EP4143292A2 - Automated integrated liposuction and tissue processing device and method - Google Patents

Abstract

The invention relates to methods and apparatus for recovery of cells from a tissue sample using a single container for processing and separation of tissue and cells. The processing unit comprises a processing compartment having a processing side wall; at least one wash fluid compartment having a washing side wall, wherein the wash fluid compartment is located radially outside of the processing compartment and separated by the processing side wall, wherein a first opening is formed at the top of the processing side wall to allow fluidic communication between the processing compartment and the wash fluid compartment; and at least one concentration compartment, wherein the concentration container is located radially outside of the wash fluid compartment and separated by the washing side wall, wherein a second opening is formed at the top of the washing side wall to allow fluidic communication between the wash fluid compartment and the concentration compartment.

Description

AUTOMATED INTEGRATED LIPOSUCTION AND TISSUE PROCESSING DEVICE

AND METHOD

PRIOR RELATED APPLICATIONS

[0001] This application claims priority to U.S. serial No. 63/018,475, filed on April 30,

2020, which is incorporated by reference in its entirety for all purposes.

FEDERALLY SPONSORED RESEARCH STATEMENT

[0002] Not applicable.

FIELD OF THE DISCLOSURE

[0003] The disclosure generally relates to a centrifuge-based processing method and device, and more particularly to a multistage centrifuge-based processing method and device for automated processing of cell extraction and recovery in a single processing unit.

BACKGROUND OF THE DISCLOSURE

[0004] With increasing molecular diagnostic and therapeutic possibilities and treatments, it is of great relevance and importance to isolate from solid tumor tissue the respective cellular fraction that can be processed to recover single tumor cells. In analyzing these cells, a subsequent analysis of the gene expression, single cell RNA and DNA gene and protein expression, cells surface markers can also be performed.

[0005] Conventionally, to extract stem cells from adipose tissues, human handling plays an important role, including extracting the adipose tissue, processing the adipose tissue with enzymes, separating the processed cells from the rest of the liquid mixture, washing the separated cells, and further adding liquid to dilute the concentration of enzymes in the final product, and finally centrifugation to concentrate the cells in a reduced amount of fluid.

[0006] However, human handling requires training to properly handle the tissues, and the more human handling is involved, the more likely it is to introduce mistakes and contaminant into the cells. [0007] Therefore, there is the need for an automated tissue processing unit and method, where the tissues can be processed, washed, and concentrated using automated centrifuge, while at the same time reducing or even avoiding completely human handling and interference in order to reduce contamination.

SUMMARY OF THE DISCLOSURE

[0008] The present disclosure describes a tissue processing unit and method for processing tissues to extract cells with minimum human handling. The processing unit comprises a processing compartment having a processing side wall; at least one wash fluid compartment having a washing side wall, wherein the wash fluid compartment is located radially outside of the processing compartment and separated by the processing side wall, wherein a first opening is formed at the top of the processing side wall to allow fluidic communication between the processing compartment and the wash fluid compartment; and at least one concentration compartment, wherein the concentration container is located radially outside of the wash fluid compartment and separated by the washing side wall, wherein a second opening is formed at the top of the washing side wall to allow fluidic communication between the wash fluid compartment and the concentration compartment.

[0009] The different slopes and shapes of the sidewalls of the chambers facilitate processing, breaking down and separation of the initial tissue into its component. By creating openings of different diameters on sidewalls of the bowls or using filters to the same extent, a part of the liquid previously present in the processing compartment can initially be separated from the processed tissue and flows into a waste or washing compartment. The separation and recovery of different components of tissues in the processing chamber is achieved through repetitive stop-and-go cycles and subsequent application of different cycles of centrifugation forces. In the washing fluid compartment, the processed tissues and cells can be further washed and the remaining enzymatic activity can be diluted. Separation and recovery of cells from debris, washing fluid including cell, erythrocyte and collagen fragments and from liquid fat is based on the different specific density and gravity of the substances and based on application of different acceleration forces applied to the tissue components in the respective chambers. By centrifugation with increasing force, the cells are moved from the processing compartment into the washing chamber and from there into the concentration compartment, which contain previously deposited clean washing fluid. The amount of clean washing fluid is preferably calculated such that after dilution the concentration of enzymatic rest activity from collagenases and proteases will be reduced to a level safe and acceptable for immediate use.

[0010] The sidewalls of the bowls are designed to have different slopes with openings placed on the sidewalls. Based on the specific mass of the processed tissue and cells and the processing fluid in the inner processing chamber, we can calculate the centrifugal g- force such that only the washing fluid would travel along the sidewall high enough to reach the openings and could enter the concentration chamber.

