receive a microfluidic chip and at least one sample chamber that receives a sample for processing; each of the plurality of carriages being rotatable around one of the plurality of axes; and the case including a main body and a cover, the main body being configured to receive a processing system, the cover being configured to receive a processing system. The bar is positioned on the main body and is configured to seal the processing system within the main body, protecting a user from the system for processing samples; the spin stand has a motor, an externally threaded rod attached to the motor, and rotation of the motor rotates the externally threaded rod; the plate has a plurality of engagement structures for holding a syringe, the plate is attached to a bearing having an internal thread, the internal thread being configured to engage with the external thread of the rod, and rotation of the motor is configured to vertically raise or lower the plate; the syringe has a chamber having an opening configured to fluidly connect to at least one sample chamber and a plunger disposed within the chamber, and advancing or retracting the plunger ejects or takes in sample for processing; the plate holds the distal end of the plunger, and the plate vertically lowers or raises the plunger within the chamber of the syringe to eject or take in sample for processing.

[0071] In some embodiments, the rotary motor is attached to a cover of the case (housing), while in other embodiments, the rotary motor is located outside the case (housing). In some embodiments, the plate is circular, while in some embodiments, the plate includes a ring attached to a bearing by multiple arms. In one embodiment, the plate includes a central circular plate and a coaxial ring. In some embodiments, the engagement structure is hook-shaped and configured to allow insertion and removal of the distal end of a plunger. In some embodiments, the system includes multiple spaced engagement structures for securing a syringe when the syringe is attached to at least one sample chamber at a first end of the microfluidic chamber or when the syringe is attached to at least one sample chamber at a second end of the microfluidic chamber. In some embodiments, each of the multiple arms further includes an engagement structure configured to engage with a corresponding structure disposed on each of the multiple carriages to hold each of the multiple carriages in a first orientation. In some embodiments, the engagement structure is configured to release and engage the corresponding structure such that the carriage is configured to move between multiple orientations about one of the multiple axes. In some embodiments, the engagement structure includes a magnet, and the corresponding structure includes a magnetically responsive material. Conversely, in some embodiments, the engagement structure includes a magnetically responsive material and the corresponding structure is a magnet. In certain embodiments, the acceleration or deceleration force is configured to move the carriage between multiple orientations. In some embodiments, the carriage includes a gear assembly, and the gear assembly is configured to move the carriage between multiple orientations. In some embodiments, the carriage includes a centripetal ratchet, and the centripetal ratchet is configured to move the carriage between multiple orientations.

[0072] A method, such as a method for processing a sample, is provided, the method including the steps of providing a sample to at least one sample chamber, inserting the sample chamber into at least one of a plurality of carriages, securing a syringe to the sample chamber, and rotating a support plate about a first axis. In the method, the sample chamber includes at least one microfluidic channel fluidly connected to the microfluidic chip, one of a plurality of carriages is attached to a support plate, the support plate is configured to rotate about a first axis, and at least one of the plurality of carriages is configured to rotate about a second axis, the second axis being parallel to the first axis, an opening of a syringe is fluidly connected to the sample chamber, a distal end of a plunger of the syringe is removably attached to the plate, the plate is attached to a motor and configured to be rotatable and move vertically, vertical movement of the plate lowers or raises the plunger in the barrel of the syringe to eject or take up the sample for processing in the sample chamber, and rotation of the support plate about the first axis is configured to drive the sample from the sample chamber through the at least one microfluidic channel in a first direction away from the sample chamber. In some embodiments, the method further includes vertically lowering the plate to lower the plunger in the barrel of the syringe to eject or take up the sample for processing into the sample chamber. In such methods, there is optionally a step of vertically raising the plate such that the plunger rises within the barrel of the syringe to remove the sample from the sample chamber for processing. In some embodiments, the method further includes rotating at least one of the plurality of carriages in a second orientation about a second axis.

[0073] As described above, a microfluidic chip includes one or more microfluidic pathways. In some embodiments, the pathways include compression regions (e.g., regions where the pathway walls are spaced a shorter distance from each other) and expansion regions (e.g., regions where the pathway walls are spaced a greater distance from each other). FIG. 16 shows a non-limiting embodiment of a chip having two inlets / outlets and a single expansion flow region disposed between two compression flow regions. In some embodiments, multiple expansion and compression regions are formed in the microfluidic pathways within the chip. FIG. 17 shows a non-limiting embodiment of a chip having two inlets / outlets and three expansion regions alternatingly disposed between compression regions. As described above, in some embodiments, the compression / expansion regions promote the activation of specific cells within a biological sample.

[0074] In some embodiments, the carriage includes a spring-like material (e.g., a coil spring, an elastomer, etc.) that urges the floor of the carriage up toward the fixed top, holding the microfluidic chip in place during operation of the device. In some examples, the floor of the carriage includes a wedge-shaped lateral extension on one side (the portion that extends out of the plane of the figure toward the viewer) that allows the floor to be pushed down (e.g., in an arcuate shape) to place and / or remove the microfluidic chip.

[0075] In yet another embodiment, there is provided a use of the processed tissue sample for the treatment of a medical condition. In some embodiments, the medical condition is a diabetic ulcer. In some embodiments, the medical condition is one that would be ameliorated or benefit from activated cells, such as stem cells. In some embodiments, there is provided a use of activated stem cells derived from adipose tissue resulting from the processing methods and systems disclosed herein for use in the manufacture of a medicament for treating a disease or condition.

[0076] In certain embodiments, treatment of a subject with the mechanically processed adipose-derived stem cells (ADSCs) described herein achieves, for example, one, two, three, four, or more of the effects listed below. (i) improvement in the severity of the disease or symptoms associated therewith; (ii) a reduction in the duration of disease-related symptoms; (iii) protection against the progression of the disease or its associated symptoms (iv) regression of the disease or its associated symptoms (v) protection against the progression or onset of disease-related symptoms (vi) protection against recurrence of disease-related symptoms (vii) a reduction in hospitalization of subjects (viii) Reduction in hospital stay (ix) increasing the survival rate of subjects with the disease (x) a reduction in the number of symptoms associated with the disease (xi) enhancing, improving, supplementing, complementing, or augmenting the prophylactic or therapeutic effects of another therapy Administration can be by a variety of routes, including, but not limited to, intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and / or local delivery to the affected tissue.

[0077] [Biological sample processing system] Systems for processing biological samples are disclosed. Figures 1A-1B, 2A-2B, 23A-23B, 24, 25A-25B, 26A-26B, and 27A-27F show non-limiting embodiments of systems for processing biological samples. In some embodiments, each of the systems for processing biological samples may include a motor coupled to a vertically oriented rotatable chuck. A support plate is attached or fixed to the rotatable chuck.

[0078] [Outline of the centrifuge] 1 illustrates one embodiment of a centrifuge device for processing biological samples. As described in more detail below, the centrifuge device includes a base with multiple carriages and at least one tip assembly attached to the base. Application of rotational and / or centrifugal forces to the at least one tip assembly allows bidirectional flow of the biological sample within the tip assembly, which can aid in processing the biological sample for subsequent therapeutic use.

[0079] 1A-1B illustrate one embodiment of a centrifuge device 100 for processing biological samples. As shown in FIGS. 1A and 1B, the centrifuge device 100 may include a base 160 having a central portion 161 and a plurality of lateral arms 163. The central portion 161 may be engageable with a portion of a motor component, which may rotate the base 160. Each of the lateral arms 163 extends radially from the central portion 161 of the base 160. As shown in FIGS. 1A-1B, each of the lateral arms 163 may include an opening 164 and a plurality of pedestal-engaging openings 166 configured to receive and retain the base portion of the carriage 130. In some embodiments, the carriage 130 may be secured to each of the lateral arms 163 using screw caps 170 and screws 132.

[0080] As shown in FIGS. 1A-1B, the centrifuge device 100 can hold multiple carriages 130. In some embodiments, the number of carriages 130 is the same as the number of lateral arms 163, although in some embodiments, a fewer number of carriages 130 than the number of lateral arms 163 may be used. As described in more detail below, each of the carriages 130 can include a clamp 110, a spring platform 140, and a base platform 150. Each of the base platforms 150 can include a first arm 156 a and a second arm 156 b. The first arm 156 a and the second arm 156 b engage corresponding first and second mating regions 142 a and 142 b of the spring platform 140. As described in more detail below, the carriage 130 can include one or more springs 134 between the base platform 150 and the spring platform 140. In some examples, the carriage 130 may include a clamp 110 that can secure the tip assembly 120 onto the carriage 130 .

[0081] Each of the carriages 130 can secure a chip assembly 120. As described in more detail below, each of the chip assemblies 120 can include a microfluidic chip 122, a sample chamber 190a secured to a first end of the microfluidic chip 122, and a sample chamber 190b secured to a second end of the microfluidic chip 122.

[0082] 2A-2B illustrate another embodiment of a centrifuge 200 for processing biological samples. The centrifuge 200 is similar to the centrifuge 100 shown in FIGS. 1A and 1B. As shown in FIGS. 2A and 2B, the centrifuge 200 may include a base 260 having a central portion 261 and a plurality of lateral arms 263. The central portion 261 is engageable with a portion of a motor component, which can rotate the base 260. Each of the lateral arms 263 extends radially from the central portion 261 of the base 260. In some embodiments, as shown in FIGS. 2A-2B, each of the lateral arms 263 may include an opening 264 and a plurality of seat-engaging openings 266 configured to receive and retain the base of the carriage 230. Similar to the centrifuge 100, the carriage 230 of the centrifuge 200 may be secured to each of the lateral arms 263 using a screw cap 270 and a screw 232. As described in more detail below, each of the lateral arms 263 of the base portion 260 may include a plurality of notches 268 that are engageable with a plurality of rotor foot inserts 280 .

[0083] As shown in FIGS. 2A and 2B, the centrifuge device 200 can hold multiple carriages 230. Similar to the carriage 130 described above, the carriage 230 includes a clamp 210, a spring platform 240, and a base platform 250. Each of the base platforms 250 can include a first arm 256a and a second arm 256b. The first arm 256a and the second arm 256b hold the spring platform 240 in a corresponding first mating region 242a and a corresponding second mating region 242b. The carriage 230 can include one or more springs 234 between the base platform 250 and the spring platform 240. The carriage 230 can also include a clamp 210 capable of securing the tip assembly 220 to the carriage 230. The clamp 210 can include a first mounting flange 212a and a second mounting flange 212b configured to engage a shoulder 236 of the base platform 250. In some embodiments, first mounting flange 212a and second mounting flange 212b each include a first opening 214a and a second opening 214b. First opening 214a and second opening 214b are aligned with first opening 236a and second opening 236b. In some examples, clamp 210 can be secured to carriage 230 by inserting multiple screws into holes in clamp 210 and base platform 250. As shown in FIGS. 2A-2B , first screw 238a is insertable into first opening 214a and first opening 236a, and second screw 238b is insertable into second opening 214b and second opening 236b.

[0084] Each of the carriages 230 can secure a chip assembly 220. Similar to the chip assembly 120, each of the chip assemblies 220 can include a microfluidic chip 222, a sample holding chamber 290a secured to a first end of the microfluidic chip 222, and a sample holding chamber 290b secured to a second end of the microfluidic chip 222.

[0085] [Carriage Assembly Overview] 3A-3B show an expanded view of one embodiment of carriage 230. Unless otherwise noted, the following description of carriage 230 and its components is also largely applicable to carriage 130 shown in FIGS. 1A-1B.

[0086] As described above, the carriage 230 may include a base platform 250, a spring platform 240, and a clamp 210 that secures the tip assembly 220. The base platform 250 may include a first arm 256a and a second arm 256b that receive the spring platform 240. The base platform 250 may include a post 258. The post 258 extends from a base portion of the base platform 250 and extends through one of the openings 264 in the lateral arms 263 of the base portion 260. In some embodiments, the post 258 may include a channel having a distal opening 258a and an opening 252 through the top surface of the base platform 250. The distal opening 258a can receive the screw 232 through the post 258 and into the base platform 250. In some embodiments, the outer periphery of the post 258 may be shaped to be received in a proximal opening 272 of a screw cap 270. Screw 232 may be configured to extend through tip (distal end) 270b of screw cap 270, tip opening 258a of post 258, and opening 252 to secure one of carriages 230 to one of lateral arms 263. In some embodiments, screw 232 is secured via a threaded fastener (e.g., a nut and bolt) that provides sufficient force to secure carriage 230 to lateral arm 263 while allowing rotation of the carriage. In some embodiments, as shown in FIG. 2B , the bottom surface of base platform 250 may include first and second pedestals 251a and 251b, each engageable with one of pedestal mating openings 266.

[0087] The carriage 230 may also include a spring platform 240. The spring platform 240 may include a first mating region 242a and a second mating region 242b. The first mating region 242a and the second mating region 242b are configured to receive the first arm 256a and the second arm 256b, respectively. Although FIGS. 3A-3B depict the spring platform 240 and the base platform 250 as separate components, in some embodiments, the spring platform 240 and the base platform 250 may be integrally formed.

