Methods and apparatus for processing tissue and cells

Abstract

Provided herein are methods and devices for processing tissues and cells. In some embodiments, it includes using a motor-driven rotating chuck to rotate one or more microfluidic chips mounted on a support plate. By rotating the one or more microfluidic chips in a controlled manner about a common center of rotation, high flow rates (and high shear forces) are imparted to the sample in a controlled manner. Each microfluidic chip can be rotated 180 on the support plate so that the sample can be passed back and forth through the microfluidic device. Because the support plate can be driven at relatively high RPMs, high flow rates are generated within the microfluidic chips. This increases the shear forces on the sample and also reduces the processing time involved, as the sample can be quickly passed through the shear-inducing devices of the one or more microfluidic chips.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201780047636.3, filed on June 7, 2017, entitled "Method and Apparatus for Processing Tissues and Cells".

[0002] Related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 347,290, filed June 8, 2016, which is incorporated herein by reference in its entirety. Priority is claimed pursuant to 35 U.S.SC § 119 and any other applicable laws. Technical Field

[0004] The technical field generally relates to apparatuses and methods for applying shear stress to living cells and / or tissues, reagents, particles, and fluids. More specifically, the technical field relates to microfluidic-based devices that utilize rotational force to apply shear stress to living cells and / or tissues, reagents, particles, and fluids. Background Technology

[0005] Various techniques and procedures are used to process tissues. In some applications, chemicals or enzymes are added to the tissue to break down larger clumps or aggregates into smaller pieces. For example, digestive enzymes such as collagenase, trypsin, or dispersants are used to digest tissues such as adipose tissue. This enzymatic treatment typically involves washing, followed by enzymatic degradation and centrifugation. This enzymatic method can be subject to variability due to the varying activity levels of the digestive enzymes. Furthermore, these methods require increased reagent costs, including expensive enzymes derived from bacteria, and take considerable time to complete, as well as additional treatment and / or washing steps to minimize the effects of enzyme contamination.

[0006] Non-enzymatic methods have also been developed for processing tissues, including adipose tissue. For example, ultrasonic cavitation has been proposed for separating stromal vascular portions from adipose tissue. See each of patent numbers 8,440,440, which are incorporated herein by reference in their entirety. Other methods involve using beads to homogenize adipose tissue, such as the method disclosed in International Patent Publication No. WO2014-036094, which is incorporated herein by reference in its entirety. U.S. Patent No. 9,580,678 (which is incorporated herein by reference in its entirety) discloses a microfluidic tumor dissociation device using multiple channels or stages arranged in series, having expansion and contraction zones for breaking up tumor tissue. An infusion pump is used to pass tumor tissue back and forth through the microfluidic device.

[0007] The manipulation of tissues such as adipose tissue is particularly important in the field of reconstructive plastic surgery, where adipose tissue is transferred from one location to another to fill soft tissue defects (i.e., fat grafting). Cell-assisted fat grafting (CAL) is a technique involving the addition of stromal vascular fractions (SVFs) to fat grafts and resulting in significant improvements in fat graft retention. Typically, SVFs are harvested from adipose tissue using the enzyme collagenase through a short digestion step. Recently, a technique called 'nanoplasty' has been developed, in which a standard adipose tissue extract is homogenized by manually passing it fully between two connected syringes and then re-injecting the homogenized adipose tissue extract into a human patient to correct surface wrinkles and pigmentation. Nanoplasty methods have also been found to be a means of mechanically dissociating SVFs while simultaneously applying stress to cells to generate subpotent stem cells or even pluripotent cell populations. For example, nanoplasty-derived SVFs are known to have a higher proportion of mesenchymal stem cells (MSCs), adipose-derived mesenchymal stem cells (ADSCs), endothelial progenitor cells (EPCs), and Muse cells. It is speculated that the stress applied to the cells is directly related to embryonic stem cell-like properties.

[0008] For example, MSCs can be used to treat diabetic foot ulcers. Current treatments for diabetic foot ulcers (like allogeneic grafts) are expensive and may be ineffective due to potential patient rejection. Without treatment, these ulcers often require amputation, leading to further health complications. An innovative solution for treating these ulcers is to directly treat them using MSCs. However, current methods for obtaining these cells are lengthy, complex, and produce variable results in terms of cell yield, quantity, and reproducibility. A rapid and cost-effective method for obtaining processed tissue is needed. Summary of the Invention

[0009] In view of the need for devices, systems and methods for processing tissues in an effective, efficient and reproducible manner, this document provides various embodiments of such devices, methods and systems, as well as their use in the development, production or otherwise preparation of cells for treating or administering to subjects.

[0010] In several embodiments, a system for processing biological samples is provided, comprising: a support plate including a central portion including a receiving element; a side portion including a plurality of interacting regions, each interacting region configured to reversibly interact with a plurality of brackets; and a plurality of brackets, wherein each of the plurality of brackets is configured to be operatively connected to the side portion of the support plate.

[0011] In several embodiments, the receiving element is configured to reversibly interact with the drive shaft of a motor, which is configured to apply centrifugal motion to a support plate. In one embodiment, the central portion lies in a plane perpendicular to the axis of rotation of the drive shaft of the motor, and the side portions extend radially from the central portion and lie at least partially in a plane parallel to the plane of the central portion.

[0012] In several embodiments, each of the plurality of brackets includes a first end and a second end, a base extending between the first and second ends, and a receiving region configured to reversibly interact with a microfluidic chip fluidly connected to at least one sample chamber configured to receive a sample for processing. In several embodiments, each of the plurality of brackets includes a post, rod, shaft, or other extension extending substantially orthogonally from the base and configured to interact (e.g., connect, attach, or otherwise interact with) one of a plurality of interaction regions on the sides. In several embodiments, each of the plurality of brackets is coaxially positioned about one of a plurality of axes, wherein during operation, each axis extends substantially parallel to the rotational axis of a motor drive shaft, and wherein each of the plurality of brackets is at least intermittently rotatable about one of the plurality of axes. Depending on the embodiment, the brackets can be rotated to different angles. For example, in several embodiments, the brackets are induced to rotate through approximately 180 degrees of arc.

[0013] In several embodiments, the system further includes at least one microfluidic chip for holding and processing samples according to the system. In several 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 and second ends, the at least one channel comprising different sizes and configured to allow sample transfer from the first end to the second end. In several embodiments, each of the first and second ends is configured to interact fluidly with a sample chamber. For ease of use, each individual microfluidic chip is sized to fit within a corresponding receiving area on a corresponding holder. In several embodiments, each microfluidic chip is reversibly fluidly connected to the sample chamber at each of the first and second ends.

[0014] Optionally, some embodiments include a sample chamber comprising a vent and a venting channel, the venting channel being fluidly connected to the interior of the sample chamber. In several embodiments, each sample chamber is reversibly fluidly connected to the microfluidic chip via a connector.

[0015] In several embodiments, each tray includes a capture element at both first and second ends of the tray, the capture element being configured to communicate with a release element on the side of a support plate, wherein the communication between the capture element and the release element allows intermittent rotation of each of the plurality of trays. In other words, the capture element is used to hold the tray in a desired position until a signal (or force, or lack thereof) is present that allows the capture element to disengage or otherwise cease interaction with the release element, which in turn allows the tray to rotate, after which the capture element re-engages to stop the tray from moving (in several embodiments, this allows the tray to rotate 180 degrees in an arc at a desired time during the tissue processing procedure). In several embodiments, the capture element includes a magnet of a first polarity, while the release element includes a magnet of the opposite polarity.

[0016] In several embodiments, the sides of the support plate include disks, with interacting areas spaced circumferentially around the disks. In some such embodiments, the sides and center are an integral structure, but in other embodiments, the support plate comprises multiple pieces that are joined or integrated before use.

[0017] In several embodiments, the sides of the support plate include multiple arms, each arm comprising a corresponding interaction area. In one embodiment, the arms and the central portion are a unified structure. In other embodiments, the arms and the central portion are separate structures connected together. In some embodiments, the arms are hinged relative to the central portion. In some such embodiments, the hinge allows the arms to move during operation into a plane of an axis substantially parallel to the axis of rotation of the motor's drive shaft. In several embodiments, this allows for gentle start-up and shutdown processes, such that the abrupt application or removal of centrifugal force does not damage the cell / tissue sample.

[0018] In several embodiments, the interaction area of ​​the side includes a through-hole that receives a post (or other structure) from the respective bracket. In several embodiments, the receiving area is located on the upper surface of the base of the bracket. In several embodiments, the post extends from the bottom surface of the base of the bracket. In such embodiments, the post (or other structure) extends from the bottom of the bracket and passes through the through-hole (receiving area) of the side (e.g., arm) and is secured, for example, by a nut, pin, clamp, or other such mechanism (although rotation relative to the side is permitted). In several embodiments, intermittent rotation of each of the brackets is achieved by the interaction of a gear located on the side with a fixed tooth that causes rotation of each bracket.

[0019] In several embodiments, the side portion includes at least three arms, each of the three arms including an interaction region configured to interact with one of at least three brackets including a first end and a second end, each of the brackets being configured to reversibly interact with one of at least three microfluidic chips, each chip including a first end, a second end and a body therebetween, each end of the microfluidic chip being fluidly connected to a sample chamber, and the body of the chip including a plurality of microfluidic channels extending between the first end and the second end, and wherein the brackets are configured to rotate intermittently between a first position and a second position, in which the first end is located at a first position at a first distance from a receiving element at the center, and in the second position at a second position at a second distance from a receiving element at the center, wherein the first distance is greater than the second distance.

[0020] Depending on the implementation, the system may optionally include a housing that isolates the system from the external environment.

[0021] Depending on the implementation, the system may optionally include a motor operatively connected to the drive shaft. In some embodiments, the motor is controlled by a controller unit that allows control of the motor's rotational speed, the controller unit including an interface that allows the user to program a protocol (or select from pre-programmed protocols) to process the organization.

[0022] This document also provides methods for processing biological samples. For example, in several embodiments, a method for processing a biological sample is provided, comprising loading a biological sample into a first sample chamber configured to be fluidly connected to a microfluidic chip, the chip including a central body portion located between a first end and a second end configured to be fluidly connected to the first sample chamber and the second end to a second sample chamber, at least one microfluidic channel extending between the first and second ends, the at least one channel including different sizes and configured to allow sample transfer from the first end to the second end, reversibly connecting the microfluidic chip to a receiving area of ​​one of a plurality of holders as part of a centrifuge apparatus, the centrifuge apparatus including a support plate including a central portion and side portions, the side portions extending radially from the central portion and lying in a plane parallel to the plane of the central portion, each of the holders being operatively connected to the side portion of the support plate and including a first... The device comprises a first end, a second end, and a base extending between the first and second ends, the base including a receiving region. Each of the holders is configured to be rotatable about an axis substantially perpendicular to a plane perpendicular to the center portion. The holder begins in a first position and is rotatable to a second position. In the first position, the first end is located at a first distance from the center portion of a support plate. In the second position, the second end is positioned such that the second end is located at the same first distance from the center portion of the support plate. A rotational force is applied to the centrifugation apparatus, thereby causing a sample to flow from a first sample chamber connected 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 holder is allowed to rotate between the first and second positions. An additional rotational force is applied to cause a sample to flow from the second sample chamber through at least one microfluidic channel extending between the second and first ends and back into the first sample chamber. In several embodiments, the biological sample includes adipose tissue, although other tissue types can be processed using the systems and methods disclosed herein. For example, adipose tissue, tumor tissue, cell preparations, adipose tissue extracts, cultured cells, etc., can be readily processed.

[0023] In several embodiments, a system for processing samples is also provided, including a motor connected to a vertically rotatable chuck; a support plate comprising a plurality of rotatable brackets radially positioned around the support plate, wherein the support plate is mounted on or fixed to the rotatable chuck; and at least one microfluidic chip disposed on one of the rotatable brackets, the at least one microfluidic chip defining a fluid path formed by one or more microfluidic channels disposed therein and extending between a first port of the microfluidic chip and a second port located at the opposite end of the microfluidic chip.

[0024] In several embodiments, such a system may further include a first sample holding chamber and a second sample holding chamber disposed in a rotatable carriage, the first sample holding chamber being fluidly connected to at least one microfluidic chip via a first port, and the second sample holding chamber being fluidly connected to at least one microfluidic chip via a second port. In several embodiments, the first and second sample holding chambers are fluidly connected to at least one microfluidic chip via respective connectors inserted between the first port and the first sample holding chamber, and between the second port and the second sample holding chamber. In several embodiments, at least one microfluidic chip includes the first and second sample holding chambers disposed within the at least one microfluidic chip.

[0025] In several embodiments, the support plate includes a plurality of arms, each of which holds a rotatable bracket. In several embodiments, the plurality of arms are fixed to a single central hub. In several embodiments, the support plate includes a first magnetic element disposed in or on the support plate and adjacent to one end of the rotatable bracket, the rotatable bracket further including a second magnetic element disposed therein or on it.

[0026] Alternatively, in several embodiments, the rotatable bracket is connected to a gear set disposed in a gear assembly mounted to a support plate, wherein the gear set includes an open gear (exposed gear) on the radially outward portion of the gear assembly. In several embodiments, the gear assembly or support plate also includes a stationary magnet disposed therein, and the rotatable bracket includes a pair of magnetic elements disposed at opposite ends thereof.

[0027] In some embodiments, the plurality of rotatable brackets are rotatable in a plane substantially parallel to the plane of rotation of the support plate. In some embodiments, the plurality of rotatable brackets are rotatable in a plane substantially orthogonal to the plane of rotation of the support plate.

[0028] In several embodiments, the system further includes an electromagnet disposed in a support plate below each of a plurality of rotatable brackets, wherein the rotatable brackets include magnetic column elements extending through through-holes formed in the support plate.