[0011] In some embodiment, additional sealable openings are provided on the lid. One of these sealable openings can be connected with a liposuction syringe for extracting adipose tissues directly from a subject for a contactless transfer of the adipose tissues into the processing chamber, thus minimizing the chance of contamination. Additional openings can be used to deposit the washing fluid into the washing and concentration chambers.

[0012] In one embodiment, the plurality of openings comprises a first array of perforations and a second array of perforations or filters, wherein the first array of perforations are formed on the side wall at a location lower than the second array of perforations. In one embodiment, the size of the first array of openings allows liquid to pass through, but not the cells in order to keep the cells inside the processing chamber. Second array of openings further up the sidewall of the processing chamber have a diameter that the cells can pass through, and centrifugation at a higher g-force can spin the cells to reach the secondary openings, where the cells will travel pass the secondary openings into the dilution chamber.

[0013] In one embodiment, the first array of openings has a diameter of 1-5 pm. In one embodiment, the second array of openings have a diameter of 40-200 pm.

[0014] In one embodiment, the processing compartment is generally triangular in shape, wherein a first distance from the bottom recess to a vertex of the triangular shape is greater than a second distance from the bottom recess to a side of the triangular shape.

[0015] In one embodiment, the processing compartment has bottom baffles to improve tissue breakdown during agitation. [0016] In one embodiment, the concentration compartment is a detachable concentration container. In one embodiment, the detachable concentration containers are threaded to the processing unit.

[0017] In one embodiment, the tissue processing unit further comprises a lid that covers and seals the first, second and third bowls. In one embodiment, the lid further comprises a first inlet that allows the injection of tissues to the processing chamber, and a second inlet that allows the introduction of washing fluid into the wash fluid chamber.

[0018] In one embodiment, a filter is provided to cover the second opening between the wash fluid compartment and the concentration compartment. The filter can be a filter mesh having mesh openings of 40-300 pm.

[0019] In one embodiment, a siphon structure is provided between the second opening and the concentration compartment to prevent fluid backflow.

[0020] In one embodiment, a concentrated cellular suspension can be recovered from a delivery device such as a syringe, for injection into the recipient by, for example, subcutaneous, intravenous, intramuscular, or intraperitoneal techniques. For example, the regenerative cells can be injected into blood vessels for systemic or local delivery, into tissue (e.g., cardiac muscle or skeletal muscle), into the dermis (subcutaneous), into tissue space (e.g., pericardium or peritoneum), or other location. Injection of the regenerative platform can result in an area near the injection site being augmented, repaired, having reduced inflammation, reduced pain, and combinations thereof. In some embodiments, one or more additives are added to the cells before administration. For example, the cells can be mixed with other cells, biologically active compounds, biologically inert compounds, demineralized bone, a matrix or other resorbable scaffold, one or more growth factors, or other additive that can enhance the delivery, efficacy, tolerability, or function of the cell population. In one embodiment, the recovery of a cellular preparation, such as from tumor or other tissues, can be used for further diagnostic measures.

[0021] In another aspect of this disclosure, a method for automated extraction of adipose- derived stem cells from adipose tissue is described. The method comprises the steps of: providing a centrifuge processing unit discussed above and a centrifuge machine; loading adipose tissue, enzymatic reagents and a fluid such as saline or calcium containing ringer solution into the processing chamber; loading washing fluid into the middle washing chamber; placing the centrifuge processing container into the centrifuge processing unit; agitating the tissue together with an enzyme-containing fluid in an inner processing compartment by applying repetitive acceleration and deceleration cycles of a first g-force of 0.5 to 3 g in order to dissociate the tissue; accelerating the processed tissue from step a) with a second g-force greater than the first g-force sufficient to move the processed tissue from the processing compartment into a washing compartment placed radially outside the processing compartment, wherein the washing compartment is prefilled with a washing fluid; allowing the processed tissue to be diluted and washed in the washing fluid; accelerating the processing unit with a third g-force greater than the first and second g-forces to transfer the cells into a concentration compartment pre-filled with an enzyme-free fluid; and optionally removing the cellular preparation from the concentration compartment.

[0022] In another aspect of this disclosure, a vibrating liporator is described. The vibrating liporator comprises: a housing having a cannula channel, a cylindrical piston space, an outlet, and a vacuum port, wherein the vacuum port is fluidically connected to the cylindrical piston space; a piston located in the cylindrical piston space; and a cannula located within the cannula channel and sealingly connected to the outlet through the piston such that a flow path from the cannula to the outlet is sealed, wherein the piston engages with a spring such that when a vacuum creates a negative pressure inside the cylindrical piston space through the vacuum port, the piston moves to compress the spring. In one embodiment, the vibrating liporator is disposable.