[0088] The carriage 230 may include a clamp 210 configured to secure the chip assembly 220 on a base platform 250 of the carriage 230. As described above, the clamp 210 may include a first mounting flange 212a having a first opening 214a and a second mounting flange 212b having a second opening 214b. The first and second mounting flanges 212a and 212b (the first and second openings 214a and 214b) are configured to align with first and second openings 236a and 236b on a shoulder 236 of the base platform 250. In some embodiments, the clamp 210 may be secured to the base platform 250 by inserting a first screw 238a into the first and first openings 214a and 236a and inserting a second screw 238b into the second and second openings 214b and 236b.

[0089] In some embodiments, the upper surface of base platform 250 has positioned thereon one or more springs (e.g., coil springs, leaf springs, etc.) or another deformable material (e.g., an elastomeric material) that at least substantially returns to its original position after a force is applied and released. As shown in FIGS. 3A and 3B , multiple springs 234 can be secured between spring platform 240 and base platform 250. As shown in FIG. 3A , base platform 250 can include distal spring holders 254 a and 254 b that can receive the base portions of springs 234, respectively. As shown in FIG. 2B , the lower surface of spring platform 240 is provided with proximal spring holders 244 a and 244 b. Proximal spring holders 244 a and 244 b can receive the upper portions of springs 234, respectively. 3B , when the chip assembly 220 is secured to the carriage 230, the plurality of springs 234 are compressed between the spring platform 240 and the base platform 250. In some embodiments, the plurality of springs 234 are configured to provide an upward force to the spring platform 240. This upward force can help press the spring platform 240 upward against the chip assembly 220, thereby securing the chip assembly 220 snugly between the spring platform 240 and the clamp 210. The spring tension action of the spring platform 240 allows the microfluidic chip 222 to be held in place by being pressed upward against the bottom surface of the clamp 210. During operation, when the center of the device is subjected to a rotational force and the carriage is rotated about an axis substantially parallel to the axis of the rotational force applied to the device, the spring pressure allows the microfluidic chip 222 to be firmly held within the carriage 230.

[0090] To insert or remove the microfluidic chip 222 from the carriage 230, the user applies a downward force to the microfluidic chip 222 or the base platform 250, which further compresses the spring 234 and increases the distance between the microfluidic chip 222 and the base of the clamp 210. This may allow movement (e.g., insertion or removal) of the microfluidic chip 222 relative to the carriage 230. As described in more detail below, the clamp 210 may have reliefs or other openings to assist in chip removal.

[0091] [Chip Assembly] 4A-4B illustrate one embodiment of chip assembly 220. As noted above, unless otherwise noted, the following description of chip assembly 220 and its components is largely applicable to chip assembly 120 shown in FIGS. 1A-1B.

[0092] The chip assembly 220 may include a microfluidic chip 222 fluidly coupled to a first sample holding chamber 290a and a second sample holding chamber 290b. The first sample holding chamber 290a is fluidly connectable to at least one microfluidic chip via a first port 221a, and the second sample holding chamber 290b is fluidly connectable to one of the microfluidic chips 222 via a second port 221b. As shown in FIGS. 4A-4B and 14A-14F, each of the first sample holding chamber 290a and the second sample holding chamber 290b may be fluidly connected to one of the microfluidic chips 222. The sample holding chamber 290a may be fluidly connected to a first end of the microfluidic chip 222 by engaging a first port 293a of the sample holding chamber 290a with a first port 221a of the microfluidic chip 222. The sample holding chamber 290b can be fluidly connected to a second end of the microfluidic chip 222 by engaging a second port 293b of the sample holding chamber 290b with a second port 221b of the microfluidic chip 222.

[0093] In some embodiments, an adapter is interposed between first port 221 a and first sample holding chamber 290 a, and between second port 221 b and second sample holding chamber 290 b. In some embodiments, at least one microfluidic chip comprises a first sample holding chamber and a second sample holding chamber disposed within the at least one microfluidic chip.

[0094] As described in more detail below, chip assembly 220 allows samples in either or both sample holding chamber 290a and sample holding chamber 290b to move bidirectionally through sample holding chamber 290a, microfluidic chip 222, and sample holding chamber 290b.

[0095] [Microfluidic chip] FIGS. 5A-5C, 7A-7C, 8A-8B, 9-10, 11A-11B, 12A-12B, and 13A-13B illustrate various embodiments of microfluidic chips for use in chip assembly 220. The disclosed microfluidic chips may include a fluid pathway having a microfluidic channel. The microfluidic channel has multiple expansion and contraction regions along the length of the channel. In some embodiments, the multiple expansion and contraction regions are defined by curved walls within the microfluidic channel. In some examples, the multiple expansion and contraction regions are defined by slanted walls within the microfluidic channel. In some embodiments, the fluid pathway may include multiple branch channels of decreasing dimensions. The branch channels recombine into multiple branch channels of increasing dimensions. Each branch channel may include a branch point. In some embodiments, the branch point may have a sharp edge. In some embodiments, the microfluidic chip may be used in a pumping system. The microfluidic chip can be fluidly connected to a luer lock and a syringe fluidly connected to the luer lock.

[0096] 5A-5C illustrate one embodiment of a microfluidic chip 222. FIG. 5A illustrates a side view of the microfluidic chip 222, FIG. 5B illustrates a top view of the microfluidic chip 222, and FIG. 5C illustrates an isometric three-dimensional view of the microfluidic chip 222. The microfluidic chip 222 may include a body 225. The body 225 has a first port 221a at a first end thereof and a second port 221b at a second end of the body 225. The first port 221a may include an opening 223a that is fluidly connected to a first end of a fluid path of the microfluidic chip 222. The second port 221b may include an opening 223b that is fluidly connected to a second end of the fluid path of the microfluidic chip 222. The fluid path of the microfluidic chip 222 extends from the opening 223a and the opening 223b. In some embodiments, the fluid path of the microfluidic chip 222 is symmetrical. However, in other embodiments, the fluid pathways of the microfluidic chip 222 may be asymmetric or a combination of symmetric and asymmetric portions. Although this disclosure describes the microfluidic chip 222 and its associated fluid pathways as extending between a "first" end and a "second" end, the orientation of the microfluidic chip 222 may be interchangeable such that flow within the chip assembly 220 and the microfluidic chip 222 may be bidirectional.

[0097] 5A-5C, the fluid pathway of the microfluidic chip 222 may include multiple expansion and compression portions. For example, as shown, the fluid pathway of the microfluidic chip 222 may include a first channel 226a, a first expansion portion 227a, a first compression portion 228a, an expansion region 229, a second compression portion 228b, a second expansion portion 227b, and a second channel 226b.

[0098] In some embodiments, the fluid pathway of the microfluidic chip 222 includes a constricted first channel 226a, which is closest to the opening 223a of the first port 221a. The first port 221a can have a length, width, or diameter of about 0.5 mm to about 4 mm. In some embodiments, the length, width, or diameter may be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, or about 4.0 mm, or may be about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, or about 3.5-4.0 mm, or any value within these ranges, inclusive. First channel 226a may expand and increase in width, height, and / or diameter such that first channel 226a is fluidly connected to first expansion portion 227a. As shown in FIG. 5C, first expansion portion 227a is three-dimensionally teardrop-shaped. First expansion portion 227a may have a width, height, and / or diameter greater than first channel 226a. First expansion portion 227a may range from about 0.3 mm to about 5 mm. In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, about 5.0 mm, or about 0.3-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, about 4.0-4.5 mm, or about 4.5-5.0 mm, or any value within these ranges, inclusive. The first expanding portion 227a may be fluidly connected to the first compressing portion 228a. The fluid pathway may be constricted at the first compressing portion 228a, and the first compressing portion 228a may have a smaller width, height, and / or diameter than the first expanding portion 227a. The first compressed portion 228a may have a width, height, and / or diameter ranging from about 0.3 mm to about 3 mm.In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, or about 0.3-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, or any value within these ranges, inclusive. The fluid pathway may expand and increase in width, height, and / or diameter such that first compressed portion 228a is fluidly connected to second expanded region 229. Second expanded region 229 may be in the range of about 0.3 mm to about 5 mm. In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, about 5.0 mm, or about 0.3-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, about 4.0-4.5 mm, or about 4.5-5.0 mm, or any value within these ranges, inclusive. In some embodiments, second expansion region 229 has a spherical or ellipsoidal expanded shape having a length, width, and / or diameter greater than that of first compressed portion 228a. Second expansion portion 229 is fluidly connected to second compressed portion 228b. The fluid pathway is constricted at second compression portion 228b such that the width, height, and / or diameter of compression portion 228b is smaller than the width, height, and / or diameter of second expansion portion 227b. Second compression portion 228b can have a length, width, and / or diameter ranging from about 0.3 mm to about 3 mm. In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, or about 0.3-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, or any value within these ranges, inclusive. Second compression portion 228b is fluidly connected to third expansion portion 227c, and the fluid pathway is expanded to third expansion portion 227b.Third expanding portion 227b expands such that its width, height, and / or diameter are greater than those of second compressing portion 228b. Third expanding portion 227b can range from about 0.3 mm to about 5 mm. In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, or about 5.0 mm, or may be about 0.3-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, about 4.0-4.5 mm, or about 4.5-5.0 mm, or any value within these ranges, inclusive. The third extension portion 227b may have a three-dimensional teardrop or other arched shape. The third extension portion 227b may be fluidly connected to the second channel 226b and may contract to the second channel 226b. The second channel 226b may have a width, height, and / or diameter that is smaller than the width, height, and / or diameter of the extension portion 227b. The second channel 226b may have a length, width, and / or diameter ranging from about 0.5 mm to about 4 mm. In some embodiments, the length, width, or diameter may be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, or about 3.5-4.0 mm, or any value within these ranges, inclusive. In some embodiments, the fluid pathways of microfluidic chip 222 are symmetrical, while in other embodiments, the fluid pathways of microfluidic chip 222 may be asymmetrical or may have a combination of symmetrical and asymmetrical portions. In some embodiments, the ratio of first channel 226a to extension portion 227a may be in the range of 1:1 to 1:17. In some examples, the ratio of extension portion 227b to extension portion 227b may be in the range of 1:1 to 1:17.In some embodiments, the ratio of compressed portion 228a or compressed portion 228b to expansion region 229 may range from 1:1 to 1:17.

[0099] 6A-6D show enlarged views of the fluid pathways of the microfluidic chip 222. FIG. 6A shows an enlarged view of the ports 221 (i.e., first port 221a and / or second port 221b) and openings 223 (i.e., openings 223a and / or openings 223b) at either end of the microfluidic chip 222. In some embodiments, the ports 221 may be luer lock adapters for use with syringes or other luer lock connections. In some embodiments, the ports 221 may instead be slip luers or any other type of luer connection. The openings 223 of the ports 221 are fluidly connected to the inlet channels 226 (i.e., first channel 226a and second channel 226b). The inlet channels 226 may increase the velocity of the fluid moving through the inlet channels 226, creating laminar flow. FIG. 6B shows the first and third extensions 227a and 227c of the extensions 227. In some embodiments, the expansion section 227 may have a "teardrop" shape. The expansion section 227 can reduce velocity and allow for turbulence, mixing and vortexing the fluid flowing through the fluid pathway of the microfluidic chip 222. FIG. 6C shows the compression zone 228 of compression sections 228a and 228b. The compression zone can change the flow to a laminar flow profile, again helping to increase the fluid velocity. The compression zone 228 can provide high shear forces due to the interaction of the sample with the walls of the fluid pathway. FIG. 6D shows the second expansion region 229. As described above, the expansion region 229 can have a spherical and / or elliptical shape. The expansion region 229 can reduce the fluid velocity by inducing turbulence and vortices. The increase or decrease in the fluid flow velocity can help break down tissue, such as adipose tissue, for reinjection.

[0100] The microfluidic chips disclosed above can be manufactured by a variety of methods. For example, they can be manufactured using additive manufacturing, subtractive manufacturing, injection molding, resin molding, urethane casting, 3D printing, or layer lamination. Microfluidic chips can be made from a variety of materials, including, for example, plastics (such as polycarbonate and acrylic), polyurethane, or metals (such as aluminum, steel, stainless steel, surgical steel, brass, and copper).

[0101] 7A-7C illustrate one embodiment of a microfluidic chip 322. FIG. 7A illustrates a top view of the microfluidic chip 322. FIG. 7B illustrates a side view of the microfluidic chip 322 in which the second expansion region 329 has a hemispherical shape. FIG. 7C illustrates a side view of the microfluidic chip 322 in which the second expansion region 329b forms a flat, channel-like extension. The microfluidic chip 322 serves a similar purpose to the microfluidic chip 222 described above, except that the shapes of the expansion and compression regions are different. The microfluidic chip 322 may include a body 325 having at least a first port 321a at a first end of the body 325 and at least a second port 321b at a second end of the body 325. The first port 321a may include an opening 323a that is fluidly connected to a first end of a fluid pathway of the microfluidic chip 322. The second port 321b may include an opening 323b fluidly connected to a second end of the fluid pathway of the microfluidic chip 322. The fluid pathway of the microfluidic chip 322 may extend from the opening 323a and the opening 323b. In some embodiments, the fluid pathway of the microfluidic chip 322 is symmetric. In other embodiments, the fluid pathway of the microfluidic chip 322 may be asymmetric or a combination of symmetric and asymmetric portions. Although this disclosure discusses the microfluidic chip 322 and the associated fluid pathway as extending between a "first" end and a "second" end, the orientation of the microfluidic chip 322 is interchangeable, as long as flow within the chip assembly 320 and the microfluidic chip 322 can be bidirectional.

