Mechanical apparatus and method for isolating stromal vascular fraction

Abstract

Provided is an apparatus and process for isolation of stromal vascular fraction (SVF) following lipoaspiration.

Claims

1. A method for enriching a cell population from a mammalian adipose tissue, the method comprising: aspirating adipose tissue into a container assembly using an aspiration tube having a proximal end detachably connected to the container assembly and a distal end inserted into at least one cavity of a mammalian body; electrically actuating a mechanical tissue disruption device, while the mechanical tissue disruption device is positioned within the container assembly, with an electrical actuator that is detachably connected to the mechanical tissue disruption device for said electrically actuating, wherein the mechanical tissue disruption device comprises at least one rotatable blade that rotates, when the mechanical tissue disruption device is electrically actuated, to disrupt/rupture the adipose tissue in the container assembly to obtain a cell suspension, wherein the disruption does not utilize an enzyme; optionally washing the suspension of cells within the container assembly; and transferring the container assembly, with the suspension of cells being within the container assembly, into a centrifuge and centrifuging the container assembly to thereby obtain a cell pellet from the suspension of cells within the container assembly, and extracting the cell pellet from within the container assembly, and removing the container assembly from the centrifuge; to thereby obtain an enriched cell population.

2. The method of claim 1, wherein the adipose tissue aspirated into the container is washed prior to mechanical disruption/rupturing.

3. The method of claim 1, wherein following mechanical disruption/rupturing the cell pellet is extracted following phase separation.

4. The method of claim 1, wherein the mechanical disruption/rupturing is carried out in lipoaspirate solution without the addition of a washing solution.

5. The method of claim 4, wherein the suspension of cells is washed following mechanical disruption/rupturing.

6. The method of claim 4, wherein the suspension of cells are not washed following mechanical disruption/rupturing and wherein the cell pellet is extracted following phase separation.

7. The method of claim 5, wherein the suspension of cells is washed with a washing solution, the washing solution containing at least one additional reagent selected from a cell specific antibody, a buffering agent, a sugar based density gradient, a non-sugar density gradient, an antibody mediated magnetic cell sorting solution and a non-magnetic antibody mediated cell sorting solution.

8. The method of claim 1, wherein the enriched cell population is further processed to enrich for a specific cell population.

9. The method of claim 1, wherein the enriched cell population is a stromal cell fraction (SVF).

10. The method of claim 9, wherein the SVF is characterized by at least one of the following: having 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; and/or having a percentage of between about 4 and about 10 of CD45() CD31() gate of monocytes(+) CD34(+) cells.

11. The method of claim 9, wherein the SVF is characterized by: having 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; and having a percentage of between about 4 and about 10 of CD45() CD31() gate of monocytes(+) CD34(+) cells.

12. The method of claim 9, wherein the SVF is characterized by having 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.

13. The method of claim 9, wherein the SVF is characterized by having a percentage of between about 4 and about 10 of CD45() CD31() gate of monocytes(+) CD34(+) cells.

14. The method of claim 9, further comprising a step of isolating fat from which the SVF was obtained.

15. The method of claim 1, wherein the adipose tissue is not a liquid.

16. The method of claim 1, wherein the container assembly is provided with the mechanical disruption device.

17. The method of claim 1, wherein the blades are in contact with the tissues.

18. The method for enriching a cell population from a mammalian adipose tissue as recited in claim 1, further comprising washing the suspension of cells within the container assembly.

19. The method for enriching a cell population from a mammalian adipose tissue as recited in claim 1, wherein the suspension of cells is not washed within the container assembly.

20. The method of claim 1, wherein the mechanical tissue disruption device comprising the at least one rotatable blade disrupts/ruptures the adipose tissue that has been directly aspirated into the container assembly via the aspiration tube, thereby obtaining a cell suspension from the aspirated adipose tissue that has a maintained cell viability.

21. The method of claim 1, wherein, prior to placing the container assembly in the centrifuge, processing of the adipose tissue in the container assembly consist of said mechanical disruption and optionally said washing.

22. The method of claim 1, wherein, prior to disrupting/rupturing the adipose tissue and optional washing in the container assembly, adipocytes collected into the container assembly are at most partially disrupted due to shear forces affected during suction and passage through the aspiration tube.

