Isolation of SVF cells from lipoaspirate through ultrasonic cavitation and mechanical processing in liquid bath

Abstract

An apparatus for isolation of stromal vascular fraction cells from lipoaspirate includes a tub; a sonotrode attached to an exterior to the tub; and a mixer configured to mix lipoaspirate within the tub. The blades may comprise sets of blades arranged at different heights, wherein each set of blades may be operated independently of the others. The tub, sonotrode, and mixer may be formed as one integral unit that is removably attachable to a motor, wherein, when attached, the motor is configured to rotate the mixer. A method of isolating stromal vascular fraction cells from lipoaspirate includes introducing the lipoaspirate into a tub; introducing fluid into the tub; while mixing the lipoaspirate, applying ultrasonic energy to the interior of the tub from a sonotrode, to thereby induce ultrasonic cavitation within the fluid; and centrifuging the lipoaspirate and fluid to thereby produce a pellet of stromal vascular fraction cells.

Description

[0001] ISOLATION OF SVF CELLS FROM LIPOASPIRATE THROUGH ULTRASONIC CAVITATION AND MECHANICAL PROCESSING IN LIQUID BATH

[0002] RELATED APPLICATIONS

[0003] This Application claims priority to U.S. Provisional Patent Application No. 63 / 659,486, filed June 13, 2024, entitled “Isolation of SVF Cells From Lipoaspirate Through Ultrasonic Cavitation in Liquid Bath,” the contents of which are hereby incorporated by reference as if fully set forth herein.

[0004] TECHNOLOGICAL FIELD

[0005] The present disclosure relates to the field of regenerative medicine, and more specifically, but not exclusively, to devices and methods for isolating stromal vascular fraction cells from lipoaspirate.

[0006] BACKGROUND OF THE INVENTION

[0007] The Stromal Vascular Fraction (SVF) is a heterogeneous mixture of cells obtained from adipose tissue, more commonly known as body fat. These cells include adipose- derived stem cells, endothelial cells, endothelial progenitor cells, pericytes, T cells, and other immune cells. More specifically the stem cell progenitor cell sub-population (defined by the following surface markers: CD45(-)CD34(+)CD31(-) within the SVF) has been shown to possess a high regenerative potential. Isolation of the SVF from body fat is desirable for many applications in the field of regenerative medicine, including joint diseases (like knee arthritis), autologous fat transplantation, pulmonary diseases and Crohn’s disease, and stem cell therapy for neurological conditions.

[0008] Factors known to affect the number of viable adipocytes and SVF cells extracted from fat cells include the manner of fat extraction; and, if laser is used, the specific configuration of the laser (wavelength of laser, location of laser fiber relative to cannula).

[0009] One important consideration influencing the quality of the preadipocytes is the specific manner of processing the extracted lipoaspirate to remove unwanted materials (e.g., enzymatic digestion or non-enzymatic methods of separation utilizing pressure, shear, and centrifugal force). Various non-enzymatic techniques have been used in order to separate SVF cells. These include mechanically disrupting with a rotatable blade or rotor [1], optionally including an integrated delivery unit within the apparatus having the rotating blade or rotor, followed by centrifuging in a container or syringe, and removing the SVF as a pellet.

[0010] Ultrasonic techniques for breaking down lipoaspirate in order to isolate the SVF are also known. Some ultrasonic techniques induce cavitation on the lipoaspirate by inserting an ultrasonic probe into the lipoaspirate [2], Other ultrasonic techniques include applying the ultrasonic waves to the exterior of a container holding the lipoaspirate. For example, publication [3] discloses a transducer coupled to a first end of a resonant horn to form an ultrasonic resonator. Specimen containers of lipoaspirate are arranged within slots in the ultrasonic resonator, and an ultrasonic wave is generated within the resonator to induce cavitation. Publication [4] discloses applying ultrasonic waves through a constant temperature bath to tissue that is held in a sterile sonication container such as a test tube or ajar.