[0102] 7A-7C, the fluid pathway of the microfluidic chip 322 may include multiple expansion and compression portions. For example, as shown, the fluid pathway of the microfluidic chip 322 may include a first channel 326a, a first expansion portion 327a, a first compression portion 328a, a second expansion region 329, a second compression portion 328b, a third expansion portion 327b, and a second channel 326b.

[0103] In some embodiments, the fluid pathway of microfluidic chip 322 includes a constricted first channel 326a, which is closest to opening 323a of first port 321a. First port 321a can have a length, width, or diameter of about 0.5 mm to about 4 mm. In some embodiments, the length, width, or diameter may be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, or about 4.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, or about 3.5-4.0 mm, or any value within these ranges, inclusive. First channel 326a may expand and increase in width, height, and / or diameter such that first channel 326a is fluidly connected to first expanding portion 327a. In some embodiments, the shape of first expanding portion 327a may be semi-teardrop or other arched (bow-like) shape. First expanding portion 327a may have a width, height, and / or diameter greater than first channel 326a. First expanding portion 327a may range from about 0.5 mm to about 5 mm. In some embodiments, the length, width, or diameter may be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, about 5.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, about 4.0-4.5 mm, about 4.5-5.0 mm, or any value within these ranges, inclusive. The first expanding portion 327a may be fluidly connected to the first compressing portion 328a. The fluid pathway is constricted at the first compressing portion 328a, and the first compressing portion 328a has a smaller width, height, and / or diameter than the first expanding portion 327a. The first compressed portion 328a may have a width, height, and / or diameter ranging from about 0.3 mm to about 3 mm.In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, or any value within these ranges, including the upper and lower limits. The fluid pathway may expand and increase in width, height, and / or diameter such that the first compressed portion 328a is fluidly connected to the second expanded region 329. As shown in FIG. 7B, the second expanded region 329 has a hemispherical or semi-elliptical expanded shape with a length, width, and / or diameter greater than the first compressed portion 328a. The second expanded region 329 may range from about 0.3 mm to about 5 mm. In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, about 5.0 mm, or about 0.3-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, about 4.0-4.5 mm, or about 4.5-5.0 mm, or any value within these ranges, inclusive. As shown in FIG. 7C, the second expansion region 329 may be in the form of a flat channel-like expansion. The second expansion region 329 may be fluidly connected to the compression portion 328b. The second expansion region 329 may be fluidly connected to the compression portion 328b. The fluid pathway may be constricted at the second constricted portion 328b such that the width, height, and / or diameter of the constricted portion 328b is smaller than the width, height, and / or diameter of the second expanded portion 328b. The second constricted portion 328b may have a length, width, and / or diameter ranging from about 0.3 mm to about 3 mm. In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 0.3-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, or any value within these ranges, inclusive.The second compression portion 328b is fluidly connected to the third expansion portion 327b such that the fluid path expands at the third expansion portion 327b. The third expansion portion 327b may have a width, height, and / or diameter that is greater than the width, height, and / or diameter of the second compression portion 328b. The first expansion portion 327a may be in the range of about 0.5 mm to about 5 mm. In some embodiments, the length, width, or diameter may be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, about 5.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, about 4.0-4.5 mm, or about 4.5-5.0 mm, or any value within these ranges, inclusive. The third extension portion 327b may have a three-dimensional semi-teardrop or other arched shape. The third extension portion 327b may be fluidly connected to contract with the second channel 326b. Second channel 326b may have a width, height, and / or diameter smaller than the width, height, and / or diameter of third extension portion 327b. Second channel 326b may have a length, width, and / or diameter ranging in size from about 0.5 mm to about 4 mm. In some embodiments, the length, width, or diameter may be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, or any value within these ranges, inclusive. In some embodiments, microfluidic chip 322 is configured like microfluidic chip 222 described above cut in half. In some examples, microfluidic chip 322 is symmetrical. In other embodiments, the microfluidic chip 322 may be asymmetric or a combination of symmetric and asymmetric portions. In some embodiments, the ratio of the first channel 326a to the extension portion 327a may range from about 1:1 to about 1:10.In some examples, the ratio of second channel 326b to second channel 326b can range from about 1:1 to about 1:10. In some embodiments, the ratio of compressed portion 328a or compressed portion 328b to expansion region 329 can range from about 1:1 to about 1:17.

[0104] 8A-8B illustrate another embodiment of a microfluidic chip 422 in which a fluid pathway includes multiple expansion and compression sections. FIG. 8A illustrates a top view of the microfluidic chip 422, and FIG. 8B illustrates a side view of the microfluidic chip 422. The microfluidic chip 422 serves a similar purpose to the microfluidic chips 222 and 322 described above, except that the expansion and compression regions of the fluid pathway differ in shape. The microfluidic chip 422 may include a body 425 having a first port 421a at a first end of the microfluidic chip 422 and a second port 421b at a second end of the body 425. The first port 421a may include an opening 423a fluidly connected to a first end of the fluid pathway of the microfluidic chip 422. The second port 421b may include an opening 423b fluidly connected to a second end of the fluid pathway of the microfluidic chip 422. The fluid pathways of the microfluidic chip 422 can extend from opening 423a to opening 423b. In some embodiments, the fluid pathways of the microfluidic chip 422 are symmetric. In other embodiments, the fluid pathways of the microfluidic chip 422 can be asymmetric or a combination of symmetric and asymmetric portions. Although this disclosure discusses the microfluidic chip 422 and associated fluid pathways as extending between a "first" end and a "second" end, the orientation of the microfluidic chip 422 is interchangeable, as long as flow within the chip assembly and within the microfluidic chip 422 can be bidirectional.

[0105] 8A-8B, the fluid path of the microfluidic chip 422 may include multiple expansion and compression portions. For example, as shown, the fluid path of the microfluidic chip 422 may include a first channel 426a, a first expansion portion 427a, a first compression portion 428a, a second expansion region 429, a second compression portion 428b, a third expansion portion 427b, and a second channel 426b.

[0106] In some embodiments, the fluid pathway of the microfluidic chip 422 includes a constricted first channel 426a, which is closest to the opening 423a of the first port 421a. The first port 421a can have a length, width, and / or diameter between about 0.5 mm and about 4 mm. In some embodiments, the length, width, or diameter may be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, or any value within these ranges, inclusive. First channel 426a may expand and increase in width, height, and / or diameter such that first channel 426a is fluidly connected to first expanding portion 427a. In some embodiments, first expanding portion 427a may be semi-teardrop shaped. First expanding portion 427a may have a width, height, and / or diameter greater than first channel 426a. First expanding portion 427a may range from about 0.5 mm to about 5 mm. In some embodiments, the length, width, or diameter may be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, about 5.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, about 4.0-4.5 mm, or about 4.5-5.0 mm, or any value within these ranges, inclusive. The first expanding portion 427a may be fluidly connected to the first compressing portion 428a. The fluid pathway is constricted at the first compressing portion 428a, and the first compressing portion 428a has a smaller width, height, and / or diameter than the first expanding portion 427a. The first compressed portion 428a may have a width, height, and / or diameter ranging from about 0.3 mm to about 3 mm.In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, or any value within these ranges, including the upper and lower limits. The fluid pathway may expand and increase in width, height, and / or diameter such that the first compressed portion 428a is fluidly connected to the second expanded region 429. As shown in FIG. 8A, the second expanded region 429 has a D-shaped configuration with a length, width, and / or diameter greater than the first compressed portion 428a. The second expanded region 429 may range from about 0.3 mm to about 5 mm. In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, about 5.0 mm, or any value between about 0.3 and 1 mm, about 1 and 1.5 mm, about 1.5 and 2.0 mm, about 2.5 and 3.0 mm, about 3.0 and 3.5 mm, about 3.5 and 4.0 mm, about 4.0 and 4.5 mm, or about 4.5 and 5.0 mm, inclusive. As shown in FIG. 8A, the D-shape of the second expansion region 429 has a right-angled end on a first side and an angled second side to provide more turbulence and vortex flow. The second expansion region 429 may be fluidly connected to the second compression portion 428b. The fluid pathway may be constricted at second compressed portion 428b such that the width, height, and / or diameter of second compressed portion 428b is smaller than the width, height, and / or diameter of second compressed portion 428b. Second compressed portion 428b may have a length, width, and / or diameter ranging from about 0.3 mm to about 3 mm. In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 0.3-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, or any value within these ranges, inclusive.The second compressed portion 428b is fluidly connected to the third expanded portion 427b such that the fluid path is expanded at the third expanded portion 427b. The third expanded portion 427b may have a width, height, and / or diameter that is greater than the width, height, and / or diameter of the second compressed portion 428b. The third expanded portion 427b ranges from about 0.5 mm to about 5 mm. In some embodiments, the length, width, or diameter may be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, about 5.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, about 4.0-4.5 mm, or about 4.5-5.0 mm, or any value within these ranges, inclusive. The third extension portion 427b may have a three-dimensional semi-teardrop shape. The third extension portion 427b may be fluidly connected to the second channel 426b so as to contract in the second channel 426b. Second channel 426b can have a width, height, and / or diameter that is smaller than the width, height, and / or diameter of third extension 427b. Second channel 426b can have a length, width, and / or diameter that ranges in size from about 0.5 mm to about 4 mm. In some embodiments, the length, width, or diameter can be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, or any value within these ranges, inclusive. In some embodiments, microfluidic chip 422 is configured like microfluidic chip 222 described above, cut in half. In some examples, microfluidic chip 422 is symmetrical. In other embodiments, the microfluidic chip 422 may be asymmetric or may have a combination of symmetric and asymmetric portions. In some embodiments, the ratio of the first channel 426a to the first extension portion 427a may range from about 1:1 to about 1:10.In some examples, the ratio of second channel 426b to third expansion portion 427b can range from about 1:1 to about 1:10. In some embodiments, the ratio of compressed portion 428a or compressed portion 428b to second expansion region 429 can range from about 1:1 to about 1:17.

[0107] FIG. 9 illustrates another embodiment of a microfluidic chip 522. As shown in FIG. 9, the microfluidic chip 522 includes a fluid path, which includes a microfluidic channel formed by a pair of tapered regions that join at a constriction in a central region of the fluid path. In some embodiments, the tapered region includes a continuous tapered region. In some examples, the tapered region includes a stepped tapered region. FIG. 9 illustrates a top view of the microfluidic chip 522. The microfluidic chip 522 serves a similar purpose to the microfluidic chips 222, 322, and 422 described above. The microfluidic chip 522 may include a body 525 having a first port 521 a at a first end of the microfluidic chip 522 and a second port 521 b at a second end of the body 525. The first port 521 a may include an opening 523 a fluidically connected to the first end of the fluid path of the microfluidic chip 522. The second port 521b may include an opening 523b that is fluidly connected to a second end of the fluid pathway of the microfluidic chip 522. The fluid pathway of the microfluidic chip 522 may extend from opening 523a to opening 523b. In some embodiments, the fluid pathway of the microfluidic chip 522 is symmetric. In other embodiments, the fluid pathway of the microfluidic chip 522 may be asymmetric or a combination of symmetric and asymmetric portions. Although this disclosure discusses the microfluidic chip 522 and the associated fluid pathway as extending between a "first" end and a "second" end, the orientation of the microfluidic chip 522 is interchangeable, as long as flow within the chip assembly and within the microfluidic chip 522 can be bidirectional.

[0108] As shown in FIG. 9 , the fluid path of the microfluidic chip 522 may include multiple expansion and compression portions. As shown in FIG. 9 , the fluid path of the microfluidic chip 522 may include a first channel 526 a, a first expansion portion 527 a, a compression portion 528, a second expansion portion 527 b, and a second channel 526 b. As shown in FIG. 9 , the fluid path of the microfluidic chip 522 may have an hourglass shape to provide a smooth increase in the velocity of the sample. This helps maintain laminar flow and slowly increase the shear stress on the sample as it interacts with the walls of the device. In some embodiments, the first channel 526 a (and the second channel 526 b) provide an initial velocity of the fluid that enables laminar flow. The diamond shape of the first expansion portion 527 a (and the second expansion portion 527 b) allows the sample to decelerate, which creates turbulence and vortexes that mix the fluid. The compression portion 528 can increase the velocity of the fluid, thereby increasing the shear force. The size of the first channel 526a and the second channel 526b can be about 0.5 mm to about 4 mm in length, width, and / or diameter. In some embodiments, the length, width, or diameter can be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, or any value within these ranges, inclusive. The size of the first expansion portion 527a and the second expansion portion 527b can be larger than the width, length, and / or diameter of the first channel 526a and the second channel 526b. The first extension portion 527a and the second extension portion 527b can range from about 0.3 mm to about 5 mm.In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, about 5.0 mm, or about 0.3-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, about 4.0-4.5 mm, about 4.5-5.0 mm, or any value within these ranges, inclusive. The size of compressed portion 528 may be about 0.3 mm to about 3 mm. In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, or about 0.3-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, or any value within these ranges, inclusive. In some embodiments, first port 521a and second port 521b are luer locks. In some embodiments, first port 521a and second port 521b may be other luer adapters, such as slip luers. In some embodiments, the ratio of compressed portion 528 to first extension portion 527a and / or the ratio of compressed portion 528 to second extension portion 527b may be in the range of about 1:1 to about 1:17.