23. The method of claim 1, wherein once adipocytes of the adipose tissue are collected in the container assembly, the mechanical disruption of the harvested adipocytes of the adipose tissue is directly carried out using the mechanical tissue disruption device controlled by the electrical actuator that is external and detachably connected to the closing cap of the container assembly, wherein the mechanical disruption of the harvested adipocytes of the adipose tissue is carried out in either fluid aspirated during liposuction, the washing solution resulting from washing the harvested adipocytes within the container assembly, or in an isolated lipoaspirate that was separated from any added solutions.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) 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:

(2) FIG. 1 depicts a device for collecting adipocytes according to some embodiments of the invention;

(3) FIG. 2 depicts a device for isolating SVF cells according to some embodiments of the invention;

(4) FIG. 3 depicts a device for isolating SVF cells according to some embodiments of the invention;

(5) FIGS. 4A-B depict mechanically and enzymatically isolated SVF cells which display a similar fat (FIG. 4A) and bone (FIG. 4B) differentiation potential. Adipose derived stem cells isolated from fat using enzymatic, mechanical or wash techniques were incubated in a bone differentiation medium or with a fat differentiation medium or with control medium for 3 weeks. The cells were then stained for bone with alizarin red or for fat with Oil red O. Human. ASC differentiation into bone and fat is shown.

(6) FIGS. 5A-B depict SVF isolated by enzymatic digestion or mechanical disruption of fat displaying an enriched population of CD45 () cells in the gate of monocyte. FIG. 5A depicts all nucleated cells and FIG. 5B depicts CD45() only cells.

(7) FIG. 6 depicts a device for the mechanical isolation of stromal vascular fraction (SVF) cells by pressure filtration. The device is constructed of two spreadable containers designated 1 and 2.

(8) FIG. 7 depicts a device for the mechanical isolation of stromal vascular fraction (SVF) cells by pressure filtration in which the two containers are separated by a shutter that has a closed and an open position.

DETAILED DESCRIPTION OF EMBODIMENTS

(9) The present invention is directed to a mechanical method for isolating adipocytes from fat tissue, using a combination of mechanical disruption, washing and centrifugation for obtaining an enriched population of stromal vascular fraction.

(10) In order to have a better understanding of the invention, the reader is now directed to the figures.

(11) Reference is first made to FIG. 1 which provides an exemplary embodiment of a device 10 having an inlet 1 communicating with the interior of said device and adapted to sealably and detachably engage a cannula for introducing cells into the device, a sealable and detachable closing cap 3, a proximal mount 4 for a rotor shaft 5 and a rotor 7 attached thereto.

(12) The closing cap 3 of the device 10 further includes two additional inlets 9 and 11 to let in a washing solution (e.g. normal saline, ringer's solution, lactated ringer's solution, hanks' balanced salt solution and any combination thereof) through cannulas inserted therethrough. The solution or liquid or fat level is typically maintained at or below level 13.

(13) Turning to device 100, shown in FIG. 2, an electrical actuator external to the closing cap 16 is depicted, said actuator being detachably connected by a shaft 21 to the rotor through a sealable opening 16a in the closing cap. Features 25, 27, 15, 16 and 29 are equivalent to features 9, 11, 1, 3 and 13 shown in FIG. 1, respectively.

(14) Additional reference is made to device 200, shown in FIG. 3, which further depicts the concave base 45 of said device adapted for collection of the cells. Features 39, 41, 31, and 43 are equivalent to features 9, 11, 1, and 13 shown in FIG. 1, respectively.

(15) It is appreciated that device 10, 100 or 200 can be of a cylindrical, rectangular, trapezoid shape and any other known suitable geometry in the art and can be made from any inert, transparent or opaque, stable and sealable (e.g. under vacuum and using a hermetically sealed closing cap) material suitable to collect cells and maintain the sterility and viability thereof such as a thermoplastic polymer (e.g. polypropylene), a synthetic aromatic polymer (polystyrene) and a metal (e.g. aluminum).

(16) Regardless of the material and shape of the device, the volume of the device may be between 0.05 liter and 2 liters.

(17) The actuator may be motor-driven (e.g. electrical) or manually operated.

(18) The cannula may be made from any flexible non-reactive material (e.g. silicone, polyvinyl chloride).

Example 1

(19) Fat was isolated from thighs of a 50 years female by liposuction. The highest body mass index (BMI) previously measured was 52.7 and from a stable BMI of 33.9 was measured over a period 5 years. Fat was kept at 4 degrees Celsius overnight and processed at the next morning.