[0023] In another aspect of this disclosure, a closed system for processing and recovering cellular component from mammalian tissue, without contacting ambient environment, is described. The system comprises a processing unit having: a processing compartment having a processing side wall; at least one wash fluid compartment having a washing side wall, wherein the wash fluid compartment is located radially outside of the processing compartment and separated by the processing side wall, wherein a first opening is formed at the top of the processing side wall to allow fluidic communication between the processing compartment and the wash fluid compartment; at least one concentration compartment, wherein the concentration container is located radially outside of the wash fluid compartment and separated by the washing side wall, wherein a second opening is formed at the top of the washing side wall to allow fluidic communication between the wash fluid compartment and the concentration compartment; a top cover having a first inlet and a second inlet, wherein the first inlet provides fluidic communication with the processing compartment, and the second inlet provides fluid communication with the wash fluid compartment. The closed system further comprises a tissue-extraction device operatively coupled to the first inlet on the top cover, such that tissues extracted by the tissue-extraction device are sterilely transferred into the processing compartment of the processing unit.

[0024] The tissue extraction device is powered by vacuum to induce a vibrating cannula that is connected to a moving piston. The piston is located within a cylinder and the vacuum retracts the piston and the cannula against a spring. By releasing the vacuum the spring moves the piston and canula forward again, thus vibrating the device. Movements of the cannula at a frequency between 5 and 50 Hz can be achieved. The same vacuum source that also applied the vacuum suction to the tip of the canula is used to drive the vibration of the piston and cannula.

[0025] In one embodiment, the tissue-extraction device is the vibrating liporator described herein.

[0026] The vacuum-driven liposuction device preferably comprises a handheld piece formed from polycarbonate, and the cannula made from stainless steel. Due to its simplified construction, material and external power source, the device can be made as a single use disposable device.

[0027] In another aspect of this disclosure, a power-assisted disposable tissue extraction device is described. The device comprises: a housing having a cannula channel, a cylindrical piston space, an outlet, and a vacuum port, wherein the vacuum port is fluidically connected to the cylindrical piston space; a piston located in the cylindrical piston space; and a cannula located within the cannula channel and through the piston, said cannula having a distal end and a proximal end, wherein the proximal end is sealingly connected to the outlet such that a flow path from the distal end of the cannula to the outlet is sealed, wherein the piston engages with the spring such that when a vacuum creates a negative pressure inside the cylindrical space on the piston through the vacuum port, the piston moves and compresses the spring, wherein the spring decompresses to push the piston and cannula back when the vacuum releases, thereby creating a vibrational movement of the cannula with repetitive cycles of vacuum and vacuum release to facilitate the recovery of tissue from the distal end of the cannula.

[0028] In another aspect of this disclosure, a method of recovering cells from a mammalian tissue using a single disposable device is described. The method comprises: filling the mammalian tissue into a processing chamber; processing the mammalian tissue in the processing chamber into cells and non-cell tissue fragments; separating the cells from the non-cell tissue fragments using multi-stage centrifugation of different centrifugal forces; and recovering the cells in a sterile container in a concentrated low fluid volume of 2 to 10 ml. [0029] As used herein, “vial”, or “bowl” refers to a bowl-like container that is capable of holding liquid with a sealed bottom.

[0030] As used herein, "processing" refers to the step of treating a tissue with an enzymatic fluid and/or mechanical force to digest and/or breakdown the tissue. Enzymes may include protease, collagenase, and mixtures thereof. Heating may also be applied to facilitate processing.

[0031] The use of the word “a” or “an” when used in conjunction with the term

“comprising” in the claims or the specification means one or more than one, unless the context dictates otherwise.

[0032] The term “about” means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated.

[0033] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.

[0034] The terms “comprise”, “have”, “include” and “contain” (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim. [0035] The phrase “consisting of’ is closed, and excludes all additional elements.

[0036] The phrase “consisting essentially of’ excludes additional material elements, but allows the inclusions of non-material elements that do not substantially change the nature of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS [0037] FIG. 1 A-C. Perspective view of the processing unit of this disclosure.

[0038] FIG. 2. Top view of the processing unit without the lid.

[0039] FIG. 3. Cross-sectional view of the processing unit at the sidewall.

[0040] FIG. 4A. Alternative view of the lid.

[0041] FIG. 4B. The lid in conjunction with a liposuction syringe. [0042] FIG. 5. Flow diagram of the method of this disclosure.

[0043] FIG. 6A. Cross-sectional view of the processing unit according to another embodiment of this disclosure.

[0044] FIG. 6B. Top view of the processing unit with the cover on according to another embodiment of this disclosure.

[0045] FIG. 6C. Top view of the processing unit without the cover according to another embodiment of this disclosure.