[0109] FIG. 10 illustrates another embodiment of a microfluidic chip 622, in which the fluid pathway includes multiple expansion and compression sections. FIG. 10 illustrates a top view of the microfluidic chip 622. The microfluidic chip 622 serves a similar purpose to the microfluidic chips 222, 322, 422, and 522 described above, except that the expansion and compression regions of the fluid pathway differ in shape. The microfluidic chip 622 may include a body 625 having a first port 621 a at a first end of the microfluidic chip 622 and a second port 621 b at a second end of the body 625. The first port 621 a may include an opening 623 a fluidically connected to a first end of the fluid pathway of the microfluidic chip 622. The second port 621 b may include an opening 623 b fluidically connected to a second end of the fluid pathway of the microfluidic chip 622. The fluid pathways of the microfluidic chip 622 can extend from opening 623a to opening 623b. In some embodiments, the fluid pathways of the microfluidic chip 622 are symmetric. In other embodiments, the fluid pathways of the microfluidic chip 622 can be asymmetric or a combination of symmetric and asymmetric portions. Although this disclosure discusses the microfluidic chip 622 and associated fluid pathways as extending between a "first" end and a "second" end, the orientation of the microfluidic chip 622 is interchangeable, as long as flow within the chip assembly and within the microfluidic chip can be bidirectional.

[0110] 10, the fluid path of the microfluidic chip 622 may include multiple expansion and compression portions. For example, as shown in FIG. 10, the fluid path of the microfluidic chip 622 may include a first channel 626a, a first expansion portion 627a, a compression portion 628, a second expansion portion 627b, and a second channel 626b. The first channel 626a, the first expansion portion 627a, the compression portion 628, the second expansion portion 627b, and the second channel 626b are fluidly connected.

[0111] In the microfluidic chip 622 shown in FIG. 10, the fluid path has a sharper hourglass shape to provide increased turbulence and vortex flow. The first channel 626a and the second channel 626b can provide an initial velocity of the sample that allows for laminar flow. In some embodiments, the size of the first channel 626a and the second channel 626b can range from about 0.5 mm to about 4 mm in length, width, and / or diameter. In some embodiments, the length, width, or diameter can be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, or any value within these ranges, inclusive. The first and second extension portions 627a and 627b may have a diamond-shaped shape that provides fluid deceleration, which can create turbulence and vortexes to mix the fluid sample. In some embodiments, the size of the diamond-shaped first and second extension portions 627a and 627b is larger than the width, length, and / or diameter of the first and second channels 626a and 626b. The first and second extension portions 627a and 627b may range from about 0.3 mm to about 5 mm. In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, about 5.0 mm, or about 0.3-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, about 3.0-3.5 mm, about 3.5-4.0 mm, about 4.0-4.5 mm, or about 4.5-5.0 mm, or any value within these ranges, inclusive. The compression section 628 may increase the velocity of the sample, thereby increasing the shear force applied to the fluid sample. Similarly, the sharp angles on either side of the compression section 628 may provide turbulence and vortices. In some embodiments, the compressed portion 628 can range in width, length, and / or diameter from about 0.3 mm to about 3 mm.In some embodiments, the length, width, or diameter may be about 0.3 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, or about 0.3-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, or any value within these ranges, inclusive. In some embodiments, first port 621a and second port 621b are luer locks. In some embodiments, first port 621a and second port 621b may be other luer adapters, such as slip luers. In some embodiments, the ratio of compressed portion 628 to extended portion 627a and / or the ratio of compressed portion 628 to extended portion 627b may range from about 1:1 to about 1:17.

[0112] Figures 11A-11B, 12A-12B, and 13A-13B show several embodiments of microfluidic chips with three-dimensional (cubic) channel structures. The three-dimensional channel structures in Figures 11A-11B, 12A-12B, and 13A-13B have fluid paths that compress / contract and expand in multiple stages. This compression and expansion allows for the generation of turbulent flow, which is useful for processing biological samples. For example, the device can be used to mechanically break down adipose tissue to reduce its size for applications such as orthopedic surgery, arthroscopic surgery, neurosurgery, gastrointestinal and related organ surgery, urological surgery, general surgery, and gynecological surgery. The disclosed microfluidic chips can be fabricated using either 3D printing, additive manufacturing, subtractive manufacturing, or injection molding. The materials used to print or fabricate the devices are biocompatible and sterilizable. The microfluidic chip includes an inlet and an outlet, which may be a luer lock, threaded, or any other form that allows for a fluid seal. When the seal is formed, a fluid pathway is created. As the sample passes through the inlet, it interacts with a compression region that is smaller than the diameter of the inlet, creating mechanical stress. The diameter of this region may range from about 0.5 mm to about 3.0 mm. In some embodiments, the length, width, or diameter may be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, or any value within these ranges, inclusive. After passing through the compression region, the fluid pathway expands in an expansion region. In some embodiments, the expansion region may generate turbulence and vortexes. In some examples, the expansion region may provide a controlled, precise laminar flow. The diameter of the expansion region can range from about 4 mm to about 25 mm.In some embodiments, the diameter of the expansion zone may be about 4 mm, about 6 mm, about 8 mm, about 10 mm, about 12 mm, about 14 mm, about 16 mm, about 18 mm, about 20 mm, about 22 mm, about 24 mm, or about 25 mm, or about 4-6 mm, about 6-8 mm, about 8-10 mm, about 10-12 mm, about 12-14 mm, about 14-16 mm, about 16-18 mm, about 18-20 mm, about 20-22 mm, about 22-24 mm, or about 24-25 mm, or any value within these ranges, inclusive. After passing through the expansion zone, the subsequent contraction zone subjects the sample to significant shear stress. As noted above, the diameter of the compression zone may range from about 0.5 mm to about 3.0 mm. In some embodiments, the length, width, or diameter may be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, or any value within these ranges, inclusive. In some embodiments, the shapes of the expansion and contraction regions, along with the overall design, may be circular, oval, square, and rectangular, although other shapes may be used.

[0113] 11A-11B illustrate one embodiment of a microfluidic chip 722 having a single extension region. FIG. 11A illustrates a side view of the microfluidic chip 722, and FIG. 11B illustrates a perspective view of the microfluidic chip 722. Fluid flow through the fluid pathways of the microfluidic chip 722 is described in more detail above. The microfluidic chip 722 includes a first port 721a at a first end of the microfluidic chip 722 and a second port 721b at a second end of the microfluidic chip 722. The first port 721a may include an opening 723a that is fluidly connected to the first end of the fluid pathway of the microfluidic chip 722. The second port 721b may include an opening 723b that is fluidly connected to the second end of the fluid pathway of the microfluidic chip 722. The fluid pathway of the microfluidic chip 722 may extend from the opening 723a to the opening 723b. In some embodiments, the fluid path of the microfluidic chip 722 is symmetrical. As shown in FIGS. 11A-11B, the fluid path of the microfluidic chip 722 may include a fluidically connected first channel 726a, an extension 727a, and a second channel 726b. As described above, the fluid sample may experience significant shear stress in the first channel 726a and the second channel 726b. In the extension 727a, the fluid sample may experience turbulence and vortex motion. In some embodiments, the ratio of the first channel 726a to the extension 727a and / or the ratio of the second channel 726b to the extension 727a ranges from about 1:1 to about 1:83.

[0114] 12A-12B illustrate one embodiment of a microfluidic chip 822 having two expansion regions and a compression region between the two expansion regions. FIG. 12A illustrates a side view of the microfluidic chip 822, and FIG. 12B illustrates a perspective view of the microfluidic chip 822. Fluid flow through the fluid pathways of the microfluidic chip 822 is described in more detail above. The microfluidic chip 822 may include a first port 821a at a first end of the microfluidic chip 822 and a second port 821b at a second end of the microfluidic chip 822. The first port 821a may include an opening 823a that is fluidly connected to the first end of the fluid pathways of the microfluidic chip 822. The second port 821b may include an opening 823b that is fluidly connected to the second end of the fluid pathways of the microfluidic chip 822. The fluid pathways of the microfluidic chip 822 may extend from the opening 823a to the opening 823b. In some embodiments, the fluid path of the microfluidic chip 822 is symmetrical. As shown in FIGS. 12A-12B, the fluid path of the microfluidic chip 822 may include a fluidically connected first channel 826a, a first expansion portion 827a, a compression portion 828, a second expansion portion 827b, and a second channel 826b. As described above, the fluid sample in the first channel 826a, the compression portion 828, and the second channel 826b may be subjected to significant shear stress. Turbulence and vortexing may occur in the fluid sample in the first expansion portion 827a and the expansion portion 827b. In some embodiments, the ratio of the channels 826a, 826b to the expansion portions 827a, 827b may range from about 1:1 to about 1:83. In some examples, the ratio of the compression portion 828 to the expansion portions 827a, 827b may range from about 1:1 to about 1:83.

[0115] 13A-13B illustrate one embodiment of a microfluidic chip 922 having three expansion regions and two compression regions between the three expansion regions. FIG. 13A illustrates a side view of the microfluidic chip 922, and FIG. 13B illustrates a perspective view of the microfluidic chip 922. Fluid flow through the fluid pathways of the microfluidic chip 922 is described in more detail above. The microfluidic chip 922 may include a first port 921a at a first end of the microfluidic chip 922 and a second port 921b at a second end of the microfluidic chip 922. The first port 921a may include an opening 923a that is fluidly connected to the first end of the fluid pathways of the microfluidic chip 922. The second port 921b may include an opening 923b that is fluidly connected to the second end of the fluid pathways of the microfluidic chip 922. The fluid pathways of the microfluidic chip 922 may extend from the opening 923a to the opening 923b. In some embodiments, the fluid path of the microfluidic chip 922 is symmetrical. As shown in FIGS. 13A-13B, the fluid path of the microfluidic chip 922 may include a fluidically connected first channel 926a, a first expansion portion 927a, a first compression portion 928a, a second expansion portion 927b, a second compression portion 928b, a third expansion portion 927c, and a second channel 926b. As described above, the fluid sample in the first channel 926a, the first compression portion 928a, the second compression portion 928b, and the second channel 926b may be subjected to significant shear stress. Turbulence and vortexing may occur in the fluid sample in the first expansion portion 927a, the second expansion portion 927b, and the third expansion portion 927c. In some embodiments, the ratio of channels 926a, 926b to extension portions 927a, 927b, 927c can range from about 1:1 to about 1:83. In some examples, the ratio of constricted portions 928a, 928b to extension portions 927a, 927b, 927c can range from about 1:1 to about 1:83.

[0116] The examples of microfluidic chips shown in Figures 11A-11B, 12A-12B, and 13A-13B are intended to be illustrative and non-limiting. The microfluidic chip may include any number of expansion and / or compression regions to achieve the intended result.

[0117] [Sample chamber] 14A-14B illustrate a sample chamber 290 capable of holding a sample during processing or for storage before or after processing (e.g., during transport). The sample chamber 290 includes a body 291a having a port 293 disposed at a first end thereof. The port 293 of the sample chamber 290 may form a luer lock connector for connection to a syringe for sample injection and extraction. The port 293 may also be fluidly connected to the microfluidic chip 222. As shown in FIGS. 4A-4B, the port 293 of the sample chamber 290 may include an engagement surface 298 configured to engage with either the first port 221a or the second port. The port 293 of the sample chamber 290 may include an opening 294 that allows the sample to flow into or out of the sample chamber 290. The size of the opening 294 may range from about 0.5 mm to about 3 mm. In some embodiments, the length, width, or diameter may be about 0.5 mm, about 1 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, or about 0.5-1 mm, about 1-1.5 mm, about 1.5-2.0 mm, about 2.5-3.0 mm, or any value within these ranges, inclusive. Port 293 may also be a different type of luer connector, such as a slip luer. As shown in FIG. 14A, port 293 of sample chamber 290 may be located near the bottom of sample chamber 290 to facilitate fluid flow into microfluidic chip 222. In some embodiments, port 293 may be located higher in sample chamber 290. 14A, the sample chamber 290 may include a vent hole 292a at the top of the sample chamber 290, which allows for smooth fluid flow when the chip and chamber are fully assembled and prevents a vacuum from forming within the sample chamber 290 and the fluidically connected microfluidic chip 222. The body 291a of the sample chamber 290 may include tapered surfaces 291b and tapered surfaces 291b disposed on either side of the port 293.In some embodiments, tapered surface 291b may provide for smoother flow and complete drainage of the fluid sample within sample chamber 290 during centrifugation and extraction. In some embodiments, tapered surface 291b may have an angle of more than 25 degrees from the front surface of the chamber.

[0118] 14C-14D show the interior of sample chamber 290. Sample chamber 290 includes a retention cavity 296 fluidly connected to vent channel 292b, vent chamber 292c, and vent hole 292a. The internal retention cavity 296 can hold a sample during use. The volume of retention cavity 296 can range from approximately 1 mL to approximately 300 mL. Sample chamber 290 can include an arrow-shaped vent chamber 292c at the top of sample chamber 290. Vent chamber 292c can capture any sample that enters the vent channel and prevent the sample from exiting sample chamber 290. During use, the fluid sample within vent chamber 292c is subjected to centrifugal force. The arrow-shaped shape of vent chamber 292c forces the fluid sample into the corners of vent chamber 292c and away from vent hole 292a at the top during use. When the direction of centrifugal force is reversed, the fluid sample may be forced back through vent channel 292b and into retention cavity 296. The volume of vent chamber 292c may range from about 0.5 mL to about 50 mL. In some embodiments, a Luer adapter may be added to the back of the chamber as an alternative extraction and filling point. In some embodiments, sample chamber 290 may include a carriage-compatible ridge 297 that may be added to securely engage a portion of a carriage.