(20) Isolation of Cells from Fat by Collagenase I (Comparative System):

(21) Lipoaspirate fat (25 ml) was washed by adding 25 ml saline followed by thorough mixing and centrifugation at 260g for 5 minutes. Washed fat (in 10 ml portions) was mixed with 0.75 ml of 5 mg/ml collagenase I suspended in DMEM. The lipoaspirate/collagenase mixture was rotated for 1 hour at 37 degrees Celsius. Following the enzymatic digestion the fat/collagenase mixture was neutralized by 1/1 volume of DMEM with 10% FCS, transferred through a 100 M strainer and followed by 15 minutes of centrifugation at 328g. Sedimented stromal vascular fraction (SVF) cells were separated from floating adipocytes, transferred through a 100 M strainer and nucleated cells were counted using 3% Acetic Acid with Methylene Blue (Stem cells technologies, Vancouver, Canada).

(22) Mechanical Isolation Cells from Fat:

(23) Lipoaspirate fat (100 ml) was washed by addition of 100 ml saline followed by thorough mixing and centrifugation at 328g for 10 minutes. Washed fat (100 ml) was taken into an ATL-604 food processor (Gold-line, Israel) and 100 ml of saline was added. The fat/saline mixture was mechanically disrupted at 1300 RPM for 15 seconds at room temperature. Mechanically disrupted fat (with saline) was centrifuged at 328 g for 10 minutes. Sedimented SVF cells were separated from floating adipocytes and transferred through a 100 M strainer. Nucleated cells were counted using 3% Acetic Acid with Methylene Blue (Stem cells technologies, Vancouver, Canada).

(24) Isolation of Cells from Fat by Thorough Mixing and Centrifugation:

(25) Lipoaspirate fat (25 ml) was washed by addition of 25 ml saline followed by thorough mixing and centrifugation at 328g for 10 minutes. Sedimented SVF cells were separated from floating adipocytes. Nucleated cells were counted using 3% Acetic Acid with Methylene Blue (Stem cells technologies, Vancouver, Canada).

(26) Cell Culturing:

(27) Following their isolation and cell counting, SVF cells from all 3 isolation protocols (collagenase, mechanical and wash), were seeded at a concentration of 5105 cells/well of a 6 well plate and cultured for 7 days. Cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, Paisley, Scotland, UK) supplemented with 10% FCS (Thermo Scientific HyClone, Tauranaga, New Zealand), 60 g/mL penicillin, 100 g/mL streptomycin, 50 g/mL kanamycin, sodium pyruvate (1 mM), L-glutamine (2 mM) and non-essential amino acids. Cultures were propagated at 37 C. in a 10% CO.sub.2 atmosphere. Medium was changed 72 hours after cell seeding. After seven days in culture, cells were harvested by trypsin and counted.

(28) Results:

(29) As can be seen in Table 1, similar quantities of nucleated cells were isolated from fat using collagenase and mechanical cell isolation methods. In spite of vigorous washing, cell isolation without enzymatic or mechanical disruption of fat resulted in significantly lower quantities of cells.

(30) Additionally, culturing for 7 days of SVF cells in identical quantities resulted in significantly higher numbers of stromal cells when collagenase and mechanical isolated cells were used compared to cells that were isolated by washing only (Table 1). Importantly, similarly to a previous report [8] the cell yield per 1 ml of fat following culture was 30 fold higher in enzymatically isolated cells compared to cells that were isolated by washing only (1.08105 cells/1 ml fat (enzymatic) and 0.036105 cells/1 ml fat (wash). In contrast, cell yield following mechanical cell isolation and culture was similar to that obtained following enzymatic isolation (1.08105 cells/1 ml fat (enzymatic) and 0.656105 cells/1 ml fat (mechanical).

(31) TABLE-US-00001 TABLE 1 Cells/well of a 6 well Cultured cells Nucleated cells Dead plate (after per 1 ml fat Isolation per 1 ml fat cells 7 days in (after 7 days in method (lipoaspirate) (percent) culture) culture) Collagenase 5.4 10.sup.5 2.5% 1 10.sup.5 1.08 10.sup.5 Mechanical 4.1 10.sup.5 0.9% 0.8 10.sup.5 0.656 10.sup.5 disruption Wash 1.4 10.sup.5 0.2% 0.13 10.sup.5 0.036 10.sup.5

Example 2

(32) Fat was isolated from the abdominal wall, hips and back of a 46 years male by liposuction. The highest BMI previously measured was 62 and was later measure to be at a stable BMI=31 for a period of over a year. Fat was kept at 4 degrees overnight and processed at the next morning.