[0011] PUBLICATIONS

[0012] [1] US Patent No. 11,891,627

[0013] [2] US Patent No. 8,440,440

[0014] [3] US Patent No. 10, 196,611

[0015] [4] U.S. Patent No. 10,913,931

[0016] SUMMARY OF THE INVENTION

[0017] The present disclosure introduces novel apparatuses and methods for ultrasonic cavitation of lipoaspirate in order to separate SVF cells. In particular, the lipoaspirate is introduced into a tub containing a stirrer component (optionally associated with one or a plurality of blades or blade arrangements) and having a unit capable of transmitting ultrasonic vibrations, e.g., an ultrasonic probe such as a sonotrode, attached to an exterior thereof, at the bottom. The lipoaspirate is mixed with a fluid. Sonication is applied to the tub from the outside of the tub, while the lipoaspirate and fluid are being mechanically treated, e.g., stirred, thereby producing a cavitation effect within the fluid. The ultrasonic cavitation energy is spread to all the lipoaspirate within the liquid fluid due to operation of the stirred. The mechanism and method described herein are effective in separating the SVF from unwanted materials, under predetermined conditions. First, cavitation energy is applied onto the lipoaspirate without direct contact of the ultrasonic probe with the lipoaspirate. Insertion of the ultrasonic probe into the lipoaspirate, while effective in separating the SVF, increases the likelihood of unintentional destruction of some SVF cells. At the same time, the ultrasonic energy is applied directly onto a tub holding the lipoaspirate. This is in contrast to other known systems of external application of the ultrasonic energy, in which the lipoaspirate is maintained within the sterile collection tube, without being introduced into a dedicated cavitation tub. As a result, in known systems, the ultrasonic energy is applied only indirectly to the aspirated fat. By contrast, in the presently described embodiments, the ultrasonic energy is applied to the exterior of a container (tub) housing the lipoaspirate. In addition, the mixing during the application of ultrasonic energy ensures that the cavitation affects the entire sample of the lipoaspirate. As a result, the SVF is properly separated from the lipoaspirate with a high yield.

[0018] In some embodiments, the ultrasonic cavitation is performed while the lipoaspirate is mechanically disrupted. The combination of mechanical disruption with the ultrasonic cavitation has shown to induce greater quantities of SVF cells than mechanical disruption or ultrasonic cavitation standing alone.

[0019] According to a first implementation, an apparatus for isolation of stromal vascular fraction (SVF) cells from lipoaspirate is disclosed. The apparatus includes a tub; a sonotrode attached to an exterior of the tub; and a mixer configured to mix (or mechanically disrupt the) lipoaspirate within the tub.

[0020] Optionally, the mixer is a rotating stirrer. The stirrer may include one or more blades configured to mechanically disrupt the lipoaspirate during rotation of the stirrer and operation of the sonotrode. The stirrer may be associated to one or plurality of sets of blades arranged at a same or at different heights on the stirrer, wherein each set of blades may be operated independently of the other sets of blades. In this manner, the apparatus may be operated to perform similarly regardless of the volume of lipoaspirate and fluid that may be present therein.

[0021] In some embodiments, the sonotrode is attached to a central portion of a bottom external end of the tub. The tub may include at least one input port for receipt of lipoaspirate and at least one output port for removal of processed lipoaspirate. The at least one input port and at least one output port may be located on sides of the tub, and may be accessed through any valve or connection, such as Luer connections. In some cases, a bottom of the tub is sloped adjacent to the output port, to thereby enable draining of processed lipoaspirate from the tub.

[0022] In advantageous embodiments, the tub, sonotrode, and mixer are formed as one integral unit, and wherein the unit is removably attachable to a motor placed above the tub, wherein, when attached, the motor is configured to actuate rotation of the mixer. In such embodiments, the tub, sonotrode, and mixer may function as a single-use item that may be removed and replaced, with the motor being optionally reusable.

[0023] The apparatus of the invention may further comprise one or more additional elements which may be associated or used when placed on an external region of the tub.

[0024] Typically, the size and shape of the tub is not limited. The tub may be configured to hold an amount of the lipoaspirate (and fluid) that ranges from several or few millimeters to hundreds of milliliters. In some embodiments, the apparatus is sized and shaped to hold and process an amount of a lipoaspirate ranging from 2 ml to 500 ml. In other cases, the apparatus may be sized and shaped to hold and process an amount of a lipoaspirate ranging from 20 ml to 1500 ml.