[0119] [filter] 15A-15B illustrate one embodiment of a filter that can be positioned between the sample chamber and the microfluidic chip in the chip assembly. As shown in FIGS. 16A-16B, a filter can be positioned upstream or before the microfluidic chip to filter the sample to prevent clogging of the microfluidic chip. As described in more detail below, the filter can include a mesh configured to cut or pulverize tissue or tissue fragments so that the sample can pass through the microfluidic chip without clogging. Cutting or pulverizing the sample can generate macroscopic aggregates for the purposes of microfluidic shearing in the microfluidic chip. As shown in FIGS. 16A-16B, a filter can be positioned downstream or after the microfluidic chip to allow only samples of a certain size to pass out of the device for collection.

[0120] 15A-15B illustrate one embodiment of a filter 1000. The filter 1000 may include a first body portion 1100 and a second body portion 1200. The first body portion 1100 may include a first side having a port 1110. The port 1110 may include an engagement surface 1120 configured to engage a first or second port of any of the disclosed microfluidic chips. The engagement surface 1120 of the port 1110 may be configured to engage a port of the sample chamber 290 (i.e., port 293). The port 1110 may include a channel 1140 that extends from a first opening 1130 on the first side of the first body portion 1100 to a second opening 1150 on the second side of the first body portion 1100.

[0121] The second body portion 1200 may include a first side having a port 1210. The port 1210 may include an engagement surface 1220 configured to engage with either the first or second port of any of the disclosed microfluidic chips. The engagement surface 1220 of the port 1210 may be configured to engage with a port of the sample chamber 290 (i.e., port 293). The port 1210 may include a channel 1240 that extends from a first opening 1230 on the first side of the second body portion 1200 to a second opening 1250 on the second side of the second body portion 1200. As shown in FIG. 15A , the second side of the second body portion 1200 may include a mesh filter 1260. As described above, the mesh filter 1260 can filter samples to prevent clogging of the microfluidic chip and / or ensure that only samples of a certain size are passed out of the microfluidic chip for collection. In some embodiments, the mesh size of mesh filter 1260 is about 1 μm to about 2000 μm, about 1 μm to about 100 μm, about 100 μm to about 200 μm, about 200 μm to about 300 μm, about 300 μm to about 400 μm, about 400 μm to about 500 μm, about 500 μm to about 600 μm, or about 600 μm to about 700 μm. μm, about 700 μm to about 800 μm, about 800 μm to about 900 μm, about 900 μm to about 1000 μm, about 1000 μm to about 1100 μm, about 1100 μm to about 1200 μm, about 1200 μm to about 1300 μm, about 1300 μm to about 1400 μm, about 1400 μm to about 1500 μm, about 1500 μm to about 1600 μm, about 1600 μm to about 1700 μm, about 1700 μm to about 1800 μm, about 1800 μm to about 1900 μm, or about 1900 to about 2000 μm, or any value within these ranges, including the upper and lower limits.

[0122] 16A-16B show diagrams of chip assembly 2020 having first filter 1000a and second filter 1000b on opposite ends of microfluidic chip 2022. FIG. 16B shows an exploded view of chip assembly 2020. As shown in FIG. 16B, in some embodiments, filter 1000a may be positioned such that port 1110a of first body portion 1100a is fluidly connected to sample chamber 2090a and port 1210a of second body portion 1200a is fluidly connected to first port 2021a of microfluidic chip 2022. As shown in FIG. 16B, filter 1000b may be positioned such that port 1110b of first body portion 1100b is fluidly connected to sample chamber 2090b and port 1210b of second body portion 1200b is fluidly connected to second port 2021b of microfluidic chip 2022. 16A-16B show only one orientation of filters 1000a, 1000b on chip assembly 2020. In some embodiments, on one or both filters 1000, filters 1000 may be oriented such that port 1110 of first body portion 1100 is connected to a port of a sample chamber and port 1210 of second body portion 1200 is connected to a port of a microfluidic chip. In some embodiments, on one or both filters 1000, filters 1000 may be oriented such that port 1210 of second body portion 1200 is connected to a port of a sample chamber and port 1110 of first body portion 1100 is connected to a port of one of the microfluidic chips. In some embodiments, chip assembly 2020 may include filters 1000 on only one end or both ends of microfluidic chip 2022.

[0123] Filter 1000 may be manufactured using a variety of methods, such as additive manufacturing, subtractive manufacturing, 3D printing, injection molding, resin molding, or urethane casting. Filter 1000 can be made from a variety of materials, such as plastic (e.g., polycarbonate, acrylic, etc.), polyurethane, or metal (e.g., aluminum, steel, stainless steel, surgical steel, brass, copper, etc.).

[0124] [carriage] As mentioned above, the carriage 230 may include the base platform 250, the spring platform 240, and the clamp 210. Assembly of the carriage 230 is described in more detail above with reference to Figures 3A-3B.

[0125] 17A-17B illustrate one embodiment of the base platform 250. The base platform 250 may include a raised wall at one or both ends of the base portion. As shown in FIGS. 17A-17B, the base platform 250 may include a first arm 256a and a second arm 256b configured to engage with mating portions on the spring platform 240. In some embodiments, the first arm 256a may include a mating slot 257a on its side, and the second arm 256b may include a mating slot 257b on its side. Additionally, the first arm 256a may include a retaining lip 259a on its top, and the second arm 256b may include a retaining lip 259b on its top, to secure the tip assembly 220 on the carriage. On the top surface of body 253 of base platform 250, body 253 includes terminal spring holder (distal spring holder) 254a and terminal spring holder (distal spring holder) 254b for holding a base portion of a spring. Body 253 further includes shoulder 236 configured to engage clamp 210. Body 253 includes first opening 236a and second opening 236b configured to receive screws for securing clamp 210 to base platform 250. A bottom surface of body 253 of base platform 250 may include post 258 having a through hole extending between terminal opening (distal opening) 258a and opening 252. The bottom surface of body 253 also includes a plurality of pedestals (i.e., pedestals 251a and 251b). The support posts 258 and the pedestal (i.e., pedestal 251a and pedestal 251b) are configured to reversibly interact and hold the carriage 230 to one of the lateral arms 263 of the base portion 260. The base platform 250 may be manufactured using a variety of methods, such as additive manufacturing, subtractive manufacturing, injection molding, resin molding, or urethane casting. The base platform 250 can be made from a variety of materials, such as plastic (e.g., polycarbonate, acrylic, etc.), polyurethane, or metal (e.g., aluminum, steel, stainless steel, surgical steel, brass, copper, etc.).

[0126] 2B, the bottom surface of base platform 250 may include a plurality of capture elements that serve to hold carriage 230 in a desired position until a signal (or force, or lack thereof) is received that allows the capture elements to disengage or cease interaction with the release elements, after which carriage 230 can rotate, followed by re-engagement of the capture elements to stop carriage 230 movement. In some embodiments, this allows carriage 230 to rotate through a 180-degree arc at a desired time in a tissue processing protocol. In some instances, the capture elements include magnets of a first polarity and the release elements include magnets of the opposite polarity.

[0127] 2B, the capture element is a plurality of pedestals. The pedestals may include pedestals 251a and 251b. Each of pedestals 251a and 251b is configured to engage a pedestal-engaging opening 266 in transverse arm 263 to allow intermittent rotation of each of carriages 230 about openings 264 in transverse arm 263. Pedestals 251a and 251b may serve to hold carriage 230 in a desired position until a signal or force is applied that disengages pedestals 251a and 251b from the plurality of pedestal-engaging openings 266, thereby allowing rotation of the carriage about opening 264 and subsequent re-engagement of pedestals 251a and 251b with the plurality of pedestal-engaging openings 266.

[0128] Each of the plurality of carriages 230 is held in an opening in a respective one of the plurality of arms using at least one pin (i.e., post 258) configured to allow out-of-plane rotation for each carriage 230. The out-of-plane rotation of each of the plurality of carriages 230 can move each of the plurality of carriages 230 between a plurality of orientations. For example, each of the plurality of carriages 230 can move between 180-degree rotations (in-plane or out-of-plane). In some embodiments, each of the plurality of carriages 230 can move between orientations in which each of the plurality of chambers lies along the plane of a respective one of the plurality of arms. Each of the plurality of carriages 230 can move between 180-degree rotations (e.g., 0-45 degrees, 45-90 degrees, 90-135 degrees, 135-180 degrees, etc.).

[0129] 18A-18B illustrate one embodiment of spring platform 240. As described above, spring platform 240 may include first mating region 242a at a first end of spring platform 240 and second mating region 242b at a second end of spring platform 240. Spring platform 240 has a top surface that includes guide platform 248 disposed on a side of spring platform 240 and tip mating tab 246 disposed on a side of spring platform 240 opposite guide platform 248. Spring platform 240 has a bottom surface that includes proximal spring holder 244a and proximal spring holder 244b configured to respectively engage with the top of spring 234. 3A-3B, the first mating region 242a and the second mating region 242b are configured to secure the first arm 256a and the second arm 256b of the base platform 250, respectively. The chip mating tabs 246 of the spring platform 240 can mate with openings in the clamp 210. The spring platform 240 may include a guide platform 248, which can be used to guide the microfluidic chip 222 onto the spring platform 240 and assist in compressing the spring platform 240 when the microfluidic chip 222 is inserted into the carriage 230. The spring platform 240 may also include a splash guard 245 that overhangs the spring platform 240 to prevent fluid from being introduced into the springs 234 below the spring platform 240. Proximal spring holder 244a and proximal spring holder 244b can help hold and secure spring 234 in place during installation and use of carriage 230. Spring platform 240 can be sized and shaped to fit any shape or size of microfluidic chip 222.The spring platform 240 may be manufactured using a variety of methods, such as additive manufacturing, subtractive manufacturing, 3D printing, injection molding, resin molding, or urethane casting. The spring platform 240 can be made from a variety of materials, such as plastic (e.g., polycarbonate, acrylic, etc.), polyurethane, or metal (e.g., aluminum, steel, stainless steel, surgical steel, brass, copper, etc.).

[0130] 19A-19B illustrate one embodiment of a clamp 210. The clamp 210 can help hold the microfluidic chip 222 in place during use. The clamp 210 can include a finger groove 211 on the top of the clamp 210, allowing a user to easily access the microfluidic chip 222 and remove it from the carriage 230 as needed. As shown in FIGS. 3A-3B, the clamp 210 can include a base platform mounting region including a first mounting flange 212a having a first opening 214a and a second mounting flange 212b having a second opening 214b. The base platform mounting region 214 can be mated to a shoulder 236 of a base platform 250 by a plurality of screws (e.g., first screw 238a and second screw 238b). The clamp 210 may include a channel 215 that forms a groove that fits over the chip mating tab 246 and helps hold and position the spring platform 240. The height of the channel 215 allows the chip mating tab 246 to move vertically within the channel 215, guiding the movement of the spring platform 240 in one direction. The clamp 210 may include guide rails 213a and 213b that use grooves on the clamp 210 to help guide and position the microfluidic chip 222 within the carriage 230. The clamp 210 may also include multiple openings 215. The openings 215 help reduce the weight of the device. The openings 215 also allow a user to view the microfluidic chip 222 and / or adjust or remove the microfluidic chip 222 from the carriage 230. Clamp 210 may be manufactured using a variety of methods, such as additive manufacturing, subtractive manufacturing, injection molding, 3D printing, resin molding, or urethane casting. Clamp 210 can be made from a variety of materials, such as plastic (e.g., polycarbonate, acrylic, etc.), polyurethane, or metal (e.g., aluminum, steel, stainless steel, surgical steel, brass, copper, etc.).

[0131] [Other components] 20A-20B illustrate a screw cap 270 configured to hold the base platform 250 of the carriage 230 in place. The screw cap 270 can include a proximal end 270a and a distal end 270b. The proximal end 270a includes a proximal opening 272. The proximal end 270a of the screw cap 270 is shaped to fit over the post 258 on the bottom surface of the base platform 250. While the screw cap 270 illustrated in FIGS. 20A-20b depicts the screw cap 270 with a hexagonal proximal opening 272, the proximal opening 272 can be any shape capable of receiving the post 258 of the base platform 250. The screw cap 270 also includes a throughbore extending between the proximal end 270a and the distal end 270b. The throughbore allows the screw 232 to secure the screw cap 270 and the base platform 250 to each other. Screw cap 270 may include a circular proximal end 270a. As described above, proximal end 270a of screw cap 270 may be configured to extend through opening 264 in base portion 260. Screw cap 270 may be manufactured using a variety of methods, such as additive manufacturing, subtractive manufacturing, 3D printing, injection molding, resin molding, or urethane casting. Screw cap 270 may be made from a variety of materials, such as plastic (e.g., polycarbonate, acrylic, etc.), polyurethane, or metal (e.g., aluminum, steel, stainless steel, surgical steel, brass, copper, etc.).