(33) Isolation of Cells from Fat by Collagenase I (Comparative System):

(34) Lipoaspirate fat (80 ml) was washed by addition of 80 ml saline followed by thorough mixing and centrifugation at 260g for 5 minutes. Washed fat (in 10 ml portions) was mixed with 0.75 ml of 5 mg/ml collagenase I suspended in DMEM. The lipoaspirate/collagenase mixture was rotated for 1 hour at 37 degrees. Following the enzymatic digestion the fat/collagenase mixture was neutralized by 1/1 volume of DMEM with 10% FCS, followed by 15 minutes of centrifugation in 328 g. Sedimented stromal vascular fraction (SVF) cells were separated from floating adipocytes transferred through 100 M strainer and, nucleated cells were counted using 3% Acetic Acid with Methylene Blue (Stem cells technologies, Vancouver, Canada).

(35) Mechanical Isolation Cells from Fat:

(36) Lipoaspirate fat (25 ml) was washed by addition of 25 ml saline followed by thorough mixing and centrifugation 260 g for 5 minutes. Washed fat (25 ml) was taken into a Philips HR1611 food processor and 25 ml of saline was added. The fat/saline mixture was mechanically disrupted at 5240 RPM for 8 seconds at room temperature. Mechanically disrupted fat (with saline) was centrifuged at 328g for 10 minutes. Sedimented SVF cells were separated from floating adipocytes and transferred through 100 M strainer. Nucleated cells were counted using 3% Acetic Acid with Methylene Blue (Stem cells technologies, Vancouver, Canada).

(37) Cell Culturing:

(38) Following their isolation and cell counting, SVF cells from the two isolation protocols (collagenase and mechanical), were seeded at a concentration of 510.sup.5 cells/6 cm plate and cultured for 14 days. Cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, Paisley, Scotland, UK) supplemented with 10% FCS (Thermo Scientific HyClone, Tauranaga, New Zealand), 60 g/1 mL penicillin, 100 g/1 mL streptomycin, 50 g/1 mL kanamycin, sodium pyruvate (1 mM), L-glutamine (2 mM) and non-essential amino acids. Cultures were propagated at 37 C. in a 10% CO.sub.2 atmosphere. Medium was changed 72 hours after cell seeding and then every 3 days. After 14 days in culture cells were harvested by trypsin and counted.

(39) Cell Surface Marker Staining:

(40) Following cell harvest cells were stained by a panel of cell markers including: anti human CD45 (BD horizon), anti human CD31 (eBioscience), anti human CD105, anti human CD34, anti human CD29, anti human CD235, anti human CD73 (Biolegend, San Diego, CA, USA) and live/dead-vivid dye (Invitrogen Molecular Probes, Eugene, OR, USA). Labeled cells were analyzed using a BD FACS Canto II flow cytometer (Becton Dickinson, San Jose, CA, USA). Data analysis was performed by the flow-Jo software (Tree star, Ashland, OR USA).

(41) Results:

(42) In accordance with example 2, similar quantities of cells were isolated from fat using collagenase digestion or mechanical isolation (Table 2).

(43) Additionally, culturing of SVF cells for 14 days resulted in the expansion of similar numbers of stromal cells when collagenase and mechanical isolated cells were used (Table 2).

(44) Evaluation of the surface marker expression pattern of cultured stromal cells at passage 0 after 14 days of tissue expansion revealed the expected expression pattern in both the enzymatically and mechanically isolated cells. The pattern included a negligible percent of CD45 (+) hematopoietic cells and high expression of mesenchymal stem cells markers (100% CD73 expression and >90% expression of CD29 and CD105) in most of the cultured cells (Table 3).

(45) Thus, mechanical isolation of SVF cells from fat allows the extraction of high quantities of nucleated cells similarly to enzymatic extraction. In addition, mechanically isolated cells harbor similar properties to enzymatically isolated cells as they produce similar quantities of cells following 14 days propagation in culture with a similar surface marker expression pattern.