[0046] FIG. 7A. Cross-sectional view of the processing unit illustrating step 1 of the method of processing: preparation. [0047] FIG. 7B. Cross-sectional view of the processing unit illustrating step 2 of the method of processing: processing cells.

[0048] FIG. 7C. Cross-sectional view of the processing unit illustrating step 3 of the method of processing: washing and concentrating.

[0049] FIG. 8A. Perspective view of an extraction device for extracting adipocytes. [0050] FIG. 8B. Cross-sectional view of the extraction device of FIG. 8 A.

[0051] FIG. 9. Illustration of the method of this disclosure to extract adipose tissue and transfer it into the processing unit of this disclosure without contacting the atmosphere.

DETAILED DESCRIPTION

[0052] The disclosure provides novel centrifuge processing container to be used in an automated system to extract stem cells from adipose tissue.

[0053] This disclosure describes methods and apparatus for recovery of cells from tissues, including human and animal tissues such as canine, feline, equine, bovine, ovine, or porcine tissues. The methods and apparatus described herein are particularly useful for recovery of cells from adipose tissue obtained from, for example, liposuction (i.e., lipoaspirate), including suction assisted, laser or pressure assisted, or ultrasound assisted liposuction, and combinations thereof. For instance, the methods and apparatus described herein can be used to isolate stem cells, progenitor cells, hematopoietic cells, or fully differentiated cells from adipose tissue. Additionally, the method and apparatus of this disclosure can extract adipose tissue from a subject and transfer it into the processing unit of this disclosure in a completely sterile fashion, thereby ensuring the thus-extracted adipose tissue is free of contamination.

[0054] Once inside the processing unit, the extracted tissue can be processed, washed, and concentrated all within the same unit through sequential introduction/removal of buffers and automated centrifugation. The resulting cells are thus ready for further therapeutic use on site.

[0055] The methods and apparatus described herein can be used on site to prepare cellular compositions for administration to a patient (e.g., autologous administration). For example, the methods and apparatus described herein can be used to recover regenerative cells from a patient, referred to herein as a regenerative platform, that can be prepared for administration and then administered (e.g., injected or surgically implanted) back to the patient from which the cells were recovered.

[0056] The methods and apparatus also can be used to prepare cellular compositions for growth studies, gene expression studies, differentiation studies, or other research purposes. In addition, the methods and apparatus described herein can be used to recover regenerative cell populations (e.g., stem cells) such that the cells can be banked, for example, by cry opreserving the cells with an appropriate medium. For further reference, see U.S. Patent Application Publication No. 20100285588-A1.

[0057] In one embodiment, the single container for processing and separating mammal tissue is shown in Figure 1A. The single container 1 for processing consists of three separate bowls 11, 12, 13 aligned in a concentric fashion, with bowl 11 in the inner most surrounded by intermediate bowl 12 that is in turn surrounded by the outermost bowl 13. In this configuration, multiple chambers are created individual chambers, 2, 3, 4 in between each bowl, including the processing chamber 2 inside the innermost bowl 11, the washing chamber 3 between the outer wall of the innermost bowl 11 and the inner wall of the intermediate bowl 12, and the dilution/concentration chambers 4 between the intermediate bowl 12 and the outermost bowl 13. The concentric configuration puts all three bowls 11, 12, 13 revolving around a rotational axis 8 that may adapt to a shaft of a centrifugation machine (not shown). The processing chamber 2 is surrounded by the washing chamber 3, and the washing chamber 3 is surrounded by the dilution/concentration chamber 4. [0058] In one embodiment, the inner wall of the processing chamber 2 has uneven surface, or ribs, such that when the processing unit is being rotated during centrifugation, the uneven surface can facilitate breaking up of tissues.

[0059] In an embodiment, a predetermined amount of washing fluid is deposited in the washing chamber 3. A predetermined amount of dilution fluid is deposited in the dilution/concentration chambers 4.

[0060] The overall shape of the integrated chamber and unit is triangular with three distal ends. Each of the distal ends has a point of maximal acceleration upon centrifugation, such that the openings 9 are experiencing the highest gravitational force based on the distance to the rotational axis 8. The processing unit is firmly covered by a lid 5 that covers the walls of the intermediate bowl 12, the washing chamber number 3, the walls of the outermost bowl 13 and the dilution/concentration chamber 4. The upper wall of the inner most bowl 11 and the processing chamber 2 are covered by and connected to the lid 5.

[0061] On the outer wall of the distal ends of each of the three concentration chambers

4, and most distant from the rotational axis 8, there are three openings 9, which are closed by a valve or a swappable luer lock, to which a syringe can be connected in order to remove the cells after the cellular preparation further detailed below.