[0029] In several embodiments, the microfluidic chip has a fluid path comprising a microfluidic channel formed by a pair of tapered regions connected in a narrow constriction at the center of the fluid path. In several embodiments, the tapered regions comprise continuous tapered regions. In one embodiment, the tapered regions comprise continuous stepped regions. In another embodiment, the fluid path comprises a microfluidic channel having a plurality of expanding and contracting regions exposed along the length of the channel. In several embodiments, the plurality of expanding and contracting regions are defined by curved walls in the microfluidic channel. In several embodiments, the plurality of expanding and contracting regions are defined by angled walls in the microfluidic channel. In several embodiments, the fluid path comprises a microfluidic channel having a plurality of fin-like pouches disposed along the length of the microfluidic channel. In several embodiments, the fluid path comprises a plurality of decreasing-size branch channels, which are recombine with a plurality of increasing-size branch channels. In one embodiment, each branch channel includes a branch portion. In an additional embodiment, each branch portion includes a sharp edge.

[0030] In several embodiments, at least one of the first and second sample holding chambers includes a syringe barrel, such as a standard 2 mL, 5 mL, 10 mL, 20 mL, or 60 mL syringe barrel. In several embodiments, a filter is additionally included between at least one microfluidic chip and one of the first or second sample holding chambers. In some embodiments, the filter is located upstream or upstream of 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 micronize tissue or tissue fragments to allow the sample to pass through the microfluidic chip without clogging. For the purpose of microfluidic shearing in the microfluidic chip, the cutting or micronization of the sample is configured to produce macroscopic aggregates. In some embodiments, the filter is located downstream or downstream of the microfluidic chip to allow only samples of a specific size to exit the device for collection.

[0031] In several embodiments, the system further includes a sample holding chamber and a syringe. The sample holding chamber is disposed in a rotatable carriage and connected to a first port of the microfluidic chip, and the syringe is connected to a second port of the microfluidic chip, wherein the syringe is typically mounted perpendicular to the plane of rotation of the microfluidic chip. In several embodiments, the system disclosed herein may optionally include a vertically movable plate or ring connected to a plunger of the syringe. In several embodiments, the vertically movable plate or ring includes an internally threaded bearing mounted on a rotatable threaded rod. Optionally, some embodiments also include a second motor connected to the threaded rod.

[0032] In some embodiments, the system has at least one of a first sample holding chamber and a second sample holding chamber, each including an inlet having a one-way valve disposed therein.

[0033] In several embodiments, a method of using the system disclosed herein is provided, comprising rotating a support plate to move a sample via a first port into one or more microfluidic channels of at least one microfluidic chip and out of a second port; rotating a rotatable carriage containing at least one microfluidic chip by approximately 180°; rotating the support plate to move the sample via the second port into one or more microfluidic channels and out of the first port; rotating the rotatable carriage containing at least one microfluidic chip by approximately 180°; and repeating these steps multiple times until the sample is processed to a desired degree.

[0034] In several such methods, a sample is moved between a first sample holding chamber fluidly connected to a first port and a second sample holding chamber fluidly connected to a second port. In several embodiments, at least one of the first and second sample holding chambers includes a syringe barrel.

[0035] In several embodiments, the sample includes tumor tissue. In several embodiments, the sample includes adipose tissue. In several embodiments, the sample includes a fluid containing one or more reagents. In several embodiments, the sample includes particles (e.g., nanoparticles, magnetic particles, particles coated with reagents or antibodies, etc.). In several embodiments, the sample includes cells containing liquid.

[0036] In some embodiments, after processing the tissue, the methods disclosed herein also include injecting the processed tissue (e.g., adipose tissue) into a subject.

[0037] In addition to the systems, apparatus, and methods described above, this document also provides a system for processing samples, comprising a support plate including a plurality of arms extending radially from the support plate; a motor connected to the support plate and configured to rotate the support plate; and a plurality of brackets, each of which is disposed on one of the plurality of arms on the support plate, wherein each of the plurality of brackets is coaxially positioned about one of a plurality of axes, wherein each axis extends perpendicularly from the arm on which the bracket is disposed, wherein each of the plurality of brackets is configured to receive a microfluidic chip and at least one sample chamber for receiving a sample for processing, wherein the at least one sample chamber includes an opening fluidly connected to the microfluidic chip, and wherein each of the plurality of brackets is rotatable about one of the plurality of axes.

[0038] In several embodiments, the system further includes a controller configured to drive a motor, wherein the controller is configured to adjust the rotational speed or revolutions per minute (RPM) of the motor. In several embodiments, the controller is adjustable or programmable with a predetermined rotation program or sequence of operations. In several embodiments, the controller is configured (or configurable) to gradually increase the rotational rate of the motor to an RPM rate, such that the sample is configured to flow from a first end of the microfluidic chip to a second end of the microfluidic chip. In several embodiments, the controller is configured to accelerate or decelerate the RPM of the motor, such that each of the plurality of carriages is configured to rotate about one of a plurality of axes.

[0039] In several embodiments, each of the plurality of holders is configured to rotate 180 degrees about one of a plurality of axes. Furthermore, in several embodiments, each of the plurality of holders is configured to receive a first sample chamber and a second sample chamber, wherein the first sample chamber is located at a first end of the microfluidic chamber, and wherein the second sample chamber is located at a second end of the microfluidic chamber.

[0040] Some implementations include at least one sample chamber attached to one end of the microfluidic chip using a connector. The connector may include any one of a Luer slip, a slip tip connector, a Luer lock, and a rotating collar. Depending on the implementation, and also depending on whether the microfluidic chip is disposable or reusable (e.g., sterilizable), the connector may include a metallic or polymeric material.

[0041] In several embodiments, a support plate is placed inside an outer casing configured to protect the user from the effects of the system on the sample being processed. The outer casing may be made of any material, such as plastic or metal, with a thickness sufficient to prevent penetration or breakage due to reduced vacuum pressure, low temperature, thermal changes, or debris generated by the centrifugal rotation of the support plate. In several embodiments, the outer casing is configured to open and close, if desired, to place or remove samples, or to manually rotate one or more trays. In several embodiments, the outer casing is optically transparent and configured to allow monitoring of the operation of the system used for sample processing.

[0042] In some embodiments, the system includes multiple arms, and in some such embodiments, each of the multiple arms further includes a first engagement structure; and a second engagement structure located at a distance from the first engagement structure, wherein the first and second engagement structures are each configured to engage with one of a first structure located at a first end of the bracket and a second structure located at a second end of the bracket. In several such embodiments, the first and second engagement structures are configured to interchangeably release and engage the first and second structures, such that the bracket is configured to move between multiple directions about one of multiple axes.

[0043] In several embodiments, the first and second engagement structures are magnets, and both structures include magnetically responsive materials. In several embodiments, the first and second engagement structures include magnetically responsive materials, and both structures are magnets. In several embodiments, the first engagement structure is located distal to the second engagement structure along the length of each of the plurality of arms. To move the carriage, in several embodiments, an acceleration or deceleration force is configured to move the carriage between multiple directions. In several embodiments, the carriage includes a gear assembly configured to move the carriage between multiple directions. In some embodiments, the carriage includes a radial ratchet configured to move the carriage between multiple directions.

[0044] To allow the sample to flow properly through the microfluidic chip, in several embodiments, at least one sample chamber includes a vent and a vent channel, the vent channel being fluidly connected to the interior of the sample channel, wherein the vent is configured to (e.g., by preventing a vacuum) provide laminar flow through the sample chamber. In several embodiments, the vent is located at the end of the sample chamber opposite the opening. Depending on the embodiment, the sample chamber can be any desired shape, including rectangular, square, elliptical, cylindrical, oval, or other polygonal shapes. In one embodiment, the sample chamber is rectangular. In some alternative embodiments, the sample chamber is a syringe. In some such embodiments, the syringe includes a chamber having a connector end with an opening configured for fluid connection to the microfluidic chip; a plunger including a seal disposed within the chamber; and a depressor attached to the distal end of the plunger and configured to advance and retract the plunger. In some embodiments, the syringe includes a vent and a vent channel, the vent channel being fluidly connected to the interior of the chamber, wherein the vent is configured to provide laminar flow through the syringe. Furthermore, in several embodiments, the syringe may optionally include a secondary syringe plunger disposed within a venting channel and configured to selectively open and close the venting channel. In such embodiments, the secondary syringe may be optionally connected to a depressor, such that movement of the depressor is configured to advance and retract both the plunger and the secondary plunger. In several embodiments, the connector end of the syringe is configured to receive a needle. In several embodiments, the syringe is configured to be removable from the microfluidic chip, and the sample is configured to be injected directly into the injection site.

[0045] In several embodiments, each of a plurality of chambers (e.g., chambers for holding a microfluidic chip) is held in an opening of each of a plurality of arms, wherein each of the plurality of chambers extends through the opening of each of the plurality of arms. In several embodiments, each of the plurality of chambers is held along a plane of each of the plurality of arms. In several embodiments, each of the plurality of chambers is held in an opening of each of the plurality of arms by at least one pin, the at least one pin being configured to allow out-of-plane rotation of each of the plurality of chambers. In several 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 directions. In several embodiments, each of the plurality of chambers moves between 180-degree rotations (in-plane or out-of-plane). In several embodiments, each of the plurality of chambers moves between a plurality of directions, wherein each of the plurality of chambers is positioned along a plane of each of the plurality of arms.

[0046] In several embodiments, the system disclosed herein also includes at least one filter configured to prevent larger sample components from entering and clogging the microfluidic chip. In several embodiments, the filter is attached to a sample chamber to filter the sample before it enters the microfluidic channels of the microfluidic chip. In some embodiments, the filter is located upstream or upstream of the microfluidic chip and configured to filter the sample to prevent clogging of the microfluidic chip. In some instances, the upstream filter may include a mesh configured to cut or micronize tissue or tissue fragments to allow the sample to pass through the microfluidic chip without clogging. For the purpose of microfluidic shearing in the microfluidic chip, the cutting or micronization of the sample is configured to produce macroscopic aggregates. In some embodiments, the filter is located downstream or after the microfluidic chip to allow only samples of a specific size to exit the device for collection.

[0047] This document also provides other systems. For example, a system for processing samples is provided, comprising a support plate, a motor connected to the support plate and configured to rotate the support plate; and at least one bracket disposed on the support plate, wherein the at least one bracket is configured to receive a microfluidic chip and at least one sample chamber for receiving the sample for processing, and wherein the at least one bracket is configured to rotate in a plane parallel to the plane of the support plate.

[0048] Furthermore, a system for processing a sample is provided, comprising a support plate, a motor connected to the support plate and configured to rotate the support plate; and at least one bracket disposed on the support plate, wherein the at least one bracket is configured to rotate in a plane parallel to the plane of the support plate, a microfluidic chip received within the at least one bracket, wherein the microfluidic chip includes a port and at least one microfluidic channel extending along the 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 and along the length of the microfluidic chip.

[0049] In some embodiments, the length of the microfluidic chip is approximately 10 mm to 100 mm. 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 in the range of 5 μm to 8 mm. It should be understood that in some embodiments, the microfluidic chip is removable.

[0050] Depending on the implementation method and the tissue to be processed, the microfluidic channel can have various configurations. For example, in one embodiment, the microfluidic channel has an hourglass-shaped configuration. In several embodiments, at least one microfluidic channel includes a first region comprising a stepped conical portion whose width gradually decreases along the length of the at least one microfluidic channel; a constricted region; and a second region comprising a stepped conical portion whose width gradually increases along the length of the at least one microfluidic channel. In another embodiment, at least one microfluidic channel has a series of regions with increasing width and regions with decreasing width. In several embodiments, at least one microfluidic channel has a diamond pattern. In several embodiments, at least one microfluidic channel includes a plurality of pouches, optionally fin-shaped. In several embodiments, at least one microfluidic channel includes a first region and a second region, the first region including a series of branches, and in the second region, the channels of each pair of branches are recombined. In several embodiments, at least one microfluidic channel includes a plurality of pores, wherein the plurality of pores are configured to classify portions of samples of predetermined sizes.

[0051] This document provides alternative methods, such as, for example, a method for processing a sample, comprising: providing a sample in a sample chamber; inserting the sample chamber into at least one of a plurality of holders, wherein the sample chamber is fluidly connected to a microfluidic chip including at least one microfluidic channel, wherein one of the holders is attached to a support plate configured to rotate about a first axis, and wherein at least one of the holders is configured to rotate about a second axis parallel to the first axis; and rotating the support plate about the first axis, wherein the rotation 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 several embodiments, the method further includes rotating at least one of the holders about the second axis to a second direction. In several embodiments, the method further includes rotating the support plate about the first axis, wherein the rotation is configured to drive the sample through the at least one microfluidic channel chamber in a second direction toward the sample chamber. Furthermore, the method optionally includes removing the sample chamber from at least one of the holders.

[0052] An additional system is provided, including a support plate comprising a plurality of arms extending radially from the support plate; a motor connected to the support plate and configured to rotate the support plate; a plurality of brackets, each of which is disposed on one of the plurality of arms on the support plate, each of which is coaxially positioned about one of a plurality of axes extending perpendicularly from the arm on which the bracket is disposed, each of which is configured to receive a microfluidic chip and at least one sample chamber for receiving a sample for processing, and each of which is rotatable about one of the plurality of axes; and an outer casing configured to receive the support plate, the casing configured to protect a user from the system for processing the sample, wherein the casing includes an opening configured to provide an inlet to the sample chamber. In several embodiments, at least one sample chamber includes a vent and a vent channel fluidly connected to the interior of the sample channel, wherein the vent is configured to provide laminar flow through the sample chamber. In several embodiments, at least one sample chamber includes an opening fluidly connected to the microfluidic chip. In several embodiments, at least one sample chamber includes an inlet configured to allow insertion or removal of a sample for processing from at least one sample chamber. In one embodiment, the inlet is located opposite the opening, although other locations may be optionally used. In several embodiments, at least one sample chamber includes a one-way valve configured to ensure that the sample remains inside the chamber during processing. In several embodiments, the interior of the sample chamber has an inclined, angled, or other shaped surface adjacent to the inlet, configured to cause the sample to accumulate near the inlet for easy removal after processing. In one embodiment, a vent is located at the end of the sample chamber opposite the opening. In some embodiments, the inlet is configured to engage with a syringe configured to remove the sample and allow direct injection of the sample into the target site.