[0025] According to a second aspect, a method of isolating stromal vascular fraction (SVF) cells from lipoaspirate is disclosed. The method includes applying ultrasonic energy to an interior of a tub comprising a lipoaspirate and a fluid, while the lipoaspirate and the fluid are mixed or stirred or mechanically disrupted, wherein said ultrasonic energy is optionally applied by an ultrasonic probe, e.g., a sonotrode, attached to an exterior of the tub, to thereby induce ultrasonic cavitation within the fluid; and optionally centrifuging the lipoaspirate and fluid to thereby produce a pellet of stromal vascular fraction (SVF) cells.

[0026] In some embodiments, the method comprises introducing lipoaspirate into a tub; and introducing fluid into the tub.

[0027] In some embodiments, the method comprising introducing lipoaspirate into a tub; introducing fluid into the tub; while mixing the lipoaspirate, applying ultrasonic energy to the interior of the tub from a sonotrode attached to an exterior of the tub, to thereby induce ultrasonic cavitation within the fluid; and centrifuging the lipoaspirate and fluid to thereby produce a pellet of stromal vascular fraction cells.

[0028] In some embodiments, during application of the ultrasonic energy, the fluid is present relative to the lipoaspirate in a ratio of between 1 : 1 and 10: 1.

[0029] Optionally, the mixing step comprises mixing the lipoaspirate with a rotating stirrer. The mixing step may comprise mechanically disrupting the lipoaspirate with blades attached to the stirrer. The blades, or blade arrangements may be as disclosed herein.

[0030] Optionally, the method may be performed within 24 hours of aspiration of the lipoaspirate. In some cases, the method is carried out immediately after the lipoaspirate has been obtained. In other cases, the method is performed within a time period sufficient to generate a substantial amount of the cells.

[0031] Various process parameters may be optimized for performing steps of this method. The step of applying ultrasonic energy may include applying said ultrasonic energy at frequencies of 10-50 KHz, optionally with power consumption of 20-60 watts. In some embodiments, ultrasonic energy is applied at frequencies of 10-50 KHz or 20-40 KHz or 15-60 KHz.

[0032] The mixing step may include mixing at between 1,000 - 5,000 rpm, and for a duration of 10 to 90 seconds. In some embodiments, the mixing step may include mixing at between 1,000 - 5,000 rpm or between 2,000 - 3,000 rpm or between 1,500 and 2,500 rpm. In some embodiments, the mixing is continued for a period of 10 to 90 seconds or between 30 and 90 seconds or for a period of at least 30 seconds.

[0033] The mixing step may be performed with a stirrer having a plurality of sets of blades arranged at different heights on the stirrer, wherein each set of blades may be operated independent of the other sets of blades, and the method may comprise selecting one or more sets of blades to stir based on a volume of lipoaspirate and fluid in the tub.

[0034] The method may be carried out at ambient conditions, typically at room temperature (23 -33 °C) or at any suitable temperature at which the fat tissue and the produced SVF cells are unaffected. Generally, the temperature used when carrying out the method may be between 4°C and 33°C.

[0035] In some embodiments, the method comprises centrifuging the lipoaspirate and fluid to produce a pellet of stromal vascular fraction (SVF) cells, which can be isolated. In alternative embodiments, the fluid containing the SVF cells may be removed from the tub and centrifuged separately. In some embodiments, the tub or apparatus may be centrifuged at between about 200 x g and about 1,500 x g, or at between about 250 x g and between about 500 x g, or at any other speed sufficient to generate a pellet or to separate the SVF from floating adipocyte tissue.

[0036] In some embodiments, the method may comprise obtaining a lipoaspirate.

[0037] The lipoaspirate treated accoridng to methods of the invention, or using an apparatus or a device accoridng to the invention, is generally a material that is removed from a body, typically a human body, during liposuction or any convention operation or medical treatment involving separation of fat. Thus, the term lipoaspirate should not be construed to mean a material that is obtained solely by liposuction. The lipoaspirate may contain a mixture of fat cells, fluids, and other tissues that are suctioned out or removed from the body through e.g., a liposuction procedure. As is known in the art, the composition of the lipoaspirate may contain Adipocytes (fat cells), blood, stem cells (such as but not limited to adipose-derived stem cells (ADSCs)) and other cell populations.