[0062] The inner center of the processing unit is connected to the unsterile outside by an opening 15 in lid 5. In one embodiment, as shown in Figure 4A, the flow of operations occurs in a way that processing chamber number 2 is filled through the luer locked entry 16, and the washing chamber is filled through an opening 17. Each of those openings can be initially closed by a luer stopcock. Filling of the inner processing chamber 2 occurs through opening number 16 with tissue material, preferably liposuction of adipose tissue through the opening, the liposuction material is agitated together with an enzyme and fluid within the chamber number by repetitive low g force accelerations in a range from 0.5 to 3 g.

[0063] Figure 4B shows the lid 5 used in conjunction with a liposuction syringe and a three-way valve. During liposuction, the three-way valve is turned to allow vacuum suction between the needle and the syringe. Once sufficient amount of adipose tissue has been extracted from the needle, the three-way valve is turned to allow direct transfer of the adipose tissue from the syringe into the processing chamber through opening 16 on the lid 5. This configuration removes the need for any transportation or handling between liposuction and tissue processing, thus reducing the chance of contamination and time.

[0064] The amount of liposuction or tissue material for filling of the processing chamber

2 is in a range between 10 and 250 grams. An equal or higher amount of a calcium containing fluid is added to the processing chamber 2 together with an enzyme, preferably consisting of an enzyme mixture of collagenase I, collagenase II, and a protease.

[0065] To effectively digest and breakdown the tissue, the processing unit is placed within a centrifugation system that is contained in a controlled heated enclosure, such as previously described in the U.S. Pat. No. 8951513. The processing of tissue by agitation can be effected in about 20 to 40 minutes. Also helpful for better mixing and agitation of the processed fluid and tissue are elements 85 that are helpful to create vertical forces that complement the differential horizontal accelerations as represented by the distances di between 82-83 and d2 between 82-84.

[0066] The method for processing tissues to isolate cells of this disclosure will now be discussed with reference to Figures 3 and 5. Figure 3 is a side view cut through the unit depicting the left half part of the processing chamber 2, the washing chamber 3, and the dilution chamber 4 on top. The side walls and the whole unit is made preferably of polycarbonate, however other materials may also be used, as long as they are easy to manufacture and/or biocompatible. In the grey area in the middle is the unsterile outside field, through which the unit is firmly attached to the motor. The sidewall 21 of the processing chamber 2 has a slope that changes from the bottom to the top. This slope at sidewall 21 is different comparing to the slope at sidewall 31 of the washing chamber 3 that allows a higher acceleration force; first due to the greater distance to the axis and second due to the lower or less steep incline of the wall, resulting in a relatively higher vertical vector as part of the centrifugal vector.

[0067] FIGS. 6A-C depict an alternative processing unit of this disclosure. FIG. 6A is a cross-sectional view of the processing unit, whereas FIGS. 6B, 6C are top views of the processing unit with and without the cover, respectively.

[0068] In FIG. 6 A, the processing unit 100 is engaged on a rotor 700 of a centrifuge as discussed above. The processing unit 100 of this embodiment is different from the one shown in FIGS. 1-4 above. The processing unit 100 has a processing compartment 101 for holding a process fluid and tissues for processing, a wash fluid compartment 200 for holding wash fluid, and a cover 600 that complete covers the processing unit 100 to provide an isolated space within the processing unit 100, except for two inlets. A first inlet 601 is located on the cover 600 corresponding to the processing compartment 101, and a second inlet 602 is located on the cover 600 corresponding to the wash fluid compartment 200. See also FIG. 6B. The first inlet 601 enables a sterile connection with a lipoaspirator (further discussed below with regard to FIGS. 8A-B) for transferring the extracted tissue into the processing compartment 101. Similarly, the second inlet 602 enables a sterile injection of wash fluid into the wash fluid compartment 200.

[0069] The processing compartment 101 has multiple baffles 103 at the bottom of the space to increase the efficiency of physically breaking down the tissue by shear force. See also FIG. 6C. The processing compartment 101 has a first wall 105 of a first slope, and a second wall 107 of a second slope, wherein the first slope is steeper than the second slope. The wash fluid compartment 200 is separated from the processing compartment 101 by the first wall 105, and an opening 109 is provided at the top of the first wall 105 to allow fluidic communication between the two compartments 101, 200.

[0070] A concentration container 500 is removably attached to the processing unit 100 through, for example, thread 501. However, other mechanism to connect the concentration container 500 to the processing unit 100 may also be used without deviating from this disclosure.

[0071] The concentration container 500 has a fluid path from the wash fluid compartment 200 through an opening 201, a siphon structure 400, and a one-way valve 502. A filter mesh 300 is provided at the opening 201, wherein the filter mesh 300 has mesh openings large enough for cells to pass through and small enough to block the digested tissue and debris. For example, a filter mesh having mesh opening of about 40 to 200 microns can be used. The one-way valve 502 serve to prevent leakage from the processing unit 100 after the concentration container 500 is detached.