[0053] In several embodiments, each of the plurality of chambers is held in an opening of each of the plurality of arms, wherein each of the plurality of chambers extends through the opening of each of the plurality of arms. In such embodiments, each of the plurality of chambers is optionally held along a plane of each of the plurality of arms. In several embodiments, each of the plurality of chambers is optionally held in an opening of each of the plurality of arms by at least one pin, the at least one pin being configured to allow out-of-plane rotation of each of the plurality of chambers. In several 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 multiple directions. In several embodiments, each of the plurality of chambers moves between 180-degree rotations (e.g., 0 to 45 degrees, 45 to 90 degrees, 90 to 135 degrees, 135 to 180 degrees, etc.). In several embodiments, each of the plurality of chambers moves between various directions, wherein each of the plurality of chambers is positioned along a plane of each of the plurality of arms. In several embodiments, the system further includes a filter configured to prevent larger sample components from entering and clogging the microfluidic chip. In one embodiment, the filter is attached to a sample chamber. In some embodiments, filters are located upstream or upstream of the microfluidic chip and configured to filter samples to prevent clogging of the microfluidic chip. In some instances, the upstream filter may include a mesh configured to cut or micronize tissue or tissue fragments to allow samples to pass through the microfluidic chip without clogging. For the purpose of microfluidic shearing in the microfluidic chip, the cutting or micronization of the sample is configured to produce macroscopic aggregates. In some embodiments, filters are located downstream or downstream of the microfluidic chip to allow only samples of a specific size to exit the device for collection.

[0054] In several embodiments, a system for processing samples is provided, comprising a support plate including a plurality of arms extending radially from the support plate; a motor connected to the support plate and configured to rotate the support plate; a plurality of brackets, each of which is disposed on one of the plurality of arms on the support plate, wherein each of the plurality of brackets is coaxially positioned about one of a plurality of axes, wherein each axis extends perpendicularly from the arm on which the bracket is disposed, wherein each of the plurality of brackets is configured to receive a microfluidic chip and at least one sample chamber for receiving a sample for processing, and wherein each of the plurality of brackets is rotatable about one of the plurality of axes; and an outer casing including a body portion and a cover, wherein the body portion is configured to receive the processing system, and wherein the cover is disposed above the body portion and configured to... To seal the processing system within the main body and protect the user from the influence of the system used for sample processing; a rotating support including a motor; an externally threaded rod attached to the motor, wherein rotation of the motor causes the externally threaded rod to rotate; a plate including multiple engagement structures for holding the syringe, wherein the plate is attached to a bearing having internal threads configured to engage with the external threads of the rod, wherein rotation of the motor is configured to raise or lower the plate in a vertical direction, wherein the syringe includes: a chamber having an opening configured to be fluidly connected to at least one sample chamber, and a plunger disposed within the chamber, wherein advancing and retracting the plunger displaces and draws in the sample for processing, and wherein the plate holds the distal end of the plunger, and vertical movement of the plate causes the plunger to be lowered or raised within the syringe chamber to displace or draw in the sample for processing.

[0055] In several embodiments, a rotary motor is attached to the cover of the housing, while in other embodiments, the rotary motor is located outside the housing. In several embodiments, the plate is circular, while in other embodiments, the plate includes a ring attached to a bearing by a plurality of 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 the plunger. In several embodiments, the system includes a plurality of spaced-apart engagement structures to secure the syringe when attached to at least one sample chamber at a first end of the microfluidic chamber or when attached to at least one sample chamber at a second end of the microfluidic chamber. In several embodiments, each of the plurality of arms further includes an engagement structure configured to engage with a corresponding structure located on each of the plurality of brackets to hold each of the plurality of brackets in a first direction. In several such embodiments, the engagement structure is configured to release and engage the corresponding structure such that the bracket is configured to move between multiple directions about one of a plurality of axes. In several embodiments, the engagement structure includes a magnet, and the corresponding structure includes a magnetically responsive material. Conversely, in several embodiments, the engagement structure includes a magnetically responsive material, and the corresponding structure is a magnet. In some embodiments, the acceleration or deceleration force is configured to move the carriage between multiple directions. In several embodiments, the carriage includes a gear assembly configured to move the carriage between multiple directions. In several embodiments, the carriage includes a radial ratchet configured to move the carriage between multiple directions.

[0056] Methods are provided, such as a method for processing a sample, comprising: providing a sample in at least one sample chamber; inserting the sample chamber into at least one of a plurality of holders, wherein the sample chamber is fluidly connected to a microfluidic chip including at least one microfluidic channel, wherein one of the plurality of holders is attached to a support plate configured to rotate about a first axis, and wherein at least one of the plurality of holders is configured to rotate about a second axis parallel to the first axis; and securing a syringe to the sample chamber, wherein the opening of the syringe is fluidly connected to the sample chamber, and the distal end of the plunger of the syringe is removably attached to a plate, wherein the plate is attached to a motor and configured to rotate and move in a vertical direction, and wherein movement of the plate in the vertical direction causes the plunger to lower or raise within the barrel of the syringe to expel or draw in the sample for processing within the sample chamber; and rotating the support plate about the first axis, wherein the rotation is configured to drive the sample from the sample chamber through at least one microfluidic channel in a first direction away from the sample chamber. In several embodiments, the method further includes lowering the plate in the vertical direction such that the plunger is lowered within the barrel of the syringe to expel the sample for processing into the sample chamber. In some embodiments, the method may optionally include the step of raising the plate in a vertical direction such that the plunger rises within the syringe barrel to remove the sample for processing from the sample chamber. In some embodiments, the method further includes rotating at least one of the plurality of carriages about a second axis to a second direction.

[0057] In other embodiments, the use of processed tissue samples for treating medical conditions is provided. In several embodiments, the condition is diabetic ulcers. In several embodiments, the condition is one that is improved or benefited by activated cells such as stem cells. In several embodiments, the use of activated stem cells from adipose tissue is provided, the activated stem cells from adipose tissue being generated by the processing method and system disclosed herein for a device for treating diseases or ailments. Attached Figure Description

[0058] Figure 1 An exploded view of one embodiment of a system for processing samples using multiple rotatable microfluidic chips mounted on a rotating support plate is shown.

[0059] Figure 2 An embodiment of the microfluidic chip, along with optional connectors and a relatively modular sample chamber, is shown.

[0060] Figure 3 One embodiment of the housing device is shown that can be used optionally with the systems and apparatus disclosed herein.

[0061] Figure 4 An exemplary graph is shown, representing revolutions per minute (RPM) as a function of the time of several cycles of rotation or rotation of a microfluidic chip used in the systems and apparatuses disclosed herein.

[0062] Figure 5 A partial exploded view of one embodiment of a support plate having three arms that hold a rotatable bracket and a microfluidic chip therein is shown.

[0063] Figure 6 yes Figure 5 Side view of a single arm of the support plate.

[0064] Figure 7A A perspective view of a sample chamber according to one embodiment is shown.

[0065] Figure 7B A perspective view of a sample chamber according to one embodiment is shown.

[0066] Figure 7C A cross-sectional view of a sample chamber according to one embodiment is shown.

[0067] Figure 8A A perspective view of a syringe-type sample holder that can be loaded into a tray is shown.

[0068] Figure 8B It shows Figure 8A A cross-sectional view of the syringe.

[0069] Figure 9 A top plan view of one embodiment of a support plate with three arms on which a rotatable bracket is mounted is shown; each arm contains a corresponding microfluidic chip. The bracket and the microfluidic chip are shown rotated or spun 180°.

[0070] Figure 10A An embodiment is shown with a lower plate or housing incorporated into an electromagnet for rotating the carriage and the microfluidic chip.

[0071] Figure 10B An embodiment is shown with a lower plate or housing incorporated into a radial ratchet for rotating the carriage and microfluidic chip.

[0072] Figure 11 Another embodiment of a support plate utilizing a bracket and / or an out-of-plane flip-up microfluidic chip is shown.

[0073] Figure 12A A partially exploded view of another embodiment of a support plate with three arms is shown, in which a rotatable carriage and a microfluidic chip are held. This embodiment incorporates a gear assembly and an open gear that engages with a gear-shaped or toothed surface to rotate the carriage and the microfluidic chip.

[0074] Figure 12BA top plan view is shown of a gear or toothed surface that engages with an open gear extending from a gear assembly on one of the arms of a support plate.

[0075] Figure 13 A perspective view of one embodiment of an optional filter element is shown.

[0076] Figure 14A-14J Different non-limiting embodiments of microfluidic channels included in the microfluidic chip disclosed herein are shown.

[0077] Figure 15 Microfluidic chips (e.g., those using the systems disclosed herein) are shown at different RPMs. Figure 11 A graph showing the average flow rates (mL / min) obtained is also provided. For comparison purposes, the maximum flow rate achievable by using a standard syringe pump to pass the fluid back and forth through the fat treatment chip is also shown.

[0078] Figure 16 Microfluidic chips (e.g., those using the systems disclosed herein) are shown at different RPMs. Figure 11 The average shear force obtained (dynes / cm) 2 The chart shows the maximum shear force achieved by using a standard syringe pump to pass fluid back and forth through the fat treatment chip. For comparison, the maximum shear force is also shown.

[0079] Figure 17 The system disclosed herein is shown as an example. Figure 5 The graph depicts the flow rate as a function of RPM for two different microfluidic chips in the system described.

[0080] Figure 18 The display shows Figure 17 A graph of flow rate as a function of shear rate for one of the microfluidic chips.

[0081] Figure 19 These are microscopic images (4X) of tumor tissue (MCF7) processed three times at 300 RPM using a non-limiting embodiment of the system described herein.

[0082] Figure 20 Microscopic images (4X) obtained from untreated harvested tumor cells (MCF7) used as a control.

[0083] Figure 21 A microfluidic chip is shown, which has multiple reservoirs located on its surface for capturing cells or cell aggregates therein.

[0084] Figure 22 A cross-sectional view of a sample holding chamber incorporating a check valve according to another embodiment is shown.

[0085] Figure 23 A system for processing samples according to another embodiment is shown, which uses an access port to load / retrieve samples from a sample chamber mounted on a rotating support plate or disk.

[0086] Figure 24 Another embodiment of a system for processing samples, according to another embodiment, is shown. In this embodiment, one or more syringes (or other variable-volume sample containers) rotate or tumble together with one or more microfluidic chips, and rotate or tumble 180° to process the sample passing back and forth through the one or more microfluidic chips. A movable top plate or ring is fixed to the plunger of the syringe and is used to eject or retrieve the sample according to the direction of movement of the plate.

[0087] Figure 25 It shows Figure 24 Bottom plan view of the movable top plate or ring of the embodiment.

[0088] Figure 26 It shows Figure 24 A perspective view of the embodiment, in which the microfluidic chip and syringe have been rotated 180° and the movable top plate or ring has been partially lowered to depress the syringe plunger.

[0089] Figure 27A It shows the use of Figure 14B The type of microfluidic chip shown herein is used in tissue processing systems (e.g., such as...) Figure 5 A graph showing the cell count results of adipose tissue (“processed adipose tissue extract”) from the tissue processing system shown. Figure 27A Cell counting results for a standard adipose tissue extract (i.e., untreated) are also shown.

[0090] Figure 27B It shows the use of Figure 14B The type of microfluidic chip shown is through Figure 5 A graph showing the cell viability results of adipose tissue (“processed adipose tissue extract”) from the tissue processing system. Figure 27A Cell viability results for standard adipose tissue extract (i.e., untreated) are also shown.

[0091] Figure 28 This is a graph showing the improved stem cell enrichment of the treated adipose tissue extract compared to the standard adipose tissue extract. "*" indicates statistical significance when p < 0.05.

[0092] Figure 29A graph showing stem cell markers (CD45, CD31, CD34, CD73, CD146, MSC, DPP4 / CD55) for processed adipose tissue extract and standard adipose tissue extract is presented. Detailed Implementation

[0093] Figure 1 A system 10 for processing sample 12 according to one embodiment is shown. System 10 includes one or more microfluidic chips 14 for processing sample 12 as described herein. In one embodiment, sample 12 may include a biological sample. For example, in a particularly preferred embodiment, sample 12 may include a tissue sample obtained from a mammalian organism, such as fat or adipose tissue or tumor tissue. In another instance, sample 12 may contain cells subsequently processed or passed through microfluidic chip 14. In another embodiment, sample 12 may include particles such as beads. In yet another embodiment, sample 12 may contain fluids or one or more fluid reagents or reactants. In yet another different embodiment, sample 12 may contain water or a water-based sample.

[0094] In several embodiments, the microfluidic chip 14 includes one or more microfluidic channels 16 formed in the substrate 8, for example, such as Figure 2As shown in the diagram. The length (along its longest side) of the microfluidic chip 14 is typically from about 10 mm to 100 mm, for example, about 10 mm to about 20 mm, about 20 mm to about 30 mm, about 30 mm to about 40 mm, about 40 mm to about 50 mm, about 50 mm to about 60 mm, about 60 mm to about 70 mm, about 70 mm to about 80 mm, about 80 mm to about 90 mm, about 90 mm to about 100 mm, and any length in between (including the endpoints). One or more microfluidic channels 16 generally extend along the longitudinal (i.e., long) axis of the microfluidic chip 14 and typically traverse from one end of the microfluidic chip 14 to the opposite end. In some embodiments, the length of the microfluidic channel 16 is less than the total length of the microfluidic chip 14. Depending on the embodiment, the width and depth of the microfluidic channel 16 can fall within the range of about 5 μm to about 8 mm. For example, the length (or width) can range from about 5 μm to about 10 μm, about 10 μm to about 20 μm, about 20 μm to about 50 μm, about 50 μm to about 100 μm, about 100 μm to about 200 μm, about 200 μm to about 500 μm, about 500 μm to about 750 μm, about 750 μm to about 1000 μm, about 1 mm to about 2 mm, about 2 mm to about 3 mm, about 3 mm to about 4 mm, about 4 mm to about 5 mm, about 5 mm to about 6 mm, about 6 mm to about 7 mm, about 7 mm to about 8 mm, and any size in between (including the endpoints). In additional embodiments, the length-to-width ratio of the microfluidic channel ranges from approximately 1000:1, approximately 750:1, approximately 500:1, approximately 250:1, approximately 100:1, approximately 50:1, approximately 25:1, approximately 10:1, approximately 2:1, approximately 1:1, approximately 1:2, approximately 1:10, approximately 1:25, approximately 1:50, approximately 1:100, approximately 1:250, approximately 1:500, approximately 1:750, approximately 1:1000, and any ratio between those listed. Furthermore, in many microfluidic chip 14 designs, the width and depth vary due to the presence of contraction regions, expansion regions, etc.