(46) TABLE-US-00002 TABLE 2 Nucleated Dead Cells/6 cm plate Isolation cells/1 ml cells (after 14 days in method lipoaspirate (percent) culture) Collagenase 2.3 10.sup.5 17% 6.7 10.sup.5 Mechanical 1.5 10.sup.5 3% 5.8 10.sup.5 disruption

(47) TABLE-US-00003 TABLE 3 Collagenase Mechanical Surface markers dissociation dissociation expression P0 P0 CD45(+) 0.1% 0.4% CD31(+) CD34() .sup.1% 0.6% CD31() CD34(+) 0.5% 0.9% CD29(+) CD105(+) 95.6% 93% CD73(+) 100% 100%

Example 3

(48) Fat was isolated from the abdominal wall and hips of a 60 years female by liposuction. The highest BMI previously measured was 40 and was followed by a measurement of a stable BMI 29 for a period of over a year. Fat was kept at 4 degrees Celsius overnight and processed at the next morning.

(49) SVF cells were isolated from Lipoaspirate fat as was previously described in example 2 either by mechanical dissociation or by washing. Isolated cells were stained by the indicated markers and the proportion of cells positive for each marker was determined from the total population excluding dead cells and erythrocytes.

(50) Results:

(51) As can be seen, the proportion of CD45(+) hematopoietic cells was much higher in cells that were isolated by washing (91%) compared to mechanically isolated cells (29%) (Table 4). The increased proportion of CD45(+) in cells that were isolated by washing is in accordance with a previous report that found 81% of CD45(+) cells using similar extraction protocols. In spite of the difficulty to identify mesenchymal stem cell progenitors by specific surface markers [12], it is generally accepted that these cells do not originate from the CD45(+) population. Thus, the increased proportion of CD45(+) cells in cells isolated by washing can explain their poor culture expansion capacity compared to both mechanically and enzymatically isolated cells (Table 1).

(52) TABLE-US-00004 TABLE 4 Mechanical dissociation Wash CD45 (+) 29% 91% CD31(+) 8.2% 19% CD34(+) 8.2% 9%

Example 4

(53) Differentiation Potential:

(54) The multipotent potential of adipose derived stem cells isolated by enzymatic, mechanical and washing techniques was examined. As can be seen in FIG. 4, cultured cells that were isolated both by mechanical and enzymatic isolations demonstrated similar differentiation potential to both fat (FIG. 4A) and bone (FIG. 4B). Cells isolated by washing displayed a reduced fat differentiation potential compared to both mechanically and enzymatically isolated cells. Thus, mechanically isolated cells demonstrate comparable multipotent properties to enzymatically isolated cells and superior multipotency as compared to cells isolated by washing.

(55) Furthermore, subcutaneous fat was isolated from the stomach of a 58 years female by liposuction. The highest body mass index (BMI) previously measured was and from a stable BMI of was measured over a period 5 years. Fat was kept at 4 degrees Celsius overnight and processed at the next morning.

(56) Isolation of Cells from Fat by Collagenase I (Comparative System):

(57) Lipoaspirate fat (25 ml) was mixed with 0.75 ml of 5 mg/ml collagenase I suspended in DMEM. The lipoaspirate/collagenase mixture was rotated for 1 hour at 37 degrees Celsius. Following the enzymatic digestion the fat/collagenase mixture was neutralized by 1/1 volume of DMEM with 10% FCS, transferred through a 100 M strainer and followed by 15 minutes of centrifugation at 400g. Sedimented stromal vascular fraction (SVF) cells were separated from floating adipocytes, transferred through a 100 M strainer and nucleated cells were counted using 3% Acetic Acid with Methylene Blue (Stem cells technologies, Vancouver, Canada).

(58) Mechanical Isolation Cells from Fat:

(59) Lipoaspirate fat (100 ml), warmed to 37 degrees Celsius was taken into an ATL-604 food processor (Gold-line, Israel) and 100 ml of saline which was pre-warmed to 37 degrees Celsius was added. The fat/saline mixture was mechanically disrupted at 1300 RPM for 15 seconds at room temperature. Mechanically disrupted fat (with saline) was centrifuged at 400g for 15 minutes. Sedimented SVF cells were separated from floating adipocytes and transferred through a 100 M strainer. Nucleated cells were counted using 3% Acetic Acid with Methylene Blue (Stem cells technologies, Vancouver, Canada).

(60) Isolation of Cells from Fat by Thorough Mixing and Centrifugation:

(61) Lipoaspirate fat (10 ml), warmed to 37 degrees Celsius was washed by addition of 25 ml saline warmed to 37 degrees Celsius followed by thorough mixing and centrifugation at 400g for 15 minutes. Sedimented SVF cells were separated from floating adipocytes. Nucleated cells were counted using 3% Acetic Acid with Methylene Blue (Stem cells technologies, Vancouver, Canada).