[0072] The detachable concentration container is advantageous in that once the processing/washing/concentrating steps are completed, the resulting cells are collected within the concentration container 500 and is ready for the next procedure or being stored away for future use. There is no further need for the processing unit 100, as it is intended for single-use only.

[0073] The slope of the first wall 105 is steeper than the slope of the second wall 107 such that during the processing step with lower centrifugal speed, the processing fluid and tissue will stay in the processing compartment 101 without overflowing into the wash fluid compartment 200 through the opening 109. See FIG. 6C. It is only when sufficient centrifugal speed is reached can the processing fluid as well as the tissue reach high enough on the first wall 105 to pass through the opening 109 into the wash fluid compartment 200.

[0074] The following discussion describes the method of extracting adipose tissue from a subject, processing the adipose tissue to release cells from the tissue, washing the cells, and increasing the concentration of the cells. The method will be illustrated with reference to FIGS. 5-9. In step 501, the adipose tissues are extracted by a syringe or liposuction handpiece 800, as shown in FIG. 8A. In one embodiment, the liposuction handpiece is disposable. FIG. 8B shows the cross section of the liposuction handpiece 800, where a liposuction cannula 802 is connected to a lipoaspirate outlet 806 through a piston 803 and tubing 805. The piston 803 sits inside a cylindrical space 810 and engages a spring 804. The cylindrical space 810 that is fluidically connected to a vacuum port 807 that can be connected to a vacuum 809 through a vacuum modulating valve 808 (see FIG. 9). The vacuum 809 creates a negative pressure that pulls the piston 803 towards the vacuum port 807 and compresses the spring 804, which then pushes the piston 803 back to its original position inside the cylindrical space 810. The back-and -forth motion of the piston 803 creates a vibration onto the liposuction handpiece 800 and the cannula 802, thereby allowing easier extraction of adipose tissue from the patient.

[0075] In one embodiment, during liposuction, the bodily fluid and tissues extracted from a subject will first flow through the lipoaspirate outlet 806 and into a fat harvesting apparatus 900, such as LipiVage® by Genesis Biosystems, Inc. The fat harvesting apparatus separates fat tissue from unwanted fluid. The fat tissue is then transferred into the processing unit 100 through the first inlet 601 discussed above. The unwanted fluid portion is transferred into a waste container 901 for proper disposal. This creates a disposable closed system to effectively extract tissues and cells from a patient, process the tissues and wash the cells, followed by concentrating the cells in a suspension that can then be readily administered to the patient, or conserved for further diagnostic measures. The entire process is performed in the closed system such that the chance of contamination is greatly reduced or minimized. The disposable aspect makes it easy to use without having to clean and sterilize the system every time after use. [0076] The processing chamber 101 has been pre-filled with processing fluid that contains the enzyme mixture discussed above. As seen in FIG. 7A, tissue, enzymes and processing fluid are introduced inside the processing compartment 101 through the first inlet 601. Additionally, enzyme-free washing fluid is also introduced into the washing fluid compartment 200 through the second inlet 602.

[0077] The content of the washing fluid chamber 200 is initially filled to a level reflecting not more than the height of the upper limit of wall 105. However, the amount of washing fluid can vary depending on the tissues and amount thereof being processed.

[0078] In step 503, by repetitive application of rotational low g-force (1^st g-force), the fluid is moved within and around the processing compartment 101. Repetitive cycles of subsequent accelerations and decelerations agitates the to be processed tissue and fluid inside the heated or pre-heated processing compartment 101. The elevated temperature facilitates the breaking down of the solid tissue and dissociation of cells from the tissue. As discussed above, the sidewalls of processing unit 100 are not round but triangularly shaped as show in FIG. 6C, resulting in different distances from the sidewall to the middle axis of the driving axis of the centrifuge, the acceleration and deceleration at different sites inside the processing compartment 101 is not homogeneous, in contrast to a fully round shaped chamber. The distance, for example, dl from the perimeter of the middle axis to one of the three vertices is greater than the distance d2 from the perimeter of the middle axis to the wall 105. Therefore, different acceleration forces act on the tissue that don’t allow a concentric vortex, but effect a better and more intense agitation. This in turns facilitates processing and action of the enzyme to better break down of the tissues.

[0079] In step 505, also referring to FIG. 7B, with pulsatile acceleration at a 2^nd g-force, clean washing fluid from wash fluid compartment 200 is driven through opening 201 and the siphon structure 400 into the concentration container 500 to nearly fill up the concentration container. This is done before the processed tissue and cells enter the wash fluid compartment 200 and mix with the washing fluid. The siphon structure 400 prevents washing fluid from spontaneously flowing back into the wash fluid chamber 200.