[0038] The adipocytes generally refer to any type of cells and / or tissue fragments that can be isolated from the lipoaspirate which can be used to isolate the stromal cell fraction (SVF) therefrom. In accordance with the present invention, the SVF can be derived from both the fatty and fluid portions of lipoaspirate and typically comprise a heterogeneous mix of multiple cell populations having different degrees of maturity and function. Accordingly, the SVF is generally composed of a hematopoietic cell portion (e.g. granulocytes, monocytes and lymphocytes) together with pre-adipocytes, endothelial cells, pericytes, fibroblasts and stem progenitor cells.

[0039] The composition of the SVF may vary, but can generally comprise a percentage of CD45+ cell of between about 40% and between about 91%; a percentage of between about 5 and about 50 of CD45(-) CD31(-) CD34(+) and between about 3 and between about 40 of CD45(-) CD73(+) CD29(+) cells; a percentage of between about 7 and about 40 of CD45(-) CD31(-) CD34(+) and between about 6 and about 30 of CD45(-) CD73(+) CD29(+) cells; a percentage of between about 2 and between about 15 of CD45(-) CD31(- ) gate of monocytes (+) CD34 (+) cells; and / or between about 4 and about 10 of CD45(- ) CD31(-) gate of monocytes (+) CD34 (+) cells.

[0040] In some embodiments, within the scope of the present invention, the term ‘lipoaspirate’ may be exchangeable with the term ‘fat’. In some embodiments, the lipoaspirate may be a mixture of different batches or samples obtained from a subject by different ways or from different parts of the subject’s body.

[0041] In some embodiments, the lipoaspirate is obtained by liposuction. In other embodiments, the lipoaspirate is obtained following a surgical procedure involving removal of fat from the body.

[0042] The consistency of the lipoaspirate may vary and is unlimited. As disclosed herein, processing of the lipoaspirate involves addition of a fluid to allow a more efficient processing thereof. The fluid may be water-based and may contain one or more additives, stabilizers, salts, etc. Typically, the fluid is free of enzymes.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0045] FIG. 1 is a perspective view of a first embodiment of a device for processing lipoaspirate in order to separate SVF from lipoaspirate, according to embodiments of the present disclosure;

[0046] FIG. 2 is a cross-section view of the device of Fig. 1;

[0047] FIG. 3 is a different cross-section view of the device of Fig. 1, taken at a 90 degree angle to that of Fig. 2;

[0048] FIG. 4 is a top view of the device of Fig. 1;

[0049] FIG. 5 is a perspective view of a second embodiment of a device for processing lipoaspirate;

[0050] FIG. 6 is a side view of the device of Fig. 5;

[0051] FIG. 7 is a cross-section view of the device of Fig. 5;

[0052] FIG. 8 is a different cross section view of the device of Fig. 5, taken at a 90 degree angle to that of Fig. 7;

[0053] FIG. 9 illustrates steps in a method of separating SVF from lipoaspirate using the ultrasonic cavitation device of Fig. 1, according to embodiments of the present disclosure; and FIG. 10 illustrates experimental results showing the improved separation of the SVF from lipoaspirate when performed using the apparatuses and methods of the present disclosure.

[0054] DETAILED DESCRIPTION OF EMBODIMENTS

[0055] The present disclosure relates to the field of regenerative medicine, and more specifically, but not exclusively, to a device and method for isolating stromal vascular fraction cells from lipoaspirate.

[0056] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and / or methods set forth in the following description and / or illustrated in the drawings and / or the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. For example, the device described herein may be used for extraction of SVF cells for therapeutic applications other than direct reinsertion.

[0057] Figs. 1-4 illustrate a first embodiment of a device for isolation of SVF cells from lipoaspirate. Fig. 1 illustrates a perspective view; Fig. 2 illustrates a cross-section view; Fig. 3 illustrates a cross-section view taken at a 90 degree angle compared to the crosssection view of Fig. 2, and Fig. 4 illustrates a top view. Apparatus 10 includes a tub 12. Tub 12 may be made of a metallic material. Alternatively, tub 12 may be made of any solid material with low impedance, such as plastic or silicone. In preferred embodiments, tub 12 is made of stainless steel.