[0095] As in Figure 2 As shown in one non-limiting embodiment depicted herein, a first port 20 is located at one end of the microfluidic chip 14 and serves as an inlet (or an outlet as explained herein) for the sample 12. A second port 22 is located at the other opposite end of the microfluidic chip 14. As explained herein, one or more microfluidic channels 16 may be provided in any number of configurations.

[0096] The microfluidic chip 14 can be formed from any number of materials. For example, the microfluidic chip 14 can be formed from polymeric or plastic materials (e.g., polycarbonate, polymethyl methacrylate (PMMA), polyoxymethylene, polylactic acid (PLA), or acrylonitrile-butadiene-styrene (ABS) materials). The microfluidic chip 14 can be formed from other materials such as metals (e.g., aluminum, steel, titanium, alloys, etc.). The microfluidic chip 14 can be made from one or more layers bonded together to form a fully enclosed microfluidic chip 14. Therefore, the microfluidic chip 14 can be fabricated as a laminated structure formed from one or more layers or a substrate 8. For example, the first layer of the microfluidic chip 14 can be formed from polycarbonate or PMMA, and microfluidic channels 16 can be formed therein using CNC milling or laser etching (or chemical etching), wherein a second thin overlay layer is bonded or otherwise adhered to the first layer to form a complete microfluidic chip 14. The two layers can be bonded together using double-sided pressure adhesive. Ultrasonic welding can also be used to complete the bonding. Alternatively, the microfluidic chip 14 can be made from a monolithic substrate 8. The microfluidic chip 14 can be formed using any number of manufacturing methods, including 3D printing, injection molding, CNC milling, or laser etching.

[0097] Return to reference Figure 1 System 10 includes a support plate 30, which in a preferred embodiment has a plurality of arms or wings 32 extending outward from a central region or hub 31 of the support plate 30. In alternative embodiments, the support plate is simply a disc with an interior and sides, with the arms replaced by the sides. In some embodiments, the arms or wings 32 are separate structures secured to the hub 31 using one or more fasteners (e.g., screws, bolts, etc.). In other embodiments, the arms or wings 32 are integrated with the hub 31 as a single, integral structure. The central region or hub 31 may be made of the same or different materials used to form the arms or wings 32. For example, the central region or hub 31 may be formed of metal or metallic materials (e.g., aluminum, stainless steel, steel, etc.) or polymeric materials (e.g., plastic, polymethyl methacrylate (PMMA), polycarbonate, etc.). The arms or wings 32 may also be made of metal, metallic materials, or polymers, as may be used for those formations of the central region or hub 31.

[0098] Figure 1The support plate shown includes three (3) such arms or wings 32, although it should be understood that the support plate may include any number of arms 32 (e.g., one to ten arms) more than a single arm 32. In a preferred embodiment of the invention, the support plate 30 is configured such that the arms 32 extend radially from the central region of the support plate 30 and are arranged symmetrically about the support plate 30. For example, in a three (3)-arm configuration, each arm 32 is oriented at approximately 120° to each adjacent arm. In several embodiments, the arms may be hinged such that during operation, the arms move in an arcuate pattern and are positioned in a plane perpendicular to or substantially perpendicular to the axis of rotation. The central region of the support plate 30 includes a hole or aperture 33 sized to accommodate a rotating chuck 34 through the hole or aperture 33. The support plate 30 is secured to the chuck 34 using fasteners 36. For example, the chuck 34 may be threaded, and the fastener 36 may be a screw, nut, clamp, press fit, magnetic engagement, etc., which engages with the threaded portion of the chuck 34 to securely lock the support plate 30 to the chuck 34. Alternatively, the fastener 36 may include a pin, magnet, etc. Furthermore, the shaft of the chuck 34 may have a geometry that matches or locks to the hole or aperture 33 of the support plate 30 to help lock the two components together. Of course, any other type of fastener or lock can be used to secure the support plate 30 to the chuck 34. The chuck 34 is securely held on the drive shaft 40 of the motor 42 using locking pins, screws, etc. (not shown). The motor 42 may include any number of motors of any type, wherein the rotational speed of the drive shaft 40 can be adjusted. As an example, the motor 42 includes a brushless servo motor (e.g., Parker motor SM232BE, Parker Hannifin Corp., Charlotte, NC), although it should be understood that a wide variety of motors 42 may be used. In another alternative embodiment, the support plate 30 and the arm 32 may be incorporated into the chuck 34 itself. That is, the arm 32 supporting the microfluidic chip 14 may extend from the chuck 34, rather than from a separate support plate 30 placed on the chuck 34.

[0099] like Figure 1 As shown, the motor 42 is fixed to the mounting device 44 such that the drive shaft 40 and the chuck 34 mounted therein are in a substantially perpendicular orientation. In this orientation, a support plate 30 can be placed on top of the chuck 34 and locked thereto using fasteners 36. This arrangement places the support plate 30 in a substantially horizontal plane. When the motor 42 is actuated and the drive shaft 40 is rotated, the support plate 30 mounted thereon using the chuck 34 rotates about the axis of rotation of the drive shaft 40 in a substantially horizontal plane (rotation can be counterclockwise or clockwise (or both, in an alternating pattern)). Figure 3The mounting support plate 30 (and the microfluidic chip 14 mounted thereon) can optionally be placed inside the housing 46. The housing 46 protects the operator from any hazards or malfunctions that may occur while using the system 10. The housing 46 can be made of any thick plastic material (e.g., polycarbonate, polymethyl methacrylate; e.g., plexiglass) or even metal (e.g., steel, aluminum). The housing 46 can be opened and closed to provide an entry point for mounting the support plate 30, etc., to the chuck 34. One or more sections can be optically transparent, allowing for visual monitoring of the operation of the system 10. Figure 1 As an alternative to the separate mounting device 44 shown, the motor 42 can be mounted on a base or other structural support on the housing 46, such as... Figure 3 As shown in the image.

[0100] Return to reference Figure 1 Motor 42 is connected to controller 50 via one or more cables 51. As shown, one cable 51 is used to drive motor 42, while another cable 51 is used for feedback. Controller 50 (which may be a stepper / servo controller 50) is used to drive motor 42. Controller 50 can be used to adjust or program the rotational speed or RPM. In a preferred embodiment, controller 50 can be programmed or loaded with a rotational program that causes support plate 30 to rotate in a predetermined sequence of operations. For example, controller 50 can gradually increase the rotational rate of motor 42 to a desired RPM rate, causing sample 12 to pass through one or more microfluidic channels 16 of one or more microfluidic chips 14, and then, in one embodiment, reduce the RPM to rotate one or more microfluidic chips 14 half a turn or 180°. One or more microfluidic chips 14 are now in a flipped or reversed direction, and controller 50 can then gradually increase the RPM again to the desired RPM rate to push or drive sample 12 through one or more microfluidic channels 16 of one or more microfluidic chips 14 again in the reverse direction. The process can be repeated multiple times (e.g., ten to thirty 180° rotations of one or more microfluidic chips 14; such as 10-15, 15-20, 20-25, 25-30, etc.). It should be understood that more or fewer cycles can be used, depending on, for example, the viscosity of the starting sample. Figure 4 An exemplary rotating flow profile of system 10 is shown, illustrating multiple 180° cycles of one or more microfluidic chips 14.

[0101] In one implementation, the controller 50 can be programmed using software such as LabVIEW, Java, C, C++, Python, etc. The controller 50 can also be manually controlled. The actual rotational speed or RPM used can depend on the structure and configuration of the microfluidic chip 14. Typically, the RPM ranges from 0 to approximately 10,000 RPM. Higher RPMs result in higher flow rates of the sample 12 through the microfluidic chip 14. The flow rate through the microfluidic chip 14 can vary, but is typically in the range of approximately 0 mL / min to 700 mL / min. For example, the flow rate range can be approximately 0.2 mL / min to approximately 1 mL / min, approximately 1 mL / min to approximately 2 mL / min, approximately 2 mL / min to approximately 10 mL / min, approximately 10 mL / min to approximately 50 mL / min, approximately 50 mL / min to approximately 100 mL / min, approximately 100 mL / min to approximately 250 mL / min, approximately 250 mL / min to approximately 500 mL / min, approximately 500 mL / min to approximately 700 mL / min, and any rates in between (including endpoints). Depending on the rotational speed achieved using motor 42, the upper limit of the flow rate can be even greater.

[0102] Figure 4 An illustrative flow profile is shown according to one embodiment. In this embodiment, the RPM rate is gradually increased or accelerated (e.g., 2,000 RPM / s) to a maximum rotational speed of 1,600 RPM over a period of about one second. The RPM rate of 1,600 is maintained for several seconds (e.g., about 8 seconds) and then rapidly decelerated (e.g., 2,000 RPM / s), causing the microfluidic chip 14 to rotate 180°.

[0103] Return to reference Figure 1 and Figure 5 Each microfluidic chip 14 is held or otherwise secured in a rotatable bracket 60. Each rotatable bracket 60 is rotatably mounted on an arm 32 of a support plate 30. The rotatable bracket 60 includes a recess 62 ( Figure 5 As shown in the diagram, the dimensions are such that the microfluidic chip 14 is housed therein. The microfluidic chip 14 can be held therein by friction fits, tabs, clips, stops, etc. In one embodiment, the rotatable bracket 60 further includes a first end 64 and a second end 66 for holding respective sample chambers 70, 72. Sample chamber 70 is located in the first end 64, while sample chamber 72 is located in the second end 66 of the bracket 60. Each sample chamber 70, 72 is fluidly connected to the microfluidic chip 14 via ports 20, 22, for example, as shown in the diagram. Figure 2As shown in the diagram. Sample chamber 70 is fluidly connected to microfluidic chip 14 via first port 20. Sample chamber 72 is fluidly connected to microfluidic chip 14 via second port 22. Note that, optionally, one or both of sample chambers 70 and 72 can be connected to microfluidic chip 14 using connector 80. Connector 80 is located at... Figure 2 , 5 As shown in Figures 6 and 7, and may include any number of designs and configurations. By way of illustration and not limitation, these include Luer locks (e.g., sliding head connectors), Luer twist locks (e.g., rotating collars), etc. Connector 80 may be formed from any number of materials including metals (e.g., aluminum, steel, stainless steel, etc.) or polymeric materials (e.g., plastics, polycarbonate, acrylates, resin materials, etc.).

[0104] One of the sample chambers 70 and 72 contains a sample 12 to be passed through the microfluidic chip 14. The sample chambers 70 and 72 are designed to hold a volume of sample 12 or other material to be processed by one or more microfluidic chips 14. The holding volume of the sample chambers 70 and 72 can range from >0 mL to ~100 mL and can be varied by changing the size of the chambers. For example, in several embodiments, the sample chamber volume can range from about 1 ml to about 3 ml, about 3 ml to about 5 ml, about 5 ml to about 10 ml, about 10 ml to about 25 ml, about 25 ml to about 50 ml, about 50 ml to about 75 ml, about 75 ml to about 100 ml, or any volume in between (including endpoints). In one embodiment of the invention, the sample chambers 70 and 72 contain sample 12 or other material to be passed through the microfluidic chip 14. Reference Figures 7A-7C Sample chambers 70 and 72 are designed with connectors 74, which can be any type of syringe connector, such as Luer locks (e.g., slider connectors), Luer twist locks (e.g., rotary collars), etc. Sample chambers 70 and 72 are also designed with... Figure 7C The ventilation channel 76 shown can have a diameter ranging from 0 mm to approximately 10 mm (e.g., approximately 1 mm to approximately 2 mm, approximately 2 mm to approximately 4 mm, approximately 4 mm to approximately 6 mm, approximately 6 mm to approximately 8 mm, or approximately 8 mm to approximately 10 mm) to allow laminar flow but not turbulent flow through the microfluidic chip 14. Depending on the embodiment, multiple ventilation holes may be used. The ventilation channel 76 opens to the atmosphere, and; in one embodiment, as Figure 7C As shown, via vent 77 ( Figures 7A-7CThe sample chambers 70 and 72 have outlets near their ends and are connected to the interior of the sample chambers 70 and 72 near the connector head 74. Optionally, a small filter may be placed in or otherwise communicated with the vent 77 to prevent contamination or leakage. The sample chambers 70 and 74 can be made of one or more of a variety of materials such as resin, plastic, or metal. These can be manufactured by many common manufacturing methods such as injection molding, resin printing (e.g., 3D printing), blow molding, machining, etc. In one embodiment, to fill the sample chambers 70 and 72, the end or tip of a syringe (needleless) is inserted into the connector head 74 located on the sample chambers 70 and 72, and the syringe plunger is pressed down to fill the chamber volume with the sample 12 or material included in the syringe barrel. The sample chambers 70 and 72 are preferably modular components that can be inserted and / or removed from the ends 64 and 66 of the bracket 60, such as Figure 5 As shown in the diagram. For example, chamber 70 can be mounted outside the device or system 10 and then inserted into the end 64 of bracket 60.

[0105] In such Figures 8A-8B In another embodiment shown, the syringe chamber 86 serves as a sample chamber 70, 72 and can be placed in one or both ends 64, 66 of the holder 60. The ends 64, 66 are designed to accommodate the length of the syringe chamber 86. This embodiment is particularly advantageous because it allows the physician to use the syringe chamber 86 directly after treatment. For example, if fat or adipose tissue is treated using the system 10 described herein, the resulting treated tissue is loaded into the syringe chamber 86, which can be easily removed from the system 10 and used to inject the treated fat directly into the subject's application site. In this respect, the system 10 uses one or more accompanying syringe chambers 86 instead of sample chambers 70, 72. Once treatment is complete, the syringe chamber 86 is removed, a needle is added to the end, and the now-treated sample is optionally injected into the subject.