[0080] In step 507, also referring to FIG. 7C, the processing unit is accelerated at a 3^rd g-force higher than the 1^st processing force but lower than the 2^nd pulsatile acceleration force, thereby driving processed tissue and cells through the opening 109 into the wash fluid compartment 200. There the initial dissociated tissue and the enzyme is diluted and washed by the washing fluid. With the appropriate amount of clean washing fluid to be mixed with the processed tissue and cells, the concentration of enzymes can be diluted to a level that is clinically acceptable to use on a patient.

[0081] In step 509, by subsequent applying a 4^th, higher centrifugal forces, the washed tissue and fluid are driven from the wash fluid compartment 200 toward the opening 201, where the filter mesh 300 filters out the large tissue fragment and debris, while the smaller and heavier cells pass through the mesh opening and the siphon structure 400, and eventually enter the concentration container 500. This way, cells can be removed from each of the three concentration containers 500 .

[0082] The amount of the washing fluid in both the washing fluid compartment 200 and the concentration container 500 is calculated such that the concentration of any remaining processing fluid (enzymes and buffers) can be reduced below the threshold value, and that the diluted cells can be readily injected into a subject if needed, or used for further diagnostic purposes, or conserved by cryofreezing. For example, assuming originally in the processing chamber there is 250 mL of fluid, including 100 unit of enzymes in Ringer Solution and 100 mL of lipoaspirate. In step 507 the centrifugation can be controlled to transfer 100 mL of the mixture into the washing chamber, and in step 509 the washed cells are further diluted by 10X, resulting in a final concentration of less than 4 unit of enzymes.

[0083] The centrifuge machine can be programmed to automate a consecutive multi stage centrifugation, and the inventive three-chamber configuration requires no or minimal human handling between each stage; with the lid ensuring sterile inner conditions. Conventional method and system, on the contrary, requires at least one, often repetitive human handlings between each stage, which significantly increase the complexity or chance of contamination.

[0084] As can be seen in this disclosure, the processing unit can be easily used with existing a programmable centrifuge to enhance processing of adipose tissue and extract stem cells with high efficiency and low contamination.

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[00154] US8951513B2

[00155] US20160272943.

[00156] What is claimed is:

Claims

1 A processing unit for processing of mammalian tissue, the processing unit comprising: a) a processing compartment having a processing side wall; b) at least one wash fluid compartment having a washing side wall, wherein the wash fluid compartment is located radially outside of the processing compartment and separated by the processing side wall, wherein a first opening is formed at the top of the processing side wall to allow fluidic communication between the processing compartment and the wash fluid compartment; and c) at least one concentration compartment, wherein the concentration container is located radially outside of the wash fluid compartment and separated by the washing side wall, wherein a second opening is formed at the top of the washing side wall to allow fluidic communication between the wash fluid compartment and the concentration compartment.

2. The processing unit of claim 1, wherein the processing compartment has a bottom recess shaped to engaged with a centrifuge.

3. The processing unit of claim 2, wherein the processing compartment is generally triangular in shape, wherein a first distance from the bottom recess to a vertex of the triangular shape is greater than a second distance from the bottom recess to a side of the triangular shape.

4. The processing unit of claim 1, wherein the processing compartment has bottom baffles.

5. The processing unit of claim 1, wherein the concentration compartment is a detachable concentration container.

6. The processing unit of claim 1, wherein the processing side wall has an upper section of a first slope and a lower section of a second slope, and wherein first is greater than the second slope.

7. The processing unit of claim 1, further comprising a top cover.

8. The processing unit of claim 7, wherein the top cover comprises a first inlet and a second inlet, wherein the first inlet provides fluidic communication with the processing compartment, and the second inlet provides fluid communication with the wash fluid compartment.

9. The processing unit of claim 1, further comprising a filter covering the second opening.

10. The processing unit of claim 9, wherein the filter is a mesh filter.

11. The processing unit of claim 10, wherein the mesh filter has mesh openings of 40-300 pm.

12. The processing unit of claim 1, further comprising a siphon between the second opening and the concentration compartment.

13. The processing unit of claim 1, further comprising a tissue-extraction device operatively coupled with the first inlet, wherein tissues extracted by the tissue-extraction device are transferred directly into the processing compartment.