[0058] The tub 12 contains a volume 20 of lipoaspirate mixed with a fluid, e.g. saline. As is known to those of skill in the art, fat tissue is typically withdrawn from the body in the presence of added fluid, and this fluid is collected with the fat tissue. Regardless, an additional volume of fluid may be added to the lipoaspirate within the tub 12 in order to provide an ideal ratio of fluid to lipoaspirate for performance of ultrasonic cavitation. In exemplary embodiments, this ratio is between 1 : 1 fluid : lipoaspirate to 20: 1 fluid : lipoaspirate.

[0059] Tub 12 further includes a mixing mechanism. Any suitable mixing mechanism may be employed, such as vortex or shaking. In the illustrated embodiment, the mixing mechanism is a stirrer 16. The stirrer 16 rotates circumferentially to thereby disperse the fluid and lipoaspirate throughout the tub 12. The stirrer is rotated by a motor 11 configured above the tub 12. In the illustrated embodiment, there is a single stirrer 16; alternatively, more than one stirrer may be used.

[0060] In preferred embodiments, the stirrer 16 includes sharpened blades 18. Blades 18 have a sharpened edge to thereby mechanically disrupt the lipoaspirate while mixing. This mechanical disruption may be performed in the manner described in U.S. Patent 11,891,627, the contents of which are hereby formulated by reference as if fully set forth herein. Advantageously, in such embodiments, the ultrasound cavitation apparatus 10 combines two techniques for processing fat in order to detach SVF cells - ultrasonic cavitation and mechanical disruption. These two techniques applied simultaneously produce more SVF cells than ultrasonic cavitation alone, as will be described in the experimental results section below.

[0061] In preferred embodiments, and as illustrated in Fig. 2 and Fig. 3, the sharpened blades 18 are at different heights. The configuring of the blades 18 at different heights enables scaling of the mixing to match the volume of lipoaspirate that is introduced into apparatus 10. For example, when a smaller volume of lipoaspirate (and optionally saline) is inserted into the apparatus 10, only the lower blades may be used, whereas when a larger volume of lipoaspirate (and optionally saline) is introduced, all of the blades may be used.

[0062] Sonotrode 14 is attached to the underside of the tub 12, at a central portion thereof. The sonotrode is attached using any suitable means. Optionally, the sonotrode 14 is rigidly connected to the tub 12, via a thin layer 15 of stainless steel (or a different suitable material). Alternatively, the sonotrode is attached to the tub with a removable locking mechanism, to permit detachment of the sonotrode 14 from the tub 12 for purposes of cleaning and reuse. Sonotrode 14 is configured to deliver ultrasonic energy (generated by ultrasound generator 21) through the bottom of the tub 12, thereby causing cavitation within the fluid in tub 12, approximately in region 22 of the tub. A support 26 is attached to the bottom of the tub, to enable the tub to rest securely on a surface.

[0063] The tub 12 includes two inlet ports 22, accessible from the top of the tub, and an outlet port 24 at the bottom of the tub (best seen in Fig. 3). The inlet ports 22 receive a connection from a source of lipoaspirate and may be secured via a Luer connection or a similarly fluid-tight connection. The outlet 24 may have a removable stopper 25, as shown in Fig. 3, which, when removed, allows for the processed lipoaspirate to drain from the tub 12. System 10 includes ultrasonic generator 21, which is attached to the rest of the apparatus via cord 23. The ultrasonic generator 21 transmits ultrasonic energy to the sonotrode 14 via an electrical connection that runs through the center of the stirrer 16. The ultrasonic generator 21 also includes a power supply for controlling operation of the stirrer 16.

[0064] Figs. 5-8 depict a second embodiment of an apparatus 210 for extracting SVF cells out of lipoaspirate. Apparatus 210 includes many features similar to apparatus 10, and accordingly similar features are given similar reference numerals, except that they begin with “2.” Apparatus 210, like apparatus 10, includes a sonotrode 214 arranged below tub 212, and multiple levels of rotating blades 218 configured within the tub 212. In the illustrated embodiment, there are four levels of blades 218. Depending on the level of lipoaspirate and saline fluid within the tub 212, any or all of the blades 218 may be rotated during use.