[0106] The syringe chamber 86 includes a syringe barrel 88 that defines a three-dimensional volume for holding the sample. A standard plunger 90, including a rubber or polymer seal, is disposed within the barrel 88 and includes a proximal depressor 92 for advancing the plunger 90. One end of the syringe barrel 88 includes a connecting end 94, which may include a Luer slip lock (e.g., a sliding head connector), a Luer twist lock (e.g., a rotating collar), etc., that engages with the microfluidic chip 14. In one embodiment, the syringe chamber 86 is connected to a vent channel 96 that extends along the length of the barrel 88 and communicates with the interior of the barrel 88 via a hole 89 and terminates in a vent hole 98 open to the atmosphere (ideally as shown in the image). Figure 8B(As shown in the diagram). Ventilation channel 96 allows ventilation and a stable laminar flow through the syringe barrel 88 and the microfluidic chip 14. In one embodiment, a smaller secondary syringe plunger 100, including a rubber or polymer seal, is disposed in and movable therein within the ventilation channel 96 to selectively open / close the ventilation channel 96. Specifically, as shown in the diagram... Figure 8B As shown, when the seal of the secondary syringe plunger 100 is proximal to the vent hole 98, the vent passage 96 is open, and the interior of the syringe barrel 88 can be vented to the atmosphere. However, when the seal of the secondary syringe plunger 100 is distal to the vent hole 98, the vent passage 96 is closed, and the interior of the syringe barrel 88 is not vented; the syringe chamber 86 functions as a standard syringe. The secondary syringe plunger 100 can be connected to the same proximal depressor 92, such that movement of the depressor 92 will cause movement of both plungers 90 and 100. The syringe chamber 86 and other components such as the depressor 92 can be made of any number of materials, including polymeric materials such as resin, plastic, etc. Conventional manufacturing techniques such as injection molding, resin printing, etc., can be used. The syringe chamber 86 can also be formed of metal or metallic materials.

[0107] Now for reference Figure 5 and 6 A specific embodiment of the support plate 30 is disclosed. In this embodiment, the support plate 30 is formed using a center plate 104 having a plurality of arms 32 fixed to the center plate 104. The center plate 104 includes holes or apertures 106 sized to accommodate a chuck 34 (see [link to documentation]). Figure 1 In this embodiment, each arm 32 has a lower plate 108 and an upper plate 110. The lower plate 108 and the upper plate 110 can be fastened to each other using any number of fasteners such as screws, bolts, etc. The plates 108, 110 include holes or openings 112 formed therein, which are aligned and sized to accommodate posts 114 extending from the bracket 60. Figure 6 As best shown, post 114 extends through holes 112 in lower plate 108 and upper plate 110 and protrudes a distance below arm 32. Post 114 includes holes through which pins or clips 118 are received for securing bracket 60 to arm 32 (but still allowing rotation or turning).

[0108] In this embodiment, a magnet or magnetic element 120 is disposed in a support plate 30, which may include a central plate 104, such as... Figure 5 As shown. Figure 5 and 6As shown, two smaller magnets or magnetic elements 122 are disposed on the lower surface of the holder 60 (or incorporated into the structure of the holder 60) and are used to temporarily hold the holder 60 in one of two 180° configurations, wherein the longitudinal axis of the holder 60 is aligned along the radial length of the arm 32. For example, the magnetic element 122 located in the holder 60 may include an actual magnet (e.g., a rare-earth magnet or other type), or it may include a screw, bolt, or sheet metal susceptible to magnetic influence. As explained herein, the holder 60 is capable of switching back and forth between the two 180° directions, allowing the sample 12 to flow through the microfluidic chip 14 in either a forward or reverse direction. As explained herein, many different forms can be used to switch the holder 60 between these two directions. This includes using deceleration or acceleration forces to switch the direction of the holder 60. Alternatively, an electromagnet disposed in the arm 32 may be used to switch the holder 60 (and the microfluidic chip 14) between these two directions. In another alternative embodiment, the gear assembly can be used in conjunction with a radial set of gear teeth, enabling the bracket 60 to rotate between 180° directions. Yet another alternative embodiment uses a radial ratchet to mechanically rotate the bracket 60 in different directions based on a reduction in the RPM of the support plate 30. Yet another alternative embodiment can use a piston or shock absorber-like device instead of a ratchet.

[0109] Figure 9 A support plate 30 with three different arms 32 is schematically shown, each arm containing a rotatable bracket 60 in which a microfluidic chip 14 is contained. In a top or top view, each rotatable bracket 60 is oriented with a first end (①) radially inwardly positioned on the arm 32 and a second end (②) radially outwardly positioned on the arm 32. In this configuration, in response to rotation of the support plate 30, a sample 12 or other material passes through the microfluidic chip 14 in a radially outward direction. That is, for a sample 12 starting from sample chamber 70, the sample 12 moves from sample chamber 70 and into the microfluidic chip 14 and advances into sample chamber 72. Next, as... Figure 9 As shown in the lower part, each of the holders 60, each containing a microfluidic chip 14, is rotated 180° to reverse the orientation of the holder 60 and its microfluidic chip 14. In this configuration, the first end (①) is radially outwardly positioned on the arm 32, and the second end (②) is radially inwardly positioned on the arm 32. The sample 12 in the sample chamber 72 is now radially inwardly positioned, and the rotation of the support plate 30 now moves the sample 12 from the sample chamber 72 and into the microfluidic chip 14 and further into the sample chamber 70. This process can be repeated any number of times.

[0110] exist Figure 5 and 6In this embodiment, the deceleration of the support plate 30 applies a force to the bracket 60, which disrupts the force between magnets 120 and 122, causing the bracket 60 to rotate around the column 114 and redirect the bracket 60 by 180°. The opposing magnet 122 is then attracted to another magnet 120, fixing the bracket 60 in the new direction. The support plate 30 then accelerates again to move the sample 12 through the microfluidic chip 14 in the reverse flow direction; thus, the process of rotating the bracket 60 and their corresponding microfluidic chips 14 can be repeated.

[0111] refer to Figure 10A In an alternative embodiment, an electromagnet 130 mounted on the lower plate 108 is used to rotate or swivel the bracket 60 by 180°. The electromagnet 130 can be powered using an onboard battery or wires transmitted through the shaft 40. In this embodiment, the post 114 includes a magnet 115 that generates opposite magnetic poles on opposite sides of the post 114. The electromagnet 130 also includes two magnetic poles as shown, and when actuated, drives the post 114 to rotate such that the north pole of the electromagnet 130 is adjacent to the south pole of the magnet 115. Conversely, the south pole of the electromagnet 130 is adjacent to the north pole of the magnet 115. The bracket 60 is rotated by reversing the polarity of the electromagnet 130.

[0112] Figure 10B Another embodiment is shown, in which a radial ratchet is used to rotate or turn the bracket 60. In this embodiment, the lower plate 108 includes a ratchet-equipped gear or wheel 134 fixed to the post 114. The ratchet gear 134 engages with a set of ratchet teeth 136, which are connected at one end to a spring 138. The spring 138 is fixedly attached to the lower plate 108 at the opposite end. The opposite ends of the ratchet teeth 136 are connected to filaments, threads, cables, or ropes 140, which are connected to a weight 142. During operation of the system 10, as the support plate 30 rotates, the centripetal force causes the weight 142 to move radially outward on the lower plate 108, thereby extending the spring 138 to accommodate the force. It should be noted that the ratchet teeth 136 do not move the ratchet gear 134 during this operation due to the orientation of the teeth. When the support plate 30 decelerates or stops, the tension of the spring 138 pulls the ratchet tooth 136 (and the weight 142) radially inward (in the direction of arrow A), thereby engaging the ratchet tooth 136 with the ratchet gear 134 and causing the column 114 to rotate. The rotation of the column 114 causes the bracket 60 to rotate 180°.

[0113] Figure 11Another embodiment of the support plate 30a is shown, which includes a central hole or aperture 31a. In this embodiment, instead of the microfluidic chip 14 rotating in a horizontal plane generally parallel to the plane of the support plate 30, the microfluidic chip 14 is flipped out of plane relative to the plane of rotation of the support plate 30 (e.g., perpendicular to the radial plane where the arm is located). In this embodiment, the microfluidic chip 14 may include a pin 144 that engages with a slot 146 located on the support plate 30, allowing out-of-plane rotation. Note that in this embodiment, a holder 60 for holding the microfluidic chip 14 may be provided, which can be connected to... Figure 5 and 6 The microfluidic chip 14 shown rotates in a similar manner.

[0114] Figure 12A Another embodiment of the support plate 30b is shown. In this embodiment, the support plate 30b has a central aperture or hole 33 for supporting a chuck 34 as described herein. The support plate 30b supports a plurality of gearboxes or gear assemblies 150, such as... Figure 12A As shown, it effectively serves as the arm 32 of the support plate. In this embodiment, there are three such gear assemblies 150, each gear assembly 150 being fixed to the central hub 152 of the support plate 30b using aluminum screws or the like (not shown), as... Figure 12A As shown in the diagram. The gear assembly 150 includes a bottom housing 153 that houses two gears 154, 156. The external gear 154 rotates about a post 158 ​​located in the bottom housing 153 and is positioned such that a portion of the teeth of the gear 154 exposes beyond the radial edge of the gear assembly 150. In this respect, the teeth of the gear 154 can engage with another gear or gear surface (see...). Figure 12B This causes the external gear 154 to rotate. A second internal gear 156 is mechanically engaged with the external gear 154. As explained herein, the internal gear 156 is used to rotate the carrier 60' containing the microfluidic chip 14'.

[0115] like Figure 12AAs shown, a top cover 160 of the gear assembly 150 is present. The top cover 160 includes a aperture 162 through which a shaft or post 164 connected to the bracket 60' passes and is mechanically connected to the internal gear 156. Another aperture 166 is formed in the top cover 160, sized to receive a magnet 168 (e.g., a rare-earth magnet or other type). The magnet 168 is fixedly secured in the aperture 166 and in a partial recess formed in the support plate 30. The magnet 168 serves to hold the orientation of the microfluidic chip 14' in one of two directions during rotation of the bracket 60' (until the microfluidic chip 14' rotates 180°). The bracket 60' is sized to hold the microfluidic chip 14' thereon. The microfluidic chip 14' and the bracket 60' have holes 170 at the corners, allowing the microfluidic chip 14' to be secured to the bracket 60' using removable fasteners (e.g., screws, etc.); although the microfluidic chip 14' can be secured using any other type of fastener. Furthermore, there are two locations on the underside of the bracket 60' for receiving magnetic screws 172 (e.g., steel screws). These screws 172 are used to hold the bracket 60' (and the microfluidic chip 14') in one of two directions during rotation of the support plate 30b. To allow the bracket 60' of this embodiment to rotate, as... Figure 12B As shown, the surface with gear teeth 180 (as indicated by arrow A) is brought into contact with the external gear 154 while the support plate 30 rotates (the support plate 30' can rotate at a reduced speed for this operation). The rotation of the support plate 30 causes the gear 154 to move on a fixed surface (e.g., in the direction of arrow B), and the gear teeth 180 cause the gear 154 to rotate, thus rotating the bracket 60'. Figure 12A and 12B In one embodiment, the microfluidic chip 14' includes sample chambers 70', 72' formed directly inside the microfluidic chip 14'. In this embodiment, ports 20', 22' at the ends of one or more microfluidic channels 16 lead to the respective on-chip sample chambers 70', 72'.

[0116] Figure 13An optional filter element 190 is shown, which can be incorporated into the system 10 described herein. The filter element 190 is used to prevent larger sample components from entering the microfluidic channels 16 and clogging them. The filter element 190 uses a mesh-like filter pattern or mesh 193. In one embodiment, the filter constitutes an end member 192 (or top), which can have any type of syringe attachment, such as a Luer slide lock (e.g., a sliding head connector), a Luer twist lock (e.g., a rotating collar), etc. Another end member 194, including the mesh 193, is sealed relative to the opposing end member 192 to form the complete filter element 190. The end member 194 can also have any type of syringe attachment, such as a Luer slide lock (e.g., a sliding head connector), a Luer twist lock (e.g., a rotating collar), etc. In some embodiments, the filter element 190 is located upstream or upstream of the microfluidic chips 14, 14' and is configured to filter the sample to prevent clogging of the microfluidic chips 14, 14'. In some instances, the upstream filter element 190 may include a mesh configured to cut or micronize tissue or tissue fragments to allow samples to pass through the microfluidic chips 14, 14' without clogging. For the purpose of microfluidic shearing within the microfluidic chips 14, 14', the cutting or micronization of the sample is configured to produce macroscopic aggregates. In some embodiments, the filter element 190 is located downstream or after the microfluidic chips 14, 14' to allow only samples of a specific size to exit the device for collection.

[0117] Figure 14A-14J Several different embodiments of the microfluidic chip 14 are shown, having different configurations for the microfluidic channels 16. Figure 14A A microfluidic chip 14 with a microfluidic channel 16 featuring an hourglass design is shown. In this embodiment, the microfluidic channel 16 has tapered side profiles 200, 202 that converge to a small contraction region 204, which is approximately midway along the length of the microfluidic chip 14. Figure 14A As shown, the first stage (from left to right) of the microfluidic chip 14 has an inlet 206 defined by a cross-sectional area (x), which tapers outwards 208 to a cross-sectional area greater than x. The microfluidic channel 16 then tapers 200, 202 to a contraction region 204 with a cross-sectional area less than x. This design means that the velocity of the sample 12 gradually increases as it passes through the smaller cross-sectional area of ​​the contraction region 204. In an alternative embodiment, instead of tapering 200, 202 to the contraction region 204, the microfluidic channel 16 may have a stepped taper 212, such as... Figure 14BAs shown in the diagram. Note that the initial expansion portion 208 on either side of the microfluidic chip 14 may also be omitted, in which case the cross-sectional area of ​​the microfluidic channel 16 gradually decreases until the contraction region 204. In one particular embodiment, the contraction region 204 may have a width of about 1.5 mm and a depth of about 1.5 mm. In several embodiments, the length or width may range from about 0.1 mm to about 0.3 mm, about 0.3 mm to about 0.6 mm, about 0.6 mm to about 0.9 mm, about 0.9 mm to about 1.2 mm, about 1.2 mm to about 1.5 mm, about 1.5 mm to about 1.7 mm, or about 1.7 mm to about 2.0 mm, or any value between those listed (including the endpoints). Depending on the embodiment, larger dimensions may also be used. The inlet 206 (or outlet) may have larger dimensions, such as a depth of about 6 mm and a width of about 5 mm. Similarly, the length (or width) of the inlet can vary depending on the implementation, for example, ranging from about 2 mm to about 3 mm, about 3 mm to about 4 mm, about 4 mm to about 5 mm, about 5 mm to about 6 mm, about 6 mm to about 7 mm, about 7 mm to about 8 mm, or any value between those listed (including the endpoints). The length of the constricted region 204 of the microfluidic channel is defined by y and can be on the order of about 1.5 mm. Other dimensions can be used, such as about 0.5 mm to 0.7 mm, about 0.7 mm to about 1.0 mm, about 1.0 mm to about 1.2 mm, about 1.2 mm to about 1.5 mm, about 1.5 mm to about 1.7 mm, about 1.7 mm to 2.0 mm, or any value between those listed (including the endpoints).