[0132] 21A-21B show multiple rotor foot inserts 280. The rotor foot inserts 280 may include a ramp 282 at the base (proximal) end of the rotor foot insert 280 and a lip 284 at the base of the rotor foot insert 280. The rotor foot inserts 280 can assist in smooth rotation of the carriage 230. The ramp 282 of each rotor foot insert 280 allows the pedestals (i.e., pedestals 251a and 251b) on the bottom surface of the base platform 250 to move smoothly across the rotor base surface when the carriage 230 is rotated. Each rotor foot insert 280 may also include a lip 284 that fits into one of the notches 268 in the lateral arms 263 of the base portion 260 to help secure each rotor foot insert 280 in place during use. The rotor foot insert 280 may be manufactured using a variety of methods, such as additive manufacturing, subtractive manufacturing, 3D printing, injection molding, resin molding, or urethane casting. The rotor foot insert 280 may be made from a variety of materials, such as plastic (e.g., polycarbonate, acrylic, etc.), polyurethane, or metal (e.g., aluminum, steel, stainless steel, surgical steel, brass, copper, etc.).

[0133] FIG. 22 illustrates one embodiment of a base portion 260. As described above, the base portion 260 can include a central portion 261 and a plurality of lateral arms 263 extending radially away from the central portion 261 of the base portion 260. While the base portion 260 in FIG. 22 illustrates the base portion 260 having three lateral arms 263, the base portion 260 can include any number of lateral arms 263 configured to reversibly engage a carriage. In some embodiments, each of the lateral arms 263 is configured to lie in a plane parallel to the plane of the central portion 261. As described above, each of the lateral arms 263 can include an opening 264, at least one pedestal-engaging opening 266, and a plurality of notches 268. As shown in FIG. 2B, the opening 264 can be configured to receive the base of the post 258, and the plurality of notches 268 are configured to engage the pedestals 251 a and 251 b on the bottom surface of the base platform 250. Each of the notches 268 is configured to receive one of the rotor foot inserts 280. The base portion 260 can be configured to engage various microfluidic chips. The base portion 260 can include mounting openings 262 configured to mount to a centrifuge. In some embodiments, the mounting openings 262 are D-shaped chuck mounting openings 262 that mate with a chuck mounted to the centrifuge. The mounting openings 262 have a "D" shape that helps prevent slippage during centrifugation. The base portion 260 also has multiple notches 268 shaped to aid in mounting rotor foot inserts. The base portion 260 also includes carriage mounting holes, which allow the top of the screw cap 270 to mate with the carriage 230 and hold the carriage 230 in place. The base portion 260 also has multiple pedestal mating openings 266 that mate with pedestals on the bottom surface of the base platform 250 to keep the carriage aligned during use. The base portion 260 may be manufactured using a variety of methods, such as additive manufacturing, subtractive manufacturing, 3D printing, injection molding, resin molding, or urethane casting.The base portion 260 can be made from a variety of materials, such as plastic (eg, polycarbonate, acrylic, etc.), polyurethane, or metal (eg, aluminum, steel, stainless steel, surgical steel, brass, copper, etc.).

[0134] [Other embodiments] 23A-23B illustrate one embodiment of a centrifuge device, in which each carriage 330 includes a clamping and locking mechanism for holding a chip assembly 220 in place during processing. As shown in FIG. 23A, the carriage 330 includes a base platform 350 having a first side 355a and a second side 355b for forming an opening 353. The opening 353 can secure and hold the microfluidic chip 222 of the chip assembly 220.

[0135] The base platform 350 may include a first arm 356a at a first end of the base platform 350 and a second arm 356a at a second end of the base platform 350. The first arm 356a and the second arm 356a form clamps on either end of the carriage 330 to secure the tip assembly 220. The clamps formed by the first arm 356a and the second arm 356a can be separated and pulled apart with one hand to allow the tip assembly 220 to be removed from the base platform 350. In some embodiments, the carriage 330 may be positioned on each of the lateral arms 263 of the base portion 260 to allow it to rotate 360 ​​degrees for reprocessing. The carriage 330 may include pedestals at both ends of the carriage 330 to help secure the carriage 330 in an orientation parallel to the wings (as described above). The carriage 330 is otherwise similar to the carriages described elsewhere herein.

[0136] FIG. 24 illustrates a centrifuge device having an alternative fastener to the clamp 210 described above. As shown in FIG. 24, the carriage 430 utilizes a clamp and locking mechanism to hold the chip assembly 420 in place during processing. As shown, the base platform 450 of the carriage 430 may include a first side 455a and a second side 455b. A hinge 457 may be disposed on the second side 455b of the base platform 450, and an engagement portion 456 may be disposed on the first side 455a of the base platform 450. The hinge 457 may movably connect a lid 452 to the base platform 450. The lid 452 may include a window 459 that allows viewing of the microfluidic chip of the chip assembly 420 during processing. The engagement portion 456 may include a slide lock that passes through a latch hole on the side of the lid and engages with a lock stop to hold the chip assembly 420 in place on the carriage 430. In some embodiments, the latch can be released with one hand by pulling the slide lock away from the carriage to release the latch. The slide lock is then slid back through the latch hole in the lid to unlock the device. The lid 452 can swing open using hinge 457 on the second side 455b. The carriage 430 in FIG. 24 can rotate 360 ​​degrees for reprocessing. The carriage 430 may include a base on each end of the carriage 430 to help secure the carriage 430 in an orientation parallel to the wings (as described above). The carriage 430 is otherwise similar to the carriages described elsewhere herein.

[0137] 25A-25B show an embodiment of a centrifuge device further including a motor 3000a attached to the bottom of the rotor base that connects to a screw cap 270. The motor 3000a can be battery powered or connected to a power source via the base 260.

[0138] 26A-26B show one embodiment of a centrifuge device that includes a motor 3000b mounted to the bottom of a base 260 that connects to a post 258 on the underside of a base platform 250. The motor 3000b can be battery powered or connected to a power source through the base 260.

[0139] FIGS. 27A-27D show another embodiment of a centrifuge 500 for processing biological samples. The centrifuge 500 shown in FIG. 28 provides a multi-purpose centrifuge 500 for use in various stages of a processing protocol. The base 560 of the centrifuge 500 can hold multiple syringes. As shown in FIG. 27C, the centrifuge 500 includes a base 560 having a central portion 561 with a mounting opening 562 and multiple lateral arms 563. Each of the lateral arms 563 is similar to the lateral arms 263 of the base 260 described above. Like the lateral arms 263 of the base 260, the lateral arms 563 of the base 560 include openings 564 for receiving the posts 258 of the base platform 250 of the carriage 230, allowing the carriage 230 to rotate around the openings 564. The transverse arms 563 also include a plurality of base-engaging openings 566 that hold the bases (e.g., bases 251a and 251b) of the carriage 230 in a desired position until a force is applied or removed to release the base. The base 560 further includes a plurality of syringe-retaining arms 565a disposed between adjacent transverse arms 563. The syringe-retaining arms 565a may include a flared distal end 568 having an opening 565b. The distal end 568 may be angled or chamfered so that the opening 565b is not perpendicular to the syringe-retaining arms 565a. The flared distal end 568 may angle the opening 565b at an angle relative to the plane of the syringe-retaining arms 565a. In some examples, the opening 565b is angled at the distal end 568 between about 100 and 130 degrees. In some embodiments, end portion 568 may include opening 565b at a 90-degree angle. The configuration shown in Figures 27A-27D allows multiple samples to be processed to activate cells, and a syringe can be used to subject additional samples to gradient separation. This can be important because specific subpopulations of cells from a sample can be isolated and then activated by processing via a microfluidic chip located on a carriage on one of lateral arms 263 of base portion 260.

[0140] As shown in FIG. 27D , multiple openings 565b allow for placement of multiple syringes 567a through openings 565b. Each opening 565b is positioned with needle hub 567e distal to plunger 567d. In some embodiments, syringe 567a includes a flange 567c that rests against flared end 568 to retain syringe 567a during centrifugal force. In some embodiments, opening 565b in end 568 can retain syringe 567a at an angle between 0 and 80 degrees from a horizontal position. In some examples, opening 565b in end 568 can retain syringe 567a at an angle between 0 and 45 degrees from a vertical position. End 568 of syringe retaining arm 565a is configured to fit multiple (e.g., three) syringes 567a simultaneously and provide maximum centrifugal force on the syringes. 27E-27F, distal end 568 of syringe holding arm 565a allows each of syringes 567a to slide into place. Opening 565b of distal end 568 may include gap 569 that allows distal end 568 of syringe holding arm 565a to expand to accommodate a variety of syringes. For example, opening 565b of distal end 568 of syringe holding arm 565a may be used with syringes holding volumes ranging from 1 mL to 100 mL.

[0141] In some embodiments, the distal end 568 of the syringe holding arm 565a may be machined or grooved to approximate the shape of the particular barrel 567b of the syringe 567a. In some embodiments, the distal end 568 of the syringe holding arm 565a may receive and hold an insert, which is sized to hold any number of additional syringes. The insert may be configured such that the number of additional syringes is inversely proportional to the respective diameters of the openings for holding the syringes.

[0142] Use of centrifuges to process biological samples. The centrifuge devices disclosed herein process biological samples so that the tissue can be reinfused or reapplied to a patient to provide repair and / or regeneration. In some embodiments, the biological sample may be adipose tissue, although other types of tissue may be processed using the systems and methods disclosed herein. In some embodiments, adipose tissue, tumor tissue, cell preparations, lipoaspirates, cultured cells, and other similar biological samples may be processed. In some instances, the biological sample contains particles (e.g., nanoparticles, magnetic particles, reagent- or antibody-coated particles, etc.). In some embodiments, the sample contains a cell-containing fluid.

[0143] 28A illustrates one embodiment of a method 4000 for processing a biological sample. In the method for processing the biological sample 4000, in step 4100, the biological sample is inserted into a first sample chamber. Referring to the centrifuge device 200 shown in FIGS. 2A-2B as a non-limiting example, in some embodiments, the biological sample may be loaded into one of the sample retention chambers 290a or 290b, which are fluidly coupled to the microfluidic chip 222 at either end of the microfluidic chip 222. As described above, the fluid pathways of the microfluidic chip 222 are configured to allow the biological sample to pass bidirectionally between the sample retention chamber 290a, the microfluidic chip 222, and the sample retention chamber 290b.

[0144] In the method 4000 for processing a biological sample, in step 4200, the carriage of the centrifuge device is placed in a first position. The carriage 230 of the centrifuge device 200 may be fixed to the lateral arm 263 of the base portion 260 so as to be rotatable about an axis perpendicular or substantially perpendicular to the plane of the central portion 261 of the base portion 260. During operation of the centrifuge device 200, the carriage 230 starts from the first position in which the first end of the carriage 230 (i.e., the sample holding chamber 290a) is located furthest from the central portion 261. The carriage 230 may be rotated to a second position in which the second end of the carriage 230 (i.e., the sample holding chamber 290b) is located furthest from the central portion 261.

[0145] The method 4000 for processing a biological sample may include a step 4300 in which a rotational force is applied to the centrifuge device. The rotational force may be applied to the centrifuge device 200 such that the sample in the sample retention chamber 290a can pass from the sample retention chamber 290a through the first port 221a to the microfluidic chip 222. The sample can then pass through a fluid path in the microfluidic chip 222 from a first end of the microfluidic chip 222 to a second end of the microfluidic chip 222. The sample can then exit the microfluidic chip 222 and enter the sample retention chamber 290b through the second port 221b.

[0146] The method 4000 for processing a biological sample may include a step 4400 in which the carriage is moved from a first position to a second position. The carriage 230 may then be rotated (i.e., 180 degrees) so that the carriage 230 is moved from the first position to the second position. In some embodiments, the rotation is automated.

[0147] The method 4000 for processing a biological sample may include a step 4500 in which a rotational force is again applied to the centrifuge device. In this second position, a rotational force may be applied to the centrifuge device 200 such that the sample in the sample retention chamber 290b can pass from the sample retention chamber 290b through the second port 221b and into the fluid path of the microfluidic chip 222. The sample can then pass through the fluid path of the microfluidic chip 222 from the first end to the second end of the microfluidic chip 222. The sample can then exit the first port 221a and enter the sample retention chamber 290a. The above steps of the method 4000 for processing a biological sample may be repeated until the sample has been processed to the desired extent.

[0148] The method 4000 for processing biological samples may be used with the centrifuge device 500 shown in Figures 27A-27D. In the centrifuge device 500, each syringe 567a held by the syringe holding arm 565a contains a biological sample in its barrel 567b. When a rotational force is applied to the base portion 560, a centrifugal force is also applied to the biological sample in the syringe 567a, forming a gradient separation of the biological sample. This can be important because it allows for the initial isolation of specific subpopulations of cells from the sample before they undergo activation through processing via the microfluidic chip. Each syringe 567a can be easily detached from the base portion 560, allowing the biological material to be removed from the syringe 567a (or from any sample chamber attached to the microfluidic chip) and reattached to the chip assembly and carriage before further processing of the biological material continues. Thus, the method 4000 for processing biological samples can be tailored to a variety of applications and uses.

[0149] In some embodiments, after processing the biological sample according to method 4000 for processing a biological sample, the processed biological sample may be injected into a patient.