[0065] The tub 212 in this embodiment is a single-use, closed upside down plastic cup with a metallic floor. Unlike the first embodiment, in which the input to the tub was from the top, in this embodiment the input is from the side, via two Luer connectors 222. Outlet port 224 is also configured on the side of the tub 212 and includes a Luer connection 225. The floor of the tub 212 is sloped adjacent to the outlet port 224, to enable the processed lipoaspirate to exit the tub 212 without residual loss of lipoaspirate on the floor of the tub. Optionally, the apparatus 210 may include a tilting handle for tilting the apparatus when emptying it, further ensuring that a maximal amount of the processed lipoaspirate is removed.

[0066] Motor 211 is an electric motor with a quick connection (e.g., via click or magnets) to the stirrer 216 for the blades 218. The ultrasonic horn 214 at the bottom of the tub 212 is rigidly and externally connected to the metallic bottom 215 of the tub 212, for example via bolt 227 (shown in Fig. 7 and Fig. 8). Thus, the tub 212, sonotrode 214, and stand 226 constitute a single unit, which may be attached to the motor 211 for operation, when desired.

[0067] Fig. 9 depicts a method 100 of treating lipoaspirate in order to isolate the SVF cells, using either of the apparatuses 10, 210, described above, or any other apparatus with the same functionalities. At step 101, the lipoaspirate is delivered to the tub. As discussed above, the lipoaspirate may be removed from the body using any suitable apparatus and technique. At step 102, fluid is optionally added to the tub, in order to achieve the preferred ratio of fluid to lipoaspirate, as discussed.

[0068] At step 103, the mixture of lipoaspirate and fluid is sonicated while being stirred. The sonication induces cavitation of bubbles within the mixture. The cavitation energy breaks apart the lipoaspirate, while preserving the integrity of SVF cells. The stirring helps ensure that this cavitation energy is distributed to all parts of the lipoaspirate. Without wishing to be bound by a particular theory, the cavitation generally causes solid material to disperse from the center to the edges of tub 12, while the blades 18 of stirrer 16 bring the solid material back from the edges to the center, to thereby enable the cavitation to continue to act on the lipoaspirate.

[0069] Suitable process parameters that have been shown to be effective in achieving high yield of SVF cells are as follows. The ultrasonic energy is applied at a frequency of between 15-60 KHz, and preferably between 10-50 KHz, with a power consumption of 20-60 watts. The rotating blades are rotated at a frequency of 1,000 to 5,000 revolutions per minute (rpm), and preferably at approximately 2,000 rpm. The time of operation of the sonication and stirring may be approximately 10 - 90 seconds, and preferably approximately 30 seconds.

[0070] Following completion of the sonication, the entire mixture is delivered to a centrifuge, and the mixture is centrifuged to produce a pellet of SVF cells, in a manner known to those of skill in the art.

[0071] Experimental Results

[0072] Materials and methods

[0073] Human fat tissue received 24 hours post liposuction and phosphate-buffered saline (PBS IX) were warmed in a water bath for 37°C for 30 minutes. 100 mL of the prewarmed fat and 100 mL of the prewarmed PBS were transferred to the fat processing canister. The canister lid was closed, and the canister was activated as follows:

[0074] • 30 seconds of rotating blades (RBs) at 2000 RPM, or

[0075] • 30 seconds of ultrasonic (ULS) transducer at 20 kHz, or

[0076] • 30 seconds of RBs at 2000 RPM and ULS transducer at 20 kHz.

[0077] The processed fat was then transferred into 4X 50 mL tubes and taken to centrifugation for 10 minutes at 650 xg. Following centrifugation, the fat debris and liquids in each of the four tubes were discarded and the remaining pellet was resuspended in a total of 10 mL of Dulbecco's Modified Eagle Medium (DMEM). The suspension was passed through a 100 pm cell strainer into one 50 mL tube. The SVF cells suspension was then taken for cell counting in NucleoCounter (NC)- 202 (ChemoTech).