(62) Cell Culturing:

(63) Following their isolation and cell counting, SVF cells from all 3 isolation protocols (collagenase, mechanical and wash), were seeded at a concentration of 0.075 10.sup.6 cells/well of a 6 well plate and cultured for 7 days. Cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, Paisley, Scotland, UK) supplemented with 10% FCS (Thermo Scientific HyClone, Tauranaga, New Zealand), 60 g/mL penicillin, 100 g/mL streptomycin, 50 g/mL kanamycin, sodium pyruvate (1 mM), L-glutamine (2 mM) and non-essential amino acids. Cultures were propagated at 37 C. in a 10% CO.sub.2 atmosphere. Medium was changed 72 hours after cell seeding. After seven days in culture cells were harvested by trypsin and counted.

(64) Cell Surface Marker Staining:

(65) Following cell harvest cells were stained by a panel of cell markers including: anti human CD45 (BD horizon), anti-human CD31 (eBioscience), anti-human CD105, anti-human CD34, anti-human CD29, anti-human CD235, anti-human CD73 (Biolegend, San Diego, CA, USA) and live/dead-vivid dye (Invitrogen Molecular Probes, Eugene, OR, USA). Labeled cells were analyzed using a BD FACS Canto II flow cytometer (Becton Dickinson, San Jose, CA, USA). Data analysis was performed by the flow-Jo software (Tree star, Ashland, OR USA). The gradual gating strategy isolated a distinct population containing only singlets and live cells (determined by low ViViD staining (an amine reactive viability dye) while discarding RBCs (specific staining of RBC). Gating for CD45 () cells was than performed to eliminate CD45-positive immune cells. An additional gating of CD45-negative cells was performed to define the populations positive for CD31, CD34, CD73 and CD29. It is widely accepted that plotting forward scatter (FSC) data against side scatter data can reveal morphology information about a peripheral blood sample. FSC data plotted on the x-axis allows for size distinction with smaller lymphocytes appearing to the far left, while SSC data (y-axis) makes differentiating between the comparably sized monocytes and neutrophils possible based on cell granularity. Using the same parameters and whole blood as a positive control a distinct population was identified within the SVF having the same characteristic as monocytes. This area was termed the gate of immunocytes. Since monocytes are approximately 15 to 25 m in diameter the SVF population within this gate are most probably of the same size range.

(66) As can be seen in Table 5 similar quantities of nucleated cells were isolated from fat using enzymatic and mechanical cell isolation methods. In spite of vigorous washing, cell isolation without enzymatic or mechanical disruption of fat resulted in significantly lower quantities of cells.

(67) Additionally, seeding of identical quantities of SVF cells at day 0 resulted in significantly higher numbers of adipose derived stromal cells after 7 days of culture when enzymatic and mechanical isolated cells were used compared to cells that were isolated by washing only (Table 5).

(68) TABLE-US-00005 TABLE 5 Cells/well of a 6 well Cultured cells Nucleated cells Dead plate (after per 1 ml fat Isolation per 1 ml fat cells 7 days in (after 7 days in method (lipoaspirate) (percent) culture) culture) Enzymatic 2.3 10.sup.5 10% 1.33 10.sup.5 4.07 10.sup.5 Mechanical 1.33 10.sup.5 15% 1.56 10.sup.5 2.76 10.sup.5 disruption Wash 0.19 10.sup.5 0.1% 0.36 10.sup.5 0.09 10.sup.5

(69) Importantly, cell yield per 1 ml of fat following culture was 45 and 30 fold higher in enzymatically and in mechanically isolated cells compared to cells that were isolated by washing only (Table 5).

(70) Seeding of identical quantities of SVF cells resulted in significantly higher yield of stromal cells following 7 days culturing when enzymatic and mechanical isolated SVF cells were used compared to cells that were isolated by washing only. This indicates that a different composition of cell populations was isolated by the different cell isolation techniques.