[0118] Figure 14CA microfluidic chip 14 is shown having a single microfluidic channel 16 with a series of expansion regions 220 and contraction regions 222. In this design, the microfluidic channel 16 may have an initial cross-sectional area x at an inlet 224. The channel may then expand outward at the expansion regions 220 to a cross-sectional area greater than x. The width of the expansion regions 220 (measured at their maximum width) may range from about 1 mm to about 5 mm. For example, an exemplary width of the expansion regions 220 is 1.3 mm. Other embodiments may employ other widths, such as about 1 mm to about 1.2 mm, about 1.2 mm to about 1.4 mm, about 1.4 mm to about 1.7 mm, about 1.7 mm to about 2.0 mm, about 2.0 mm to about 2.5 mm, about 2.5 mm to about 3.0 mm, about 3.0 mm to about 4.0 mm, 4.0 mm to about 5.0 mm, or any value between those listed (including the endpoints). After the microfluidic channel 16 expands, it then shrinks to a cross-sectional area less than or equal to x at the contraction region 222. The width of the contraction region 220 can range from about 100 μm to about 3 mm. A specific exemplary width of the contraction region 220 is 400 μm. Other embodiments employ widths such as about 100 μm to about 150 μm, about 150 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 500 μm, about 500 μm to about 750 μm, about 750 μm to about 1000 μm, about 1000 μm to about 1.5 mm, about 1.5 mm to about 2 mm, about 2 mm to about 2.5 mm, about 2.5 mm to about 3.0 mm, and any value in between (including endpoints).

[0119] Figure 14D A microfluidic chip 14 with a microfluidic channel 16 is shown, the microfluidic channel including a series of branches 230, whereby the microfluidic channel 16 branches into two smaller microfluidic channels. For example... Figure 14DAs shown, multi-level branching may exist. Smaller microfluidic channels 16 recombine two microfluidic channels into a single microfluidic channel. This process is completed when the microfluidic channels 16 combine to form a single channel on opposite sides of the microfluidic chip 14. In this embodiment, the microfluidic chip 14 may be designed to have an initial cross-sectional area x at the inlet 232 (or outlet). The microfluidic channels 16 may then expand, contract, or remain equal to the inlet cross-sectional area x at the first region 234. The single microfluidic channel 16 then branches into two microfluidic channels 236. These branched microfluidic channels 236 are now smaller than the initial cross-sectional area x of the first region 234. The branched microfluidic channels 236 then branch again to form four microfluidic channels 238. The cross-sectional areas of these microfluidic channels 238 are even smaller than the initial cross-sectional area x. Typically, the cross-sectional area of ​​the branched channels may be determined by x / y or smaller, where x is the initial cross-sectional area and y is the number of parallel channels in that section. Each subsequent stage of the microfluidic channels (236, 238) is smaller than the upstream microfluidic channel. In this embodiment, at each branch point, an edge 240 is formed, which is a sharp tip or point, formed at the apex of the tapering wall of the converging microfluidic channel. This forms a blade that can cut through fat or other tissue as it passes through.

[0120] Figure 14E Another embodiment of the microfluidic chip 14 is shown, wherein the microfluidic channel 16 has a diamond pattern, and the inlet 250 has a cross-sectional area x. The channel has a portion that narrows sharply towards a contraction region 252, such that the cross-sectional area is less than or equal to the initial cross-sectional area x. After the contraction region 252, the microfluidic channel expands sharply outward to reach an expansion region 254 having a cross-sectional area greater than or equal to x. This embodiment may have multiple contraction regions 252 and expansion regions 254. The width of the expansion region 254 (measured at its maximum width) can range from about 1 mm to about 3 mm, for example, about 1 mm to about 1.25 mm, about 1.25 mm to about 1.5 mm, about 1.5 mm to about 1.75 mm, about 1.75 mm to about 2.0 mm, about 2.0 mm to about 2.25 mm, about 2.25 mm to about 2.5 mm, about 2.5 mm to about 2.75 mm, about 2.75 mm to about 3.0 mm, and any value in between (including the endpoints). The width of the contraction region 252 can be in the range of about 100 μm to about 1 mm, for example, a width of 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, about 600 μm to about 700 μm, about 700 μm to about 800 μm, about 800 μm to about 900 μm, about 900 μm to about 1000 μm, or any value in between (including the endpoints).

[0121] Figure 14F Another embodiment of the microfluidic chip 14 is shown, which uses a single microfluidic channel 16 having a plurality of fin-shaped pouches 260 disposed along the length of the microfluidic channel 16. The fin-shaped pouches 260 induce turbulence in the fluid flow through the microfluidic chip 14, which imposes high shear stress on the sample 12. For example, a sample 12 containing cells or tissue can pass through the microfluidic channel 16 with the fin-shaped pouches 260, and the cells can be analyzed in response to the high shear stress environment. The fin-shaped pouches 260 may have a maximum cross-sectional area at their openings equal to or less than the cross-sectional area of ​​the inlet 262. The fin-shaped pouches 260 taper to a point.

[0122] Figure 14G Another embodiment of the microfluidic chip 14 is shown. In this embodiment, the microfluidic chip 14 incorporates blade branches 240 and multiple expansion regions 220 and contraction regions 222. Figure 14H Another embodiment of the microfluidic chip 14 is shown, which includes branches 270 without any blades and multiple expansion regions 220 and contraction regions 222. (See diagram below.) Figure 14HAs shown, rounded or non-acute corners are formed at each of the branches 270. As a specific example, the contraction region 222 may have a width of about 400 μm and a depth of about 300 μm. In several embodiments, the width of the contraction region ranges from about 200 μm to about 500 μm, including about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 450 μm, about 450 μm to about 500 μm, and any width in between (including the endpoints). Similarly, the depth of the contraction region can range from about 200 μm to about 500 μm, including about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 450 μm, about 450 μm to about 500 μm, and any width (including endpoints) in between. In some embodiments, the expansion region 220 may have a depth of about 300 μm and a width of about 1.3 mm. In another embodiment, the width expansion region can range from about 200 μm to about 500 μm, including about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 450 μm, about 450 μm to about 500 μm, and any width (including endpoints) in between. Depending on the implementation, the width can range from about 0.5 mm to about 3 mm, including about 0.5 mm to about 0.75 mm, about 0.75 mm to about 1.0 mm, about 1.0 mm to about 1.1 mm, about 1.1 mm to about 1.2 mm, about 1.2 mm to about 1.3 mm, about 1.3 mm to about 1.4 mm, about 1.4 mm to about 1.5 mm, about 1.5 mm to about 2.0 mm, about 2.0 mm to about 2.5 mm, about 2.5 mm to about 3.0 mm, and any widths in between (including endpoints). In one implementation, the maximum depth of the microfluidic channel 16 at the inlet or outlet is about 6 mm. Other implementations employ depths ranging from about 3 mm to about 7 mm, including about 3 mm to about 4 mm, about 4 mm to about 5 mm, about 5 mm to about 6 mm, about 6 mm to about 7 mm, and any depths in between (including endpoints).

[0123] Figure 14I Another embodiment of the microfluidic chip 14 is shown, which includes blade branches 240 and similar structures in the smallest branch channels. Figure 14A and 14B The hourglass section is 280. Figure 14JAnother embodiment of the microfluidic chip 14 is shown, which includes a blade branch 240 and an hourglass portion 280 located just upstream or before the blade branch 240.

[0124] In one particular embodiment, adipose tissue is processed using the system described herein. First, a physician or other healthcare professional obtains a liposuction sample 12 from the patient, which, depending on the application, is any range from 2 cc to 100 cc, such as about 2 cc to about 10 cc, about 10 cc to about 25 cc, about 25 cc to about 50 cc, about 50 cc to about 75 cc, about 75 cc to about 100 cc, or any volume in between (including endpoints). The fat is then processed first by washing the blood multiple times with a phosphate-buffered saline (PBS) buffer solution (or other acceptable buffer). Once this process is complete, the fat sample 12 is loaded into sample chambers 70, 72 using a syringe or the like, and the sample chambers are loaded together with microfluidic chips 14, 14' into holders 60, 60'. Alternatively, instead of loading the fat sample 12 into separate sample chambers 70, 72, the contents of the syringe (e.g., Figure 8A and 8B The syringe chamber 86 can be directly loaded into the carriers 60, 60' along with the microfluidic chips 14, 14'. This process can be used for a single microfluidic chip 14, 14' or for multiple microfluidic chips 14, 14' (e.g., for large sample processing or samples from different subjects).

[0125] With the sample chambers 70, 72 (or syringe chamber 86) and microfluidic chips 14, 14' loaded into their respective holders 60, 60', operation is initiated by operating motor 40 using controller 50. The rotational speed is initially gradually increased to the desired RPM rate, one non-limiting embodiment of which is... Figure 4 This is illustrated in the diagram. This causes the fat sample 12 to move from sample chamber 70 (assuming this is the radially inward-facing sample chamber) into microfluidic chip 14, where the sample is subjected to shear forces in one or more microfluidic channels 16. The sample continues into another sample chamber 72, and then the carriers 60, 60' and microfluidic chips 14, 14' are rotated 180° using any of the methods described herein (e.g., deceleration, electromagnetic, mechanical gear drive, radial ratchet, manual rotation, etc.). After the microfluidic chips 14, 14' are rotated, spun, or flipped, the support plate 30 rotates to drive the sample from sample chamber 72 into microfluidic chips 14, 14', where the sample is exposed to additional shear forces in one or more microfluidic channels 16. The sample continues to move radially outward and into sample chamber 70. The carriers 60, 60' and microfluidic chips 14, 14' are rotated 180° again, and the process is repeated for the desired number of cycles.

[0126] After the fat sample 12 has been passed through the microfluidic chips 14, 14' for the desired number of cycles or times, the now-processed fat sample 12 is removed from the sample chambers 70, 72 (or syringe chamber 86). In one embodiment, the processed fat sample 12 is then transferred from the sample chambers 70, 72 to a separate syringe attached to the head 74, whereby the processed sample 12 can be removed. The processed sample 12 is now in the syringe and can then optionally be injected into the subject. Alternatively, if the syringe chamber 86 is used as a sample chamber, the syringe chamber 86 is used to directly inject the fat sample 12 contained therein into the subject.

[0127] This design also allows for rapid and repeated cycles within relatively short time periods (e.g., less than ten minutes according to some embodiments). For example, in several embodiments, 20 cycles can be completed within 4 minutes, while 100 cycles can be completed within 20 minutes. Any flow rate from 0 mL / min to 700 mL / min (or higher) can be achieved in this platform, such as 0 mL / min to about 10 mL / min, about 10 mL / min to about 25 mL / min, about 25 mL / min to about 50 mL / min, about 50 mL / min to about 75 mL / min, about 75 mL / min to about 100 mL / min, about 100 mL / min to about 200 mL / min, about 200 mL / min to about 300 mL / min, about 300 mL / min to about 400 mL / min, about 400 mL / min to about 500 mL / min, about 500 mL / min to about 600 mL / min, about 600 mL / min to about 700 mL / min, and any rate in between (including endpoints). In previous syringe pump-based processes, the maximum flow rate achievable by the fluid pump was approximately 12.5 mL / min. For example, Figure 15 It shows the use of Figure 11 The diagram illustrates a graph of the average flow rate of system 10 as a function of RPM via microfluidic chip 14. Furthermore, the system 10 described herein is capable of generating high shear forces; significantly higher than the shear forces generated using injection pump-based processes, such as… Figure 16 As shown in the diagram. This design also has the advantage of performing a single, iterative process in approximately 10 seconds, which is significantly faster than other pump-based processes.

[0128] Figure 17 A graph showing the data obtained from the experiment is presented, illustrating the use of... Figure 5 The system 10 shown processes flow rates (mL / min) as a function of RPM through two different microfluidic chips 14. The first microfluidic chip 14 is Figure 14GThe type shown (e.g., a branched microfluidic channel with expanding and contracting regions) includes a depth of 200 μm and a width of 300 μm. The second microfluidic chip 14 is... Figure 14G The type shown (e.g., branched microfluidic channels with expansion and contraction regions) includes a depth of 1 mm and a width of 300 μm. (As shown...) Figure 17 As shown, high flow rates can be achieved even at RPMs below 1,500. Figure 18 A graph showing the calculated shear stress as a function of RPM for a microfluidic chip 14 with relatively small dimensions (e.g., 200 μm depth and 300 μm width) is presented. The shear stress is calculated using the following equation:

[0129]

[0130] Where τ is the shear stress, "6" is a constant, μ is the fluid viscosity, Q is the flow velocity, w is the channel width, and h is the channel depth. For example... Figure 18 As shown, up to 60,000 dynes / cm is achieved using microfluidic chip 14. 2 (Shear force per pass through microfluidic chip 14). Of course, using a motor 42 with a higher RPM can achieve a shear force greater than 60,000 dynes / cm. 2 The shear force. In several embodiments, approximately 10,000 dynes / cm can be achieved. 2 Approximately 100,000 dynes / cm 2 Shear forces ranging from approximately 10,000 dynes / cm 2 Approximately 20,000 dynes / cm 2 Approximately 20,000 dynes / cm 2 Approximately 30,000 dynes / cm 2 Approximately 30,000 dynes / cm 2 Approximately 40,000 dynes / cm 2 Approximately 40,000 dynes / cm² to approximately 50,000 dynes / cm² 2 Approximately 50,000 dynes / cm 2 Approximately 60,000 dynes / cm 2 Approximately 60,000 dynes / cm 2 Approximately 70,000 dynes / cm 2 Approximately 70,000 dynes / cm 2 Approximately 80,000 dynes / cm 2 Approximately 80,000 dynes / cm 2 Approximately 90,000 dynes / cm 2 Approximately 90,000 dynes / cm 2Approximately 100,000 dynes / cm 2 Or any force between them (including the endpoints).

