CA3123386A1

CA3123386A1 - Cell separation apparatus and methods of use

Info

Publication number: CA3123386A1
Application number: CA3123386A
Authority: CA
Inventors: Rolf Wolters; Jules VALENTI; Tom GURSKI
Original assignee: Tissue Genesis LLC
Current assignee: Tissue Genesis LLC
Priority date: 2018-12-13
Filing date: 2018-12-13
Publication date: 2020-06-18
Also published as: EP3893949A1; EP3893949A4; KR20210092324A; BR112021011482A2; CN113660961A; IL283930A; ZA202104197B; MX2021007080A; WO2020122916A1; JP2022522323A; AU2018452704A1

Abstract

Cell separation systems and methods of separating cells are disclosed. In an embodiment, the cell separation system comprises a non-transitory storage device comprising a plurality of logic associated with centrifugation programs, wherein execution of a centrifugation program separates a cell volume from a biologic material volume; a heating mechanism electrically coupled to a power supply; a containment mechanism; an assembly removably coupled to the containment mechanism, wherein the assembly comprises: a single-walled bowl comprising a product reservoir, plurality of cell concentration areas contained within the product reservoir, a digestion area, a waste reservoir, and a center column comprising an access tube removably coupled to the product reservoir, a mounting plate, and an alignment mechanism coupled to the bowl to restrict movement of the mounting plate when the first assembly is coupled to the containment mechanism.

Description

CELL SEPARATION APPARATUS AND METHODS OF USE
RELATED APPLICATION INFORMATION
None.
BACKGROUND
Cell therapy and tissue engineering is developing toward clinical applications for the repair and restoration of damaged or diseased tissues and organs. In particular, the development of tissue grafts promotes developments in surgeries, including cardiac and peripheral vascular surgery, limb tissue repair, dental applications, as well as veterinary surgeries. Grafts and other cell-based products may be formed by isolating and/or culturing cells from human or animal tissue.
A potential source for endothelial cell seeding is microvascular endothelial cells (MVEC). Williams et al. pioneered both freshly isolated and cultured human, canine, rabbit, rat, bovine and pig endothelial cells, specifically MVEC, in their laboratory to study cellular function. The source for human MVEC was aspirated tissue from cosmetic liposuction. Two separate protocols for human fat MVEC isolation were used depending on the end use of the cell population. The protocols differed in isolation complexity from zo a simple, operating room-compatible procedure for immediate sodding of human or animal grafts to a more elaborate procedure if the MVEC will be subsequently cultured.
Endothelial cells are of critical importance in establishing a non-thrombogenic cell lining. A need still exists for an efficient and reliable method for producing endothelial cell linings on a synthetic graft in an operating room setting. It is desirable to achieve rapid cell adhesion in or on a permeable matrix, scaffold or other permeable cell substrate material in a matter of minutes or hours with an instrument that lends itself to the operating room environment, maintains a sterile barrier, is easy to use, produces consistent results, and is inexpensive.
LISTING OF THE FIGURES
FIG. 1 is a partial isometric view of a cell separation system