(71) TABLE-US-00006 TABLE 6 Surface Marker Enzymatic Mechanical expression isolation isolation Wash CD31 CD34 11 24.4 34.3 CD31 CD34+ 60 74 64 CD31+ CD34+ 28 0.4 0.7 CD31+ CD34 0.5 0.5 0.5 CD73+ CD29+ 55 64.3 42 CD73 CD29+ 25.5 1.3 0 CD73 CD29 10 25 37

(72) To better define the cell composition of SVF cells isolated by the different techniques their surface marker expression was determined by an 8 color flow cytometry panel. Examination of the percent of CD45 (+) cells within SVF cells isolated by the different techniques revealed a lower (77%) percent in enzymatically isolated cells compared to cells that were isolated mechanically or by wash yielding 91% and 90% respectively of CD45(+) cells. Since mesenchymal stem cells arise solely from the CD45() population, representing none-hematopoietic cells, the expression of additional surface marker was examined in this population. As can be seen in Table 6 a significant percent (28%) of CD31(+) CD34(+) double positive cells was observed in enzymatically isolated cells while in both mechanically isolated cells and cells isolated by a wash this population practically did not exist.

(73) An additional population that was hardly apparent in both mechanically and wash isolated cells was CD73() CD29 (+) cells. Again a significant percent (25%) of enzymatically isolated cells expressed CD73() CD29 (+). Thus, a significant difference in the composition of cells was demonstrated between enzymatically isolated SVF cells and mechanically isolated cells or cells isolated by a washing.

(74) Table 6 demonstrates the differences in the percent of surface marker expression amongst CD45 () cells between SVF cells isolated from fat by enzymatic digestion (collagenase), mechanical disruption of fat or by washing the fat. Despite the apparent similarity in the surface marker expression between mechanically isolated and wash isolated cells a substantial difference exists between these cells as was evident in the increased culture expansion of mechanically isolated cells. Therefore, the surface marker expression between the cells isolated by these two techniques was further examined.

(75) Table 7 shows differences in the percent of surface marker expression amongst CD45 () between SVF cells isolated from fat either by a mechanical disruption of fat or by fat washing. As can be seen in Table 7 a substantial increase in both CD31() CD34 (+) (10% increase) and in CD73 (+) CD29 (+) (20% increase) populations was evident in mechanically isolated cells compared to cells isolated by washing).

(76) TABLE-US-00007 TABLE 7 Surface Marker Mechanical expression isolation Wash CD31 CD34 24.4 34.3 CD31 CD34+ 74 64 CD31+ CD34+ 0.4 0.7 CD31+ CD34 0.5 0.5 CD73+ CD29+ 64.3 42 CD73 CD29+ 1.3 0 CD73 CD29 25 37

(77) The CD45() CD31() CD34(+) population is the population that harbors most adipose derived stem cell progenitor cells. As can be seen in Table 7, the mechanically isolated cell fraction has a higher percent of CD45() CD31() CD34(+) compared to cell isolated by washing, pointing to their increased ability to form Adipose-Derived Stem Cells (ASCs). Additional surface markers suggested representing ASC progenitor cells within the SVF population are CD73 and CD29. As can be seen in Table 7, mechanically isolated cells have a 20% increase in CD45() CD31() CD73(+) CD29(+) cells compared to cells isolated by washing.

(78) Examination of the cell distribution of the entire SVF using the forward light scatter area (FSC-A) and the side scatter area (SSC-A) parameters revealed an increased proportion of cells in a gate termed gate of monocytes in enzymatic and mechanical isolation compared to washing (FIG. 5A). The superior proportion of cells in the gate of monocytes in enzymatically and mechanically isolated cells was even more pronounced in the more relevant CD45() population reaching a 5 fold increase in mechanically isolated cells compared to cells isolated by washing (FIG. 5B).

(79) The SSC-A on FCS-A distribution of all nucleated live cells (FIG. 5A) or CD45 () cells (FIG. 5B) of enzymatic, mechanical and wash isolated cells is displayed in a dot plot

(80) Importantly, as can be seen in Table 8, most of the CD45() cells within the gate of monocytes were CD31() CD34(+), which harbor adipose stem cell progenitor cells.