[0078] Results

[0079] Combination of RBs activation at 2000 RPM and ULS transducer activation at 20 kHz for 30 seconds yielded a total of 332,000 cells per mL fat. This is a substantial improvement compared to each of the methods alone (Table 1, below). Figure 3 illustrates the same results in graphical form, with the leftmost bar referring to the results obtained with a rotating blade, the middle bar describing the results obtained with ultrasonic energy, and the right bar referring to results following the combination. These results indicate an additive effect of the two methods that contribute together to a higher total SVF cell yield compared to each of the methods alone.

[0080] Table 1. The effect of the Combination of RBs and ULS Transducer on SVF

[0081] Cells Yield

[0082] *Performed on a different fat sample

[0083] This finding is only relevant when using fat that was aspirated at maximum 24 hours pre procedure.

Claims

CLAIMS:

1. An apparatus for isolation of stromal vascular fraction cells from lipoaspirate, comprising: a tub; a sonotrode attached to an exterior to the tub; and a mixer configured to mix lipoaspirate within the tub.

2. The apparatus of claim 1, wherein the mixer is a rotating stirrer.

3. The apparatus of claim 2, wherein the stirrer comprises blades configured to mechanically disrupt the lipoaspirate during rotation of the stirrer and operation of the sonotrode.

4. The apparatus of claim 3, wherein the blades comprise a plurality of sets of blades arranged at different heights on the stirrer, wherein each set of blades is optionally operated independently of the other sets of blades.

5. The apparatus of claim 1, wherein the sonotrode is attached to a central portion of a bottom of the tub.

6. The apparatus of claim 1, further comprising at least one input port for receipt of lipoaspirate and at least one output port for removal of processed lipoaspirate.

7. The apparatus of claim 6, wherein the at least one input port and at least one output port are located on sides of the tub.

8. The apparatus of claim 6, wherein a bottom of the tub is sloped adjacent to the output port, to thereby enable draining of processed lipoaspirate from the tub.

9. The apparatus of claim 1, wherein the tub, sonotrode, and mixer are formed as one integral unit, and wherein the unit is removably attachable to a motor placed above the tub, wherein, when attached, the motor is configured to rotate the mixer.

10. A method of isolating stromal vascular fraction cells from lipoaspirate, comprising: while mixing lipoaspirate and a fluid present in a tub, applying ultrasonic energy to the lipoaspirate and fluid from a sonotrode attached to an exterior of the tub, to thereby induce ultrasonic cavitation within the fluid; and optionally centrifuging the lipoaspirate and fluid to thereby produce a pellet of stromal vascular fraction cells.

11. The method of claim 10, comprising obtaining the lipoaspirate.

12. The method of claim 10, comprising adding the lipoaspirate and the fluid to the tub.

13. The method of any one of claims 10 to 12, wherein, during application of the ultrasonic energy, the fluid is present relative to the lipoaspirate in a ratio of between 1 : 1 and 10: 1.

14. The method of any one of claims 10 to 13, wherein the mixing step comprises mixing the lipoaspirate with a rotating stirrer.

15. The method of any one of claims 10 to 14, wherein the mixing step comprises mechanically disrupting the lipoaspirate with blades attached to the stirrer.

16. The method of any one of claims 10 to 15, wherein the method is performed within 24 hours of aspiration of the lipoaspirate.

17. The method of any one of claims 10 to 15, wherein the method is performed immediately after the lipoaspirate is obtained.

18. The method of any one of claims 10 to 17, wherein the step of applying ultrasonic energy comprises applying said ultrasonic energy at frequencies of 10-50 KHz.

19. The method of any one of claims 10 to 18, wherein the mixing step comprises mixing at between 1,000 - 5,000 rpm.

20. The method of any one of claims 10 to 19, wherein the mixing step comprises mixing for a duration of 10 to 90 seconds.

21. The method of any one of claims 10 to 20, wherein the mixing step is performed with a stirrer having a plurality of sets of blades arranged at different heights on the stirrer, wherein each set of blades may be operated independent of the other sets of blades, and the method comprises selecting one or more sets of blades to stir based on a volume of lipoaspirate and fluid in the tub.

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