[0150] [Treatment method] [overview] 29 provides a schematic diagram of a non-limiting embodiment of a treatment paradigm. In this example, a subject (subject) in need of tissue repair or regeneration (e.g., having a wound) undergoes a liposuction procedure (e.g., liposuction). The harvested adipose tissue is then processed according to embodiments disclosed herein and reinjected into and / or around the site of the tissue in need of repair and / or regeneration.

[0151] [Administration and Dosage] Further provided herein is a method for treating a subject (subject) with a wound (e.g., DFU), the method comprising administering to the subject a composition comprising mechanically treated ADSCs (adipose-derived stem cells) as disclosed herein. For example, some embodiments of the compositions and methods described herein relate to the use of mechanically treated ADSCs to treat diabetic patients suffering from DFU. Also provided is the use of ADSCs treated as described above to treat DFU. In certain embodiments, treatment of a subject with mechanically treated ADSCs described herein achieves, for example, one, two, three, four, or more of the effects listed below. (i) reducing or ameliorating the severity of a disease or its associated symptoms; (ii) a reduction in the duration of disease-related symptoms; (iii) protection against the progression of the disease or its associated symptoms (iv) regression of the disease or its associated symptoms (v) protection against the progression or onset of disease-related symptoms (vi) protection against recurrence of disease-related symptoms (vii) a reduction in hospitalization of subjects (viii) Reduction in hospital stay (ix) increasing the survival rate of subjects with the disease (x) a reduction in the number of symptoms associated with the disease (xi) enhancing, improving, supplementing, complementing, or augmenting the prophylactic or therapeutic effects of another therapy Each of these comparisons is against a different treatment for the disease, including, for example, performing an invasive procedure to treat deep, complex wounds such as DFUs.

[0152] Administration can be by a variety of routes, including, but not limited to, intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal, and / or local delivery to the affected tissue. The dosage of mechanically processed ADSCs can be readily determined for a given subject based on their body weight, the type and condition of their disease, and the desired aggressiveness of treatment, but depending on the embodiment, approximately 10 cells per gram can be administered. 5 ~about 10 12 range (e.g., 10 5 ~10 7 , 10 7 ~10 10 , 10 10 ~10 12 In some embodiments, the range is from about 1 x 10 cells per gram to about 1 x 10 cells per gram (and overlapping ranges therebetween). In one embodiment, a dose escalation regimen is used. In some embodiments, the range is from about 1 x 10 cells per gram to about 1 x 10 cells per gram (and overlapping ranges therebetween). 6 ~Approx. 1×10 8 ADSCs are administered in a range of 100 mg / kg / day. Depending on the embodiment, various types of wounds can be treated. In some embodiments, DFUs are treated. Further embodiments provided herein include the treatment or prevention of wounds, including, but not limited to, venous stasis ulcers, arterial ulcers, and pressure ulcers (i.e., bedsores).

[0153] The dose of mechanically processed ADSCs can be readily determined for a given subject based on their body weight, the type and condition of their disease, and the desired aggressiveness of treatment, but depending on the embodiment, a dose of approximately 10 cells per gram may be used. 5 ~about 10 12 range (e.g., 10 5 ~10 7 , 10 7 ~10 10 , 10 10 ~10 12 In one embodiment, a dose-escalation regimen is used. In some embodiments, for example, about 1 x 10 cells / g is used.6 ~Approx. 1×10 8 Depending on the embodiment, various types of wounds can be treated.

[0154] [Example] Example 1: Determining optimal CD-LOC processing parameters The primary objective of this portion of the study is to determine the optimal fully automated lab-on-a-chip (CD-LOC) processing parameters that generate the maximum proportion of CD34-DPP4+ / CD55+ and multilineage differentiated stress-resistant cells (Muse cells) with acceptable levels of overall cell viability. This will be accomplished through a block-randomized experiment in which five adipose tissue samples (10 ml each) will be collected from each of 15 diabetic patients. Body mass index (BMI) and anatomical location of adipose tissue collection will be recorded at the time of surgery. Adult diabetic patients will be defined as those with a hemoglobin A1c (HbA1c) greater than 6.5. Additionally, patients receiving any form of immunosuppressive therapy or with active systemic infection will be excluded from the study.

[0155] Based on the current design of the CD-LOC platform, shear force is adjusted by adjusting the RPM of the processing device. Adipose tissue samples are collected, stored at room temperature, and processed within 24 hours. Each sample is thoroughly washed with sterile PBS. Tissue samples per patient are randomized and are divided into 0 (baseline), 25, 50, 75, and 100 kilodynes / cm. 2 A portion of each sample was snap frozen in liquid nitrogen and stored at -80°C for subsequent immunohistochemistry.

[0156] To isolate stem cells from adipose tissue, all tissue samples are digested with 0.1% collagenase at 37°C for 30 minutes. This process is used solely for single-cell isolation and subsequent analysis and is not a factor in device processing for clinical applications. Each resulting pellet is strained through a 100 μm strainer and subjected to RBC lysis prior to final single-cell analysis. Automated cell counts and viability are determined using a dual fluorescent cell counter (Luna-STEM, Logos Biosystems, Annandale, VA). Phenotypic marker analysis is also performed by staining tissue samples for CD45, CD34, and CD31 and analyzing them by flow cytometry. Five processing parameters are therefore included to allow for depiction of the log-linear nature of the induction relationship.

[0157] To test cell activity / efficacy, cells are seeded in triplicate into 96-well plates in standard control medium, and the activity of water-soluble tetrazolium salts is assessed according to the manufacturer's protocol (Dojindo Molecular Technologies, Rockville, MD). To test population doublings, cells are seeded into 1 cm wells of 6-well plates containing control medium. 2 2 x 10 4 Cells are seeded in triplicate at a density of 1000x and placed under standard culture conditions. When the first set of cells approaches 70-80% confluency, the number of cells is counted and evaluated using the following formula (Equation 1):

[0158]

number

[0159] Phenotypic marker analysis is performed by staining cells with the following combination markers: MSC:CD13-APCVio770 / CD45-VioBlue / CD34-PerCP-Vio700 / CD31-FITC / CD73-PE / CD146-APC Muse cells: CD13-APC-Vio770 / CD45-VioBlue / CD34-PerCP-Vio700 / CD31-FITC / SSEA-3-PE DPP4+ / CD55+:CD45-VioBlue / CD34-PerCP-Vio700 / CD31-FITC / CD55-APC, DPP4-APC Apoptosis detection (Annexin-V-FITC kit) All samples underwent transcriptional analysis, treated with TRIzol reagent (Thermo Fisher Scientific) for RNA extraction, reverse transcribed, and then subjected to qRT-PCR analysis (Applied Biosystems Real-Time PCR 7300 System, TaqMan gene expression assays for angiogenesis, cell proliferation, wound healing, senescence, tumorigenesis, and cell survival). Key targets included DPP4, CD55, HIF-1α, IL-6, TNF-α, PPARγ, HGF, VEGF, CXCL2, SCUBE3, DLL1, NR4A2, ADAMTS9, AK5, SOX2, RPA1, SGK1, HGF, IGF-1, SDF-I, PDGF-B, NGF-β, SCF, bFGF, POU5F1, and REX1.

[0160] The samples were also subjected to secretome analysis. Each sample was seeded (2 × 10 cells) in serum-free medium (StemPRO MSC medium, ThermoFisher Scientific) in a T75 culture flask. 6 Cells are cultured in a 0.22 μm filter and placed under standard culture conditions until confluent. The culture medium (CM) is collected, centrifuged, and saved for subsequent assays. The conditioned medium is thawed and filtered using a 0.22 μm filter. Enzyme-linked immunosorbent assay (ELISA) kits are purchased (R&D Systems, Minneapolis, MN, or Signosis, Santa Clara, CA) and the medium is assayed for cytokines (VEGF, HGF, IGF-1, SDF-1, PDGF-BB, NGF-β, SCF, bFGF, TNF-α) according to the manufacturer's protocol. Absorbance is measured spectrophotometrically at 450 nm using an Infinity microplate reader.

[0161] The paracrine activity of cells was tested using cell migration and scratch assays. For the cell migration assay, a Boyden chamber (Neuroprobe, Gaithersburg, MD) was used. The Boyden chamber was equipped with an 8-μm pore polycarbonate filter (Nucleopore, Whatman Incorporated, Clifton, NJ) coated with 5 μg / mL gelatin solution, and 1 × 10 ADSC fibroblasts (keratinocytes) were placed in migration medium. 5 Cells are added at a concentration of 1000 μg / ml. The lower compartment of the Boyden chamber is filled with ADSC-conditioned medium, and the plate is placed under standard culture conditions. After 16 hours, the filters are removed from the Boyden chamber, fixed in 4% paraformaldehyde / PBS, stained with 0.5% crystal violet, and counted for migration under a microscope.

[0162] In the scratch assay, monolayers of keratinocytes and fibroblasts are plated in 6-well plates containing control medium and placed under standard culture conditions. Once confluent, the center of each well is scratched with a P-200 tip to create a uniform cell-free zone. Cell debris is removed with a PBS wash step, and wells are treated with CM from control and experimental groups, as well as serum-starvation medium, which serves as a negative control. Cultures are photographed at successive time points to assess migration.

[0163] Preliminary data was obtained and additional data prepared using the methods and experiments detailed above. The preliminary data were obtained using the "microfat" treatment (75 kilodynes / cm 2) results in a four-fold reduction in the number of nucleated cells per mg of tissue compared to untreated tissue. Furthermore, no significant reduction in the viability of these cells was observed. Furthermore, preliminary data indicate that automated processing of "microfat" at increasing shear stress leads to a logarithmically correlated stem cell phenotype versus a linear relationship when processed manually. Finally, based on pilot data, cell viability is optimally maintained at 75 kilodynes / cm. 2 90% at a shear force of 100 kilodynes / cm 2 The shear force reduces the adipose tissue to 50%. These results demonstrate that the shear forces involved in microfat processing lead to a significant upregulation of the regenerative phenotype. Further processing under various parameters also allows for the conversion of adipose tissue to nanofat.

[0164] [Statistical method] A linear mixed-effects regression was performed, with the unit of analysis being the tissue sample from the patient. The outcome variable was the percentage of CD34+ cells. The fixed effects included shear force. The random effects were tissue samples from the same patient. Based on this mixed-effects model, the mean percentage of CD34+ cells at each treatment rate was calculated, taking into account intra- and inter-patient heterogeneity.

[0165] [Sample size and power analysis] Based on pilot data, cell viability is optimally 75 kilodynes / cm 2 90% at a shear force of 100 kilodynes / cm 2 Thus, as shear force increases, the mean percentage of CD34+ cells increases linearly, reaching 75 kilodynes / cm 2It is hypothesized that a shear force of 1000 s will reach a maximum level, resulting in cell viability exceeding 75%. Using 15 diabetic patients and five tissue samples per patient, 82% power will be achieved to test this hypothesis based on a two-tailed paired t-test at a 5% significance level. Second, it is expected that subpopulations of MSCs, Muse, and diabetic wound-healing cells will reflect CD34 activity. Furthermore, it is expected that a non-tumorigenic transcriptional pattern similar to that observed in previous adipose-derived Muse cell populations will be observed.

[0166] Example 2: Characterization of microfragmented adipose tissue Experiments were conducted to characterize the adipose tissue resulting from processing using the methods and systems disclosed herein. The adipose tissue was processed according to the non-limiting approach disclosed in Example 1. In some embodiments, because the ultimate treatment paradigm is an autologous transplantation approach (see, e.g., FIG. 29), adipose tissue was harvested from both healthy and diabetic donors to determine whether diabetic adipose tissue behaves similarly to that of healthy subjects.

[0167] The cellular profile of the resulting microfragmented adipose tissue was assessed by determining changes in the CD26+ / CD55+ cellular composition of the processed tissue. The CD26+ / CD55+ phenotype is indicative of an adipose-derived stem cell subtype and is believed to enhance the ability of the resulting cells to contribute to tissue repair and / or regeneration. Figure 30A shows that processing adipose tissue using the systems and methods disclosed herein results in an increased percentage of CD26+CD55+ cells in the processed adipose tissue (labeled "Syntr") compared to unprocessed tissue. Furthermore, this holds true whether the processed tissue is from a healthy or diabetic subject. Thus, regardless of whether the donor adipose tissue is from a healthy or diabetic subject, processing the adipose tissue according to the methods disclosed herein and using the systems disclosed herein results in a higher percentage of the resulting population within the adipose tissue having a regenerative phenotype. Figure 30B also demonstrates a similar phenomenon for the detection of CD34+ cells (e.g., stem cells).

[0168] Cell density and cell viability within the treated adipose tissue were also assessed. These data are shown in Figures 31A and 31B. As seen in Figure 31A, processing adipose tissue significantly reduces the number of cells per milliliter of adipose tissue (whether considering healthy or diabetic adipose samples). Figure 31B shows that the viability of cells within treated adipose tissue (e.g., cells that survived the processing procedure) is maintained at a level that is not statistically significantly different from untreated tissue. Taken together with the data from Figure 30, this indicates that processing adipose tissue as disclosed herein not only reduces cell density within the treated tissue, but also demonstrates that the remaining cells are as viable as those in untreated tissue, with the resulting cell population being enriched for cells with stem cell-associated phenotypes, suggesting that the post-treated adipose tissue has an improved ability to induce, promote, or facilitate tissue repair and / or regeneration.