[0131] One primary application of System 10 is the dissociation, enrichment, and activation of stem cells present in adipose tissue. As explained herein, shear forces generated within one or more microfluidic channels 16 of the microfluidic chips 14, 14' are used to break down adipose tissue, mesenchymal stem cells, and other cells present within the adipose tissue. Various microfluidic chip 14 designs shown herein (e.g., Figure 14A-14J Increased shear stress is applied to the sample. For example, the contraction, gradient, and shaping surfaces of the microfluidic channel 16 break down adipose tissue and activate cells present therein.

[0132] System 10 can be used in other applications. For example, the system can be used for tumor cell isolation. In this sense, it can also be used in general cell dissociators or cell separators, where a sample is broken down into single cells or aggregates of several cells. Figure 19 Microscopic images of tumor cells taken using the system 10 described herein via a microfluidic chip 14 are shown. Figure 19 As shown, a large number of individual cells are present in the field of view. This contrasts with the control of untreated tumor tissue, which shows a high density of individual cells. Figure 20 As seen in the diagram, it contains far fewer individual cells. Other tissue types that can be used for system 10 include, for example, brain tissue and bone marrow. Cells can also be subjected to shear stress via microfluidic chips 14, 14', allowing them to undergo phenotypic or other changes (e.g., mesenchymal stem cells). Individually, microfluidic chips 14, 14' break down cell aggregates by applying shear stress to the aggregates to shear off individual cells for separation. These single cells can then be classified (e.g., tumor type) or their intracellular signals or secretions can be analyzed.

[0133] Figure 21One embodiment of a microfluidic chip 14 is shown, which can be used to sort single cells or cells by size for laboratory or diagnostic purposes. For example, after breaking down tumor cells or pancreatic islet cells, the microfluidic chip 14 can be used to sort and trap single tumor cells for research. The microfluidic chip 14 is designed to sort single cells or aggregates of any specific size. The microfluidic chip 14 has two inlets or outlets 292 for sample entry / exit, each having a cross-sectional area (x). The sample then flows through a main channel 294, the cross-sectional area of ​​which is greater than, less than, or equal to the cross-sectional area (x) of the inlets / outlets. As the sample flows through the main channel 294, it passes through small reservoirs 296 formed in the bottom and / or sides of the microfluidic chip 14. The reservoirs 296 may include pores with a diameter less than, for example, about 100 micrometers. These reservoirs 296 are less than or equal to the cross-sectional area of ​​the main channel 294. Depending on the application, the small reservoirs 296 may be made to a specific size for single cells or aggregates. The reservoir can also be positioned at an angle of 0 to 90° relative to the vertical relative to the main channel 294 (e.g., 0° to 10°, 10° to 30°, 30° to 45°, 45° to 60°, 60° to 90°, etc.) to enhance the effect and better capture cells. In one embodiment, the captured cells can be flushed out of the microfluidic chip 14 for downstream analysis. In an alternative embodiment, the cells can be retained in the reservoir 296 and directly imaged on the chip or otherwise analyzed.

[0134] Furthermore, the system 10 can accommodate any type of microfluidic chip 14, 14', thus allowing it to be universally applicable to virtually any microfluidic device. Because the platform can integrate any chip used for processing, future applications could include: stem cell therapy, Alzheimer's disease treatment, arthritis treatment, wound care, cosmetic procedures, spinal cord injury, fracture treatment, brain injury, ulcer treatment, organ therapy, blood therapy, repair therapy, immunotherapy, nutritional applications, hair regrowth therapy, vision therapy, neurotherapy, muscle therapy, and cartilage replacement.

[0135] Figure 22 Another embodiment of the sample chamber 300 is shown, which can be connected with... Figure 23 The outer casing 46' shown is used in the embodiment. In this embodiment, the sample chamber 300 includes an inner chamber 302, which is in fluid communication with an inlet 304 and an outlet 306. The inlet 304 is used to fill the inner chamber 302 with a sample and includes a one-way valve 308 disposed therein. The one-way valve 308 may include any type of mechanical valve and may also include a releasable diaphragm. As explained below, the inlet 304 and the one-way valve 308 are sized such that the syringe 320 containing the sample ( Figure 23 The inlet 304 can be inserted and the one-way valve 308 opened, allowing the contents of the syringe 320 to be loaded into or removed from the inner chamber 302.

[0136] The outlet 306 of chamber 300 is in fluid communication with microfluidic chip 14, for example, via ports 20, 22. Inner chamber 302 includes a sloped lower surface 310. The sloped lower surface 310 is angled to drive the contents of chamber 300 toward inlet 304, such that the entire sample is retrieved when the processed sample is extracted. Sample chamber 300 includes a venting passage 312 that communicates between inner chamber 302 and the outside of sample chamber 300 (i.e., to the atmosphere). Figure 22 As shown, the ventilation channel 312 extends from the inside of the front portion of chamber 300 (the side connected to the microfluidic chip 14) to the atmosphere at the rear portion of chamber 300 (the rear portion is where the syringe is attached to fill the inner chamber 302). This allows airflow during processing, so that the fluid moves through evenly.

[0137] Figure 23 It shows the relationship with Figure 22 An embodiment of the outer casing 46' used in conjunction with the chamber 300 shown. (As...) Figure 23 As shown, the microfluidic chip 14 is mounted on the carrier 60 together with the chamber 300. In this embodiment, the outer casing 46' includes a bowl 48, which can be used to capture any sample that may be accidentally spilled or released during the process. The outer casing 46' also includes an inlet port 52 located on one side of the outer casing 46', sized to accommodate a syringe 320, allowing samples to be loaded into or removed from the chamber 300 after processing. Figure 23 A syringe 320 is shown, which extends through an inlet hole 52 and is inserted into an inlet 304 of a chamber 300, whereby untreated samples can be loaded into or removed from the chamber 302 after processing is complete.

[0138] To load or unload chamber 300, microfluidic chip 14 is aligned with inlet port 52, and syringe 320 is then inserted into inlet port 52 and into inlet 304 to open one-way valve 308. Once complete, syringe 320 is removed, which then closes one-way valve 308 and prevents any sample leakage. Microfluidic chip 14 is then processed as described herein through multiple rotational or turning cycles of carrier 60 and its microfluidic chip 14, allowing the sample to pass back and forth through microfluidic chip 14. After processing, support plate 30 can be rotated to align microfluidic chip 14 with inlet port 52. The user attaches syringe 320 to inlet 304 of chamber 300 and opens one-way valve 308. The processed sample can then be extracted into syringe 320. Inclined lower surface 310 facilitates the complete drainage of contents from chamber 300. This unloading process can be performed on each microfluidic chip 14.

[0139] Figure 24-26Another embodiment of system 10 is shown. In this embodiment, system 10 includes a top plate or ring 350 that engages with one or more syringes 352, which are mounted vertically relative to the microfluidic chip 14. Connectors 80, etc. (e.g., Figure 2 The syringe 352 outlet is connected to port 20 of the microfluidic chip 14, the connector fluidly connecting the end of the syringe 352 to the microfluidic chip 14. As disclosed herein, the opposite end of the microfluidic chip 14 can be connected to sample chambers 70, 72. The syringe plunger 354 of the syringe 352 is held via a top plate or ring 350 using a hook or clamp 356 sized to hold the syringe plunger 354. Figure 25 As shown in the diagram. In a preferred embodiment, there is a first set of hooks or clips 356 positioned radially inward (e.g., internally) and a second set of hooks or clips 356 positioned radially outward (e.g., externally), as shown in the diagram. Figure 25 As shown in the diagram. In some instances, the first set of hooks or clips 356 and the second set of hooks or clips 356 are configured to allow the distal end of the syringe plunger 354 to be easily secured to and removed from each of the hooks or clips 356. These different hooks or clips 356 engage with the syringe plunger 354 depending on the “rotation” state of the microfluidic chip 14 and the attached syringe 352. As will be discussed in more detail below, when the carriage 60 rotates, the syringe 352 attached to the microfluidic chip 14 can rotate from a radially inward position to a radially outward position. When the carriage 60 rotates, the distal end of the syringe 352 can disengage from the radially inward first set of hooks or clips 356 and subsequently engage / secure to the radially outward positioned second set of hooks or clips 356.

[0140] Therefore, in this embodiment, one or more syringes 352 are typically held perpendicular to the plane of rotation of the microfluidic chip 14. A top plate or ring 350 is rotatably mounted on a bearing 358 having internal threads and engaging a threaded rod 360 attached to a rotary motor 362. The bearing 358 allows the top plate or ring 350 to rotate together with the microfluidic chip 14 and the attached one or more syringes 352. Actuation of the rotary motor 362 causes rotation of the threaded rod 360 and vertical movement of the top plate or ring 350. Rotation in one direction causes the top plate or ring 350 to move downward in the direction of arrow A, thereby pushing the syringe plunger 354 downward into the barrel of the syringe 352. The movement of the motor 362 in this direction is used to expel the contents of the syringe 352 into the microfluidic chip 14. Conversely, movement of the rotary motor 362 in the opposite direction causes the threaded rod 360 to rotate in the opposite direction, resulting in upward movement of the top plate or ring 350 (as indicated by arrow B), thereby pulling the syringe plunger 354 out of the syringe 352. Movement of the motor 362 in this direction is used to draw or eject the sample from the microfluidic chip 14 into the syringe 352. The rotary motor 362 can be fixed to the top of the housing 46, as... Figure 24 As shown in the image.

[0141] In this embodiment, as the microfluidic chip 14 is mounted in the bracket 60 on the support plate 30, the syringe 352 is positioned at one end (e.g., Figure 24 The sample (in a radially inward position as shown) is attached to the microstrip chip 14 via a connector 80 or at the other end via a hook or clip 356. System 10 includes a control panel 370, which can be mounted on the housing 46 and can be used to program operating parameters (e.g., RPM rate, run time, deceleration rate, number of cycles, cycle time, etc.). The control panel 370 can also be used to monitor the status of specific operations using a display 372 or similar device. Buttons 374 are provided to allow the user to interact with the control panel 370. System 10 begins with the support plate 30 rotating using a motor 42, as explained above. During rotation, the syringe 352 rotates together with the microfluidic chip 14. The rotary motor 362 is activated to move the top plate or ring 350 downwards to move the sample from the syringe 352 into the microfluidic device 14. The main motor 42, which drives the support plate 30 and the rotary motor 362, stops rotating, and the syringe 352 with the microfluidic chip 14 rotates 180° (e.g., deceleration causes rotation, although any other rotation method can also be used). Figure 26The microfluidic chip 14 and syringe 352 are shown rotated 180° to a radially outward position. As discussed above, the rotation of the carrier 60 and the attached microfluidic chip 14 and syringe 352 can be accomplished in various ways. Rotation of the syringe 352 disengages the plunger 354 from the hooks or clips 356 attached to the distal end of the plunger 354. The syringe 352 can then engage with a different set of hooks or clips 356 (i.e., radially outward hooks or clips 356). The main motor 42 (in the opposite direction) then drives the support plate 30 and the rotary motor 362, thereby moving the top plate or ring 350 away from the microfluidic chip 14 and used to pull the sample across the microfluidic chip 14 in the opposite direction. This process can be repeated any number of times or cycles. After processing, the syringe 352 containing the now-processed sample can be removed from the system 10 and the syringe can be used directly to inject the processed sample (e.g., adipose tissue) into a patient or subject.

[0142] As explained herein, a primary use of system 10 is to process adipose tissue for therapeutic and / or cosmetic applications. An advantage is that the processing can be carried out without the addition of enzymes or other digestive agents (e.g., collagenase). For example, non-chemical treatment of tissue is required for wound healing applications, thereby preserving the natural extracellular matrix components. Of course, in other embodiments, such as those where samples are evaluated in a laboratory setting, optional digestive agents, such as collagenase or other chemicals or reagents, may be added. These added agents can be used to improve the efficiency of harvesting intracellular and subcellular components.

[0143] Although adipose (or other) tissues are described as being processed using microfluidic chip 14, it should be understood that tissue processing may be accompanied by various additional processing operations, such as washing or filtering steps. These additional processing steps may be performed on-chip (e.g., on microfluidic chip 14) or they may be performed off-chip (i.e., after sample processing on microfluidic chip 14). For example, washing solutions may be loaded into chambers 70, 72 or other separate washing chambers (not shown) that may be used to wash samples in microfluidic chip 14. Similarly, solutions such as... Figure 13 The disclosed filter element 190 filters the sample as it leaves the microfluidic chip. Multiple filters can be used to perform sequential filtration.