14. The processing unit of claim 13, wherein the tissue-extraction device is a liposuction device.

15. A method for recovering a cellular preparation from solid mammalian tissue in a single processing unit, the solid mammalian tissue comprising cells, comprising the steps of: a) agitating the tissue together with an enzyme-containing fluid in an inner processing compartment by applying repetitive acceleration and deceleration cycles of a first g-force of 0.5 to 3 g in order to dissociate the tissue; b) accelerating the processed tissue from step a) with a second g-force greater than the first g-force sufficient to move the processed tissue from the processing compartment into a washing compartment placed radially outside the processing compartment, wherein the washing compartment is prefilled with a washing fluid; c) allowing the processed tissue to be diluted and washed in the washing fluid; d) accelerating the processing unit with a third g-force greater than the first and second g- forces to transfer the cells into a concentration compartment pre-filled with an enzyme-free fluid; and e) optionally removing the cellular preparation from the concentration compartment.

16. The method of claim 15, wherein step a) is heated.

17. The method of claim 15, wherein the enzyme-containing fluid comprises collagenase, protease, or combinations thereof.

18. The method of claim 15, wherein the enzyme-free fluid is Lactated Ringer’s Solution or Sterofundin solution.

19. The method of claim 15, wherein the processing unit comprises a processing compartment having a processing side wall, at least one wash fluid compartment having a washing side wall, at least one concentration compartment, and a top cover having a first inlet in fluidic communication with the processing compartment, and a second inlet in fluidic communication with the wash fluid compartment, wherein the wash fluid compartment is located radially outside of the processing compartment and separated by the processing side wall, wherein a first opening is formed at the top of the processing side wall to allow fluidic communication between the processing compartment and the wash fluid compartment, and wherein the concentration container is located radially outside of the wash fluid compartment and separated by the washing side wall, wherein a second opening is formed at the top of the washing side wall to allow fluidic communication between the wash fluid compartment and the concentration compartment.

20. The method of claim 16, wherein a tissue-extraction device is operatively coupled with the processing unit through a sterile tubing, whereby tissues extracted by the tissue-extraction device can be directly extracted from a patient and transferred into the processing unit.

21. A vibrating power enhanced liposuction device (liporator), comprising: a) a housing having a cannula channel, a cylindrical piston space, an outlet, and a vacuum port, wherein the vacuum port is fluidically connected to the cylindrical piston space; b) a piston located in the cylindrical piston space; and c) a cannula located within the cannula channel and sealingly connected to the outlet through the piston such that a flow path from the cannula to the outlet is sealed, wherein the piston engages with a spring such that when a vacuum creates a negative pressure inside the cylindrical piston space through the vacuum port, the piston moves to compress the spring.

22. The vibrating liporator of claim 21, wherein the vibrating liporator is disposable.

23. A closed system for processing and recovering a cellular component from a mammalian subject without contacting ambient environment, comprising: a) a processing unit having: i) a processing compartment having a processing side wall; ii) at least one wash fluid compartment having a washing side wall, wherein the wash fluid compartment is located radially outside of the processing compartment and separated by the processing side wall, wherein a first opening is formed at the top of the processing side wall to allow fluidic communication between the processing compartment and the wash fluid compartment; iii) at least one concentration compartment, wherein the concentration container is located radially outside of the wash fluid compartment and separated by the washing side wall, wherein a second opening is formed at the top of the washing side wall to allow fluidic communication between the wash fluid compartment and the concentration compartment; and iv) a top cover having a first inlet and a second inlet, wherein the first inlet provides fluidic communication with the processing compartment, and the second inlet provides fluid communication with the wash fluid compartment; and b) a tissue-extraction device operatively coupled to the first inlet on the top cover, such that tissues extracted by the tissue-extraction device are sterilely transferred into the processing compartment of the processing unit.

24. A power-assisted disposable tissue extraction device comprising: a) a housing having a cannula channel, a cylindrical piston space, an outlet, and a vacuum port, wherein the vacuum port is fluidically connected to the cylindrical piston space; b) a piston located in the cylindrical piston space; and c) a cannula located within the cannula channel and through the piston, said cannula having a distal end and a proximal end, wherein the proximal end is sealingly connected to the outlet such that a flow path from the distal end of the cannula to the outlet is sealed, d) wherein the piston engages with the spring such that when a vacuum creates a negative pressure inside the cylindrical space on the piston through the vacuum port, the piston moves and compresses the spring, e) wherein the spring decompresses to push the piston and cannula back when the vacuum releases, thereby creating a vibrational movement of the cannula with repetitive cycles of vacuum and vacuum release to facilitate the recovery of tissue from the distal end of the cannula.

25. A method of recovering cells from a mammalian tissue using a single disposable device, comprising the steps of: a) filling the mammalian tissue into a processing chamber; b) processing the mammalian tissue in the processing chamber into cells and non-cell tissue fragments; c) separating the cells from the non-cell tissue fragments using multi-stage centrifugation of different centrifugal forces; and d) recovering the cells in a sterile container in a concentrated low fluid volume of 2 to 10 ml.