[0169] Furthering this concept of enriched cell populations for promoting tissue repair and / or regeneration, when we examine the cell types present in the tissue after treatment, we see that various types of stem cells are classified as increasing in relative proportion in the cell population after treatment. For example, Figure 32A shows that the percentage of endothelial progenitor cells (EPCs) in the treated tissue is significantly increased, whether using healthy or diabetic adipose tissue. Similarly, Figure 32B shows a significant increase in the relative proportion of mesenchymal stem cells (MSCs) after treatment. Finally, Figure 32C shows an increase in the percentage of pro-regenerative Muse cells after adipose tissue treatment.

[0170] Similar data reflecting this overall trend of reduced cell numbers coupled with relative enrichment of regenerative cell phenotypes are shown in Figures 33A-33C. Furthermore, these figures compare different adipose processing approaches. Macrofat (MF) represents unprocessed adipose tissue. Nanofat (NF30) represents adipose tissue manually processed by passing the tissue between two syringes 30 times. Each of the remaining data points represents adipose tissue processed using the system disclosed herein for 10, 20, or 30 repetitions. As seen in Figure 33A, adipose tissue processing does not essentially alter the overall viability of the resulting cell types that survive the processing procedure. Figure 33B shows that mechanical processing of adipose tissue results in a reduction in overall cell numbers. Notably, the resulting cell numbers remain stable whether the adipose tissue processing method disclosed herein is used 10, 20, or 30 times. Figure 33C presents additional data related to specific subtypes of cell populations within adipose tissue for each of the histograms. Multiple groups are displayed, with MF, NF30, Syntr10, Syntr20, and Syntr30 repeated in the same order from left to right. The leftmost histogram in Figure 33C shows the relative percentage of CD34+ cells within the tissue sample. As can be seen, each of the treatment approaches performed results in a relative increase in the percentage of CD34+ cells within the treated tissue. In particular, adipose treatment performed according to embodiments and using the systems disclosed herein advantageously results in a treatment run-dependent increase in the percentage of CD34+ stem cells within the treated tissue with increasing numbers of treatment runs. Generally, similar results are observed for the percentage of EPCs, shown in the third histogram, as well as Muse cells, shown in the fourth histogram. Mesenchymal stem cells, shown in the second histogram, do not exhibit this treatment run-dependent increase. However, it is noteworthy that enrichment at least to the same extent occurs without exceeding the NF30 syringe-based treatment. Similar results were observed for the CD26+ / CD55+ positive cell subtype.Taken together, these data demonstrate that adipose tissue processing enriches the treated tissue with cells that exhibit a pro-regenerative phenotype and do not exhibit reduced viability, suggesting that treating adipose tissue with the methods disclosed herein and using the systems disclosed herein induces or enhances tissue repair and / or regeneration. In some embodiments, tissue repair is enhanced compared to adipose tissue processed with other methods.

[0171] Example 3: Role of processed adipose tissue in promoting wound healing This non-limiting example was conducted to compare the effects of standard, unprocessed diabetic stromal vascular fraction (SVF) with microfragmented diabetic adipose tissue (SA), which has higher levels of cellular activity, in promoting wound healing in diabetic mice. Two adipose tissue samples (10 ml each) were collected from each of 20 diabetic patients. The collection methods, parameters, and criteria were those used in non-limiting Example 1 above. Forty 6-week-old male db / db mice were obtained (The Jackson Laboratory, Sacramento, CA) and maintained under controlled environmental conditions (constant laminar airflow, 20-23°C temperature, 40-60% humidity, 12-hour light / dark cycle) in the animal resource facility. A block-randomized, paired-controlled, blinded experiment with two treatment groups was conducted.

[0172] Mice were anesthetized intraperitoneally with 12 mg / kg xylazine (Vedco, St. Joseph, MO) and 80 mg / kg ketamine (Ketathesia, Butler Scein Animal Health Supply, North Dublin, OH). A 6 mm full-thickness wound was created through the panniculus carnosus muscle layer on the back of each mouse and splinted with silicone. Mice were observed for 48-72 hours before treatment. The bilateral wounds of each mouse were randomized to receive injections of either microfragmented activated diabetic adipose tissue resulting from the use of the systems and methods disclosed herein (see, e.g., Example 1) or standard diabetic SVF on one side, and placebo saline on the other wound (paired control group). In the experimental group, a total of 1 x 10 α-glucan was injected in 125 μl of saline. 5 Cells were injected subcutaneously at four sites around the wound edge; a control group received saline injections only. Wounds were covered with occlusive dressings, and digital photographs were taken at days 0, 5, 9, 13, 17, 21, and 24. Wound area was measured using the digital photographs. At the time of earliest wound closure, wounds were excised, fixed, and cryosectioned. Slides were stained with hematoxylin and eosin (H&E), trichrome, and vimentin, and imaged using an anti-CD31 antibody for basic histology and vascular density analysis using a fluorescent microscope (Evos FL, Thermo Fisher Scientific). The study staff who performed the experiments / measurements were blinded to the treatment randomization scheme.

[0173] Data from this in vivo study are provided in Figures 34A-34C. Figure 34A shows histological results from wounds excised after closure. The leftmost panel represents a wound treated with saline, the center column represents wounds treated with adipose tissue treated according to embodiments disclosed herein, and the rightmost panel represents a negative control wound that received untreated SVF. The top row shows hematoxylin and eosin staining, the second row shows Masson's trichrome staining, the third row shows immunohistochemistry detecting vimentin, and the fourth row shows immunohistochemistry detecting CD31. In the center column, arrows indicate the presence of adipose tissue derived from treated human adipose tissue. The presence of adipose-derived stem cells is generally indicated within the circle, and rectangles generally indicate areas of increased skin regeneration. The increased vimentin staining using SA adipose tissue indicates epithelial-to-mesenchymal transition, evidence of wound healing. Increased expression of CD31 (also known as PECAM-1) has also been shown to have a positive effect on inflammatory responses and angiogenesis (notably, anti-PECAM-1 antibodies are known to block normal endothelial cell-cell contacts and affect cell migration, indicating a role for PECAM-1 in angiogenesis and wound healing).

[0174] Figure 34B shows photographic evidence of healing and a healing timeline for the induced wounds. As can be seen from these data, wounds treated with saline took the longest time to heal, with the example shown in Figure 34B taking 19 days to close. Similarly, wounds treated with SVF took over three weeks to heal. In contrast, wounds treated with adipose tissue processed as disclosed herein healed in less than two weeks. The photographic data are summarized in Figure 34C. These data demonstrated a statistically significant decrease in time to wound closure when the adipose tissue process disclosed herein was administered to wounds compared to administration of saline or SVF (p<0.0003 saline vs. SVF, p<0.00003 saline vs. SA, p<0.001 SA vs. SVF by analysis of variance or Student's t-test; error bars represent s.e.m.). These data confirm that the methods, processes, and systems disclosed herein and used to treat adipose tissue result in processed tissue with enhanced wound repair properties.

[0175] While embodiments of the present invention have been disclosed in connection with certain preferred embodiments and examples, those skilled in the art will appreciate that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the invention, as well as obvious modifications and equivalents thereof. Additionally, while many variations of the invention have been shown and described in detail, other modifications that are within the scope of the invention will be readily apparent to those skilled in the art based on this disclosure. It is also contemplated that various combinations or subcombinations of specific features and aspects of the embodiments can be made and still be included in one or more of the inventions. Furthermore, any particular feature, aspect, method, property, characteristic, quality, attribute, element, etc., disclosed herein in connection with an embodiment can be used in all other embodiments described herein. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another to form varying modes of the disclosed invention. For all of the embodiments described herein, the method steps need not be performed sequentially. Therefore, it is not intended that the scope of the invention disclosed herein should be limited by the specific embodiments disclosed above.

[0176] [Statistical method] A linear mixed-effects regression analysis was performed, with the unit of analysis being the mouse wound. The outcome variable was the percentage of wound healing area. Fixed effects included a two-category variable for treatment group (activated diabetic SVF vs. standard diabetic SVF) and an indicator for whether the wound was treated or placebo. Random effects included two wounds per mouse and two samples per patient. Based on this linear mixed-effects model, the mean increase in the percentage of healing area between treated and placebo wounds was compared between the two treatment groups, taking into account the heterogeneity of patients and mice.

[0177] [Sample size and power analysis] Using a total of 40 mice with bilateral wounds treated with activated diabetic SVF or microfragmented diabetic adipose tissue from adipose samples collected from 20 diabetic patients (two samples per patient), a power of 81% is achieved to test the hypothesis that the mean increase in wound healing area is 95% in diabetic mice treated with microfragmented diabetic adipose tissue compared to 65% in diabetic mice treated with standard diabetic SVF at day 14 after receiving treatment, based on a two-tailed paired t-test with a significance level of 5%.

[0178] The present invention is not limited to the above-described embodiments, and of course, various changes and modifications can be made without departing from the scope and spirit of the present invention.

[0179] Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and representative embodiments shown and described herein. Accordingly, various modifications may be made without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents.

Claims

1. 1. A system for processing a biological sample, comprising: a support plate and at least one carriage assembly; the support plate having a central portion configured to reversibly interact with a motor to provide a rotational force and at least one lateral arm; each of the at least one lateral arm includes a first opening and at least one fixed opening; the at least one carriage assembly is configured to reversibly interact with one of the at least one lateral arms; each of the at least one carriage assembly includes a base, a spring platform, at least one spring, and a securing element; the base comprises a top surface including at least one circular groove, a bottom surface including a post configured to extend through the first opening in one of the at least one lateral arms, a first arm, and a second arm; the spring platform includes at least one circular groove disposed on a bottom surface of the spring platform, a first engagement portion, and a second engagement portion; the spring platform is disposed adjacent the top surface of the base; the at least one spring is held between the base and the spring platform; the at least one spring is secured at a first end thereof within the at least one circular groove of the base portion and at an upper end thereof within the at least one circular groove of the spring platform; the at least one spring is configured to provide an upward force on the spring platform; the securing element is configured to secure a tip assembly on the at least one carriage assembly; wherein each of the at least one carriage assembly is rotatable 180 degrees.

2. The system of claim 1 , wherein the spring platform is configured to move between a first position and a second position in response to insertion or removal of the tip assembly.

3. the spring platform further comprising a tab; the fixation element further comprises a channel configured to receive the tab of the spring platform; The system of claim 2 , wherein the tab is configured to move within the channel of the fixation element when the spring platform moves between the first position and the second position.

4. The system of claim 1 , wherein the fixation element includes at least one guide rail configured to guide and position the tip assembly.

5. the first engagement portion of the spring platform is configured to hold the first arm of the base portion; The system of claim 1 , wherein the second engagement portion of the spring platform is configured to retain the second arm of the base portion.

6. The system of claim 1 , wherein the spring platform further comprises a platform configured to guide the tip assembly into the at least one carriage assembly.

7. the spring platform includes a splash guard on the bottom surface of the spring platform; The system of claim 1 , wherein the splash guard is configured to prevent fluid from being introduced into the at least one spring.

8. the chip assembly comprises a microfluidic chip, a first sample chamber, and a second sample chamber; the microfluidic chip has a fluid pathway extending from a first end to a second end of the microfluidic chip; the first sample chamber is fluidly connected to a first end of the microfluidic chip; The system of claim 1 , wherein the second sample chamber is fluidly connected to a second end of the microfluidic chip.

9. The system of claim 8 , wherein the fluid path has at least one expansion region and at least one compression region.

10. the at least one expansion region has an increasing radius along a first axis, a second axis, and a third axis, each axis being perpendicular to a central axis of the fluid pathway; the at least one compressed region has a diameter smaller than a diameter of the at least one expanded region; The system of claim 9 , wherein the at least one compressed region does not change diameter.

11. The system of claim 10 , wherein the first axis, the second axis, and the third axis are perpendicular to one another.

12. the at least one expansion region has an increasing radius along three or more axes, each axis being perpendicular to a central axis of the fluid pathway; The system of claim 9.

13. The system of claim 8 , wherein the fluid pathway includes a plurality of teardrop-shaped expansion regions.

14. 14. The system of claim 8, wherein the fluid path includes a spherical or elliptical expansion region.

15. 13. The system of claim 8, wherein the fluid pathway includes a plurality of semi-teardrop shaped expansion regions.

16. The system of claim 15 , wherein the fluid pathway includes a hemispherical or semi-elliptical expansion region.

17. The system of claim 15 , wherein the fluid path includes a D-shaped expansion region.

18. 18. The system of claim 8, wherein the fluid path includes an hourglass portion.

19. 13. The system of claim 8, wherein the fluid path comprises at least two expansion regions and at least one compression region disposed between the at least two expansion regions.

20. the fluid path comprises at least three expansion regions and at least two compression regions; 13. The system of claim 8, wherein each of the at least two compression regions is disposed between adjacent expansion regions.

21. 21. The system of claim 8, wherein a filter is disposed between the microfluidic chip and the first sample chamber and / or between the microfluidic chip and the second sample chamber.

22. 22. The system of any one of claims 8 to 21, wherein the microfluidic chip comprises luer locks at a first end and a second end of the microfluidic chip.

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