[0144] In another embodiment, the sample 12 passing through system 10 comprises cells. System 12 can be used to disrupt (i.e., lyse) cells by passing them through microfluidic chip 14 and exposing them to high shear forces. Cell lysis releases cellular contents, which may contain one or more subcellular organelles, cellular components, membrane-bound organelles, extracellular vesicles, proteins, nucleic acids, paracrine factors, etc. In several embodiments, these components can be released intracellularly and may have therapeutic and / or cosmetic effects. These released intracellular components may have therapeutic or cosmetic effects. For example, a patient's own cells can pass through system 10 and undergo disruption or lysis to release intracellular contents. The cellular contents can then be retrieved and used in the patient. These can be used immediately after processing in some embodiments, or they can be collected and stored for future use. In some embodiments, the extracted intracellular components can be used for the same patient (i.e., autologous) or different patients (i.e., allogeneic). In other embodiments, the treated cells may optionally be cultured, for example in an in vitro culture medium with appropriate nutrients, etc., to promote cell growth. In several embodiments, conditions are selected to enhance the production of secretory proteins by the cells. This may include certain growth factors, culture medium concentration / pH, or, in some embodiments, hypoxic culture conditions (e.g., about 0.1%, about 0.5%, about 1% O2) to optimize the production of secreted proteins. In several embodiments, the cultured (post-processed) cells secrete one or more components that can be isolated from the culture medium. In several embodiments, the cultured (post-processed) cells produce one or more membrane-integrated or membrane-maintained factors, which can also be isolated by lysing or otherwise treating the cells. In several embodiments, these isolated factors can be stored and used as readily available therapeutic agents for allogeneic or autologous therapy. Non-limiting examples of components that can be isolated from cultured (post-processed) cells include various proteins, cytokines, exogenous bodies, etc., as described above. In several embodiments, these factors include, but are not limited to, VEGF, HGF, IGF-1, SDF-1, PDGF-BB, NGF-β, SCF, bFGF, TNF-α, HGFA, MFG-E8, and combinations thereof. In several embodiments, the separation of exogenous miRNAs includes, for example, exogenous miR-223, miR-146b, miR-126, and miR-199a, which may be included alone, in combination with each other, or in combination with other microRNAs.

[0145] Figure 27A and 27B The use of each is shown separately. Figure 14B The microfluidic chip 14 of the type shown is transmitted through Figure 5The graph shows cell counts and cell viability results for adipose tissue processed by the tissue processing system and for fresh, unprocessed adipose tissue (standard adipose tissue extract). Fresh adipose tissue was kept unprocessed (standard adipose tissue extract) or treated with the tissue processing system (processed adipose tissue extract) at 1600 RPM for twenty (20) cycles (each cycle consisting of a 360° rotation of the microfluidic chip 14). Subsequently, each treated sample was combined with 0.1% collagenase to harvest the stromal vascular components.

[0146] In summary, a 0.1% enzyme digestion solution was prepared by combining type I collagenase (Sigma-Aldrich CO., St. Louis, Mo.) with phosphate-buffered saline, and then sterilized using a 0.22 μm vacuum filter (Millipore Corp., Billerica, Mass.). A 1:1 volume of the collagenase solution was incubated with adipose tissue extract in a 37°C water bath for 30 minutes, with intermittent vortexing. An equal volume of control medium (Dulbecco's Modified Eagle Medium, 10% fetal bovine serum, 500 IU penicillin, and 500 μg streptomycin) was then added to neutralize the enzyme activity, and the mixture was allowed to separate for at least 10 minutes. The supernatant containing stromal vascular components was separated, filtered through a 100 μm cell filter (Corning, Inc., Durham, NC), and centrifuged at 1800 rpm for 8 minutes. Each precipitate was then resuspended in erythrocyte lysis buffer (15.5 mM ammonium chloride, 1 mM potassium bicarbonate, and 0.01 mM EDTA) for 5 minutes to minimize erythrocyte contamination. After adding 5 ml of control medium, the suspension was centrifuged one last time. The aqueous fraction was removed by aspiration after each centrifugation step. The resulting precipitates were then resuspended in control medium and stained for analysis.

[0147] A portion of freshly isolated stromal vascular component (SVF) obtained from each sample was stained with acridine orange / propidium iodide (Logos Biosystems, Inc., Annandale, Va.) and quantified using a dual-fluorescence automated cell counter (Logos Biosystems), which depicted live cells relative to dead cells and nucleated cells relative to anucleated cells. Finally, single-cell suspensions were aliquoted into polystyrene tubes and stained with propidium iodide. Each tube was subjected to absolute cell counting function using a flow cytometer (Miltenyi Biotec, Inc., Bergisch Gladbach, Germany) to assess viability. Figure 27A Standard adipose tissue extract and processed adipose tissue extract (using...) are shown. Figure 5A graph showing cell counts from the device. Treatment of samples with this device at 1600 RPM resulted in a ~4-fold reduction in the number of recovered SVF cells. Figure 27B As shown, treatment with this device at 1600 RPM did not result in any discernible difference in the viability of the restored cells. Typically, it has been found that at different applied shear rates, this leads to increased cell damage as the accumulated shear force on the cells increases.

[0148] Flow cytometry was used to analyze stem cell subtypes and stem cell markers in standard adipose tissue extracts and treated adipose tissue extracts. First, mesenchymal stem cell (MSC) populations were identified by gating live cells (marked to the right of the 200 marker on the X-axis relative to the side-scatter window). Next, CD45-negative live cell populations were specifically gated. Finally, CD31-negative and CD45-positive cells were identified. The treated adipose tissue extract was found to contain a higher proportion of MSCs (CD45- / CD31- / CD34+) than their counterparts in the standard adipose tissue extract.

[0149] Figure 28 The images show extracts from standard adipose tissue and processed adipose tissue (using...) Figure 5 A diagram illustrating stem cell markers and subtypes identified in SVF cells obtained from the device. CD34 was a universal stem cell marker that was upregulated nearly 3-fold after device treatment. Similarly, MSC subpopulations (CD45- / CD31- / CD34+) and (CD45- / CD31- / CD34+ / DPP4+ / CD55+), which are crucial for wound healing in diabetes, were found to be enriched after treatment. Graph bars marked with '*' indicate p < 0.05.

[0150] Analysis and use Figure 5 Stem cell markers and subtypes of diabetic adipose tissue extracts processed by the device and standard diabetic adipose tissue extracts (unprocessed). Figure 29 A graph showing the stem cell markers (CD45, CD31, CD34, CD73, CD146, MSCs, DPP4 / CD55) of processed adipose tissue extract and standard adipose tissue extract is presented. CD34 is a universal stem cell marker. In this specific test patient, CD34 was upregulated nearly 2-fold after device treatment. Similarly, other MSC markers (CD73, CD146) and the endothelial marker (CD31) were upregulated after device treatment. Finally, MSC subsets (CD45- / CD31- / CD34+) and (CD45- / CD31- / CD34+ / DPP4+ / CD55+) crucial for diabetic wound healing were found to be enriched after diabetic tissue treatment.

[0151] Various combinations or sub-combinations of the specific features and aspects of the disclosed embodiments are anticipated, and such combinations or sub-combinations still fall within one or more of the inventions. Furthermore, any specific features, aspects, methods, properties, characteristics, qualities, attributes, elements, etc., disclosed herein in relation to embodiments can be used in all other embodiments set forth herein. Therefore, it should be understood that various features and aspects of the disclosed embodiments can be combined or substituted with each other to form variations of the disclosed invention. Therefore, it is expected that the scope of the invention disclosed herein should not be limited to the specific embodiments disclosed above. Furthermore, while the invention is readily adaptable to various modifications and substitutions, specific examples have been shown in the accompanying drawings and described in detail herein. However, it should be understood that the invention is not limited to the specific forms or methods disclosed; rather, the invention will cover all modifications, equivalents, and substitutions falling within the spirit and scope of the various embodiments described and the appended claims. Any methods disclosed herein need not be performed sequentially. The methods disclosed herein include certain actions taken by a practitioner; however, these actions may also explicitly or implicitly include any third-party instructions regarding these actions. For example, actions such as “managing the expanded NK cell population” include “instructing the management of the expanded NK cell population.” Furthermore, in cases where features or aspects of this disclosure are described in accordance with the Markush Group, those skilled in the art will recognize that this disclosure is therefore also described in the form of any individual member or subgroup of members of the Markush Group.

[0152] The scope disclosed herein also covers any and all overlapping, subscopes, and combinations thereof. Languages ​​such as “at most,” “at least,” “greater than,” “less than,” and “between” include the referenced numbers. Numbers beginning with terms such as “about” or “approximately” include the referenced numbers. For example, “about 10 nanometers” includes “10 nanometers.”

[0153] While embodiments of the invention have been shown and described, various modifications can be made without departing from the scope of the invention. Therefore, the invention should not be limited except for the following claims and their equivalents.

Claims

1. A system for processing biological samples, comprising: A motor, having a drive shaft; Support plate, the support plate comprising: A receiving element that reversibly interacts with the drive shaft of the motor; Multiple arms extend radially from the support plate, each arm including a through hole that reversibly interacts with one of the multiple brackets; A plurality of holders, each of the plurality of holders including a first end, a second end, and a base extending between the first end and the second end, and a receiving region configured to reversibly interact with a microfluidic chip configured to allow bidirectional flow of a sample within the microfluidic chip, wherein each of the plurality of holders includes a post extending orthogonally from the base and sized to pass through the through-hole and protrude beyond the lower surface of the respective arm, wherein the microfluidic chip includes a first end and a second end, wherein the first end of the microfluidic chip is fluidly connected to a first sample chamber configured to receive a sample for processing, wherein the second end of the microfluidic chip is fluidly connected to a second sample chamber, wherein the microfluidic chip is configured to allow bidirectional flow of a sample from the first sample chamber to the second sample chamber and back to the first sample chamber; and Each of the plurality of brackets is at least intermittently rotatable by an arc of 180 degrees and each of the plurality of brackets is movable from each arm of the support plate.

2. The system of claim 1, wherein each of the plurality of brackets rotates in or out of a horizontal plane substantially parallel to the plane of the support plate.

3. The system according to claim 1 further includes a plurality of microfluidic chips, each microfluidic chip comprising: The central main body is located between the first and second sample chambers. The central body portion includes at least one microfluidic channel extending between a first end and a second end. The at least one microfluidic channel includes different sizes and is configured to allow repeated bidirectional flow of the sample from the first chamber to the second chamber and then from the second chamber to the first chamber.

4. The system according to claim 3, wherein, The rotation of the bracket allows for bidirectional flow.

5. The system according to claim 3, wherein, Each microfluidic chip is reversibly fluidly connected to a corresponding sample chamber at each of the first and second ends.

6. The system according to claim 5, wherein, Each sample chamber is reversibly fluidly connected to the microfluidic chip via a connector.

7. The system according to claim 1, wherein, Each of the plurality of brackets includes a capture element at each of the first and second ends of the bracket, the capture element being configured to communicate with a release element on the support plate, wherein the communication between the capture element and the release element allows intermittent rotation of each of the plurality of brackets.

8. The system according to claim 7, wherein, The capturing element includes a magnet of a first polarity, and the releasing element includes a magnet of the opposite polarity.

9. The system according to any one of claims 1 to 8, wherein, The support plate and the plurality of arms are an integral structure.

10. A system for processing samples, comprising: A support plate includes a plurality of arms, each of which extends radially from the support plate, and each arm includes a through hole extending therethrough; The support plate is configured to rotate centrifugally. Multiple brackets, Each of the plurality of brackets is arranged on a corresponding one of the plurality of arms on the support plate, and each of the plurality of brackets includes a post extending orthogonally from the base and is sized to pass through the through hole and protrude beyond the lower surface of the respective arm. Each of the plurality of holders is configured to receive a microfluidic chip, a first sample chamber, and a second sample chamber, wherein the microfluidic chip is fluidly connected to the first and second sample chambers and includes at least one microfluidic channel extending between the first and second chambers, the at least one microfluidic channel comprising different sizes, wherein the respective fluid connections between the microfluidic chip and the first and second sample chambers allow bidirectional flow of the sample between the first and second sample chambers through the at least one microfluidic channel, and Each of the plurality of brackets is at least intermittently rotatable by an arc of 180 degrees and each of the plurality of brackets is movable from each arm of the support plate.

11. The system according to claim 10, wherein, At least one of the first and second sample chambers is configured to be movable from the microfluidic chip.

12. The system according to claim 10, wherein, At least one of the first and second sample chambers includes a vent and a vent channel, the vent channel being fluidly connected to the interior of the sample chamber, wherein the vent is configured to provide laminar flow of the sample through the sample chamber.

13. The system according to any one of claims 10 to 12, wherein, Each of the plurality of arms further includes a first engagement structure and a second engagement structure. The first joining structure is located at a certain distance from the second joining structure, and The first engagement structure and the second engagement structure are each configured to engage with one of the first structure located at the first end of the bracket and the second structure located at the second end of the bracket.

14. The system according to claim 13, wherein, The first engagement structure and the second engagement structure are configured to interchangeably release and engage, such that the bracket is configured to move between multiple directions about one of multiple axes.

15. The system according to claim 13, wherein, The first bonding structure and the second bonding structure are magnets, and the first structure and the second structure include magnetically responsive materials.

16. A system for processing a sample, comprising: Multiple arms, each of which extends radially from a central region, the central region interacting reversibly with the drive shaft of a motor; as well as Multiple brackets, Each of the plurality of brackets is disposed on a corresponding one of the plurality of arms, and each of the plurality of brackets includes a first end, a second end, and a base extending between the first end and the second end. Each of the plurality of brackets is configured to reversibly receive a corresponding one of a plurality of microfluidic chips, the microfluidic chip including at least one microfluidic channel extending between a first end and a second end of the microfluidic chip. Each of the plurality of arms includes an interaction region, the interaction region including a through-hole receiving a post extending from the base of a corresponding bracket arranged on each arm and extending beyond the lower surface of the respective arm. Each of the plurality of holders is intermittently rotatable about the column, thereby allowing bidirectional flow of the sample between the first and second ends of the microfluidic chip, and each of the plurality of holders is movable from its respective arm. The first end of the microfluidic chip is fluidly connected to a first sample chamber configured to receive a sample for processing, the second end of the microfluidic chip is fluidly connected to a second sample chamber, and the microfluidic chip is configured to allow bidirectional flow of the sample from the first sample chamber to the second sample chamber and back to the first sample chamber.

17. The system according to claim 16, wherein, Each bracket includes a gear assembly configured to rotate the bracket about the post.

18. The system according to claim 16, wherein, Each bracket includes a radial ratchet configured to rotate the bracket about the post.

19. The system according to any one of claims 16 to 18, wherein, Each of the plurality of microfluidic chips is reversibly fluidly connected to a corresponding first sample chamber and second sample chamber located at each of the first and second ends of the microfluidic chip.

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