(81) Therefore, the present invention provide a new cell population within SVF termed CD45() CD31() gate of monocytes (+) CD34 (+). This novel population, which takes into account both the physical properties of the cells and their surface marker expression, comprise 6.6%, 4.3% and 0.88% of the total nucleated cells in enzymatically, mechanically and wash isolated cells respectively. This population comprises the majority of adipose stem cell progenitor cells as it predicts well the ratio of ASCs yields, produced from an equal number of SVF cells, after 7 days in culture (Table 8). Thus, the most profound difference between mechanically and wash isolated cells is in their CD45() CD31() gate of monocytes (+) CD34 (+) population as is summarized in Table 9. It is generally accepted that the therapeutically relevant cell population is the one that is able to produce cultured adipose derived stem cells, namely the stem cell progenitor cells. The present invention defines a subclass of stem cell progenitor cells within SVF defined using the physical properties of the cells (gate of monocyte). Thus, the CD45() CD31() gate of monocytes (+) CD34 (+) population define the most therapeutically relevant population stem cell progenitor cells within SVF. The large increase in the CD45() CD31() gate of monocytes (+) CD34 (+) cell population in mechanically isolated SVF cells compared to wash isolated cells (Table 9) suggest the increased therapeutic efficacy of mechanically isolated cells.

(82) TABLE-US-00008 TABLE 8 Surface Marker Enzymatic Mechanical expression isolation isolation Wash CD31 CD34 3 2.7 12 CD31 CD34+ 82 94 88 CD31+ CD34+ 13 0.4 0 CD31+ CD34 0.5 0.5 0.5

(83) TABLE-US-00009 TABLE 9 Mechanical Ratio Population isolation Wash Mecha/Wash CD45 (), CD31(), 4 0.88 4.54 Gate of monocytes (+), CD34+

Conclusions

(84) In contrast to SVF cells that are isolated by washing, mechanically isolated cells demonstrate improved properties being similar to enzymatically isolated cells which are considered to date to be the gold standard. This is demonstrated in the following properties: 1. The Numbers of nucleated cells that are isolated from fat: Mechanical disruption of fat results in SVF cell quantities similar to quantities obtained using collagenase isolation. This was in contrast to SVF cells that were isolated by washing (without Mechanical disruption) that demonstrate significantly lower quantities. 2. Expansion ability: culture expansion of mechanically isolated SVF yielded cell quantities similar to the quantities obtained using collagenase isolated cells after both 7 and 14 days. This was in contrast to SVF cells that were isolated by washing which demonstrated significantly lower expansion in culture. 3. Differentiation potential: mechanically isolated cells demonstrated differentiation capabilities to both bone and fat. The fat differentiation ability of mechanically isolated cells was far superior to that of cells isolated by washing. 4. Surface markers expression of cultured SVF cells: At passage 0, mechanically isolated cells demonstrated the expected mesenchymal stem cells surface marker expression pattern. 5. The proportion of CD45() CD31() gate of monocytes (+) CD34 (+) cells in SVF: the data demonstrate a large increase in the proportion of cells within this population in mechanically isolated cells compared to wash isolated cells that correlates with their increased ability to produce adipose stem cells.

Example 5

(85) Turning to a further device of the invention depicted in FIGS. 6 and 7. A device is depicted for the mechanical isolation of stromal vascular fraction (SVF) cells by pressure filtration. The device in FIG. 6 is constructed of two spreadable containers designated 1 and 2. The two containers are separated by a shutter that has a closed and an open position (FIG. 7).

(86) According to an exemplary embodiment, the process for SVF cells isolation comprises one or more of the following steps: 1. The shutter is in a closed position and the two containers are connected. 2. A liposuction device is connected to vacuum in. Suction pump is connected to Vacuum 1 and liposuction is performed into container 1. 3. At the end of the liposuction procedure the lipoaspirate is left to settle for 5 minutes. 4. The vacuum pump is then connected to vacuum out 2 and the liposuction fluid is vacuumed out. 5. Sterile fluid is then added to the fat from the vacuum in spot, the container is turned upside down a couple of times and the lipoaspirate is left to settle 5 minutes again. 6. The liquid is again vacuumed from vacuum out 2. 7. Sterile fluid is added to the fat from the vacuum in spot and the fat is mixed with fluid. 8. Container is turned upside down a couple of time to mix the fat with fluid. 9. Shutter is changed immediately to open position and fat is passed through the metal mesh by pressuring down the plunger. 10. Once all the fat and fluid were passed through the filter into container 2 the shutter is put to a closed position. 11. Access fluid from above the closed shutter is vacuumed out. 12. Container 2 is detached from container 1 and the fat remains sterile because the top end is closed by the closed shutter. 13. Container is centrifuged and SVF cells are concentrated at the tube like part at the bottom of the container. 14. SVF cells are sterilely isolated by a needle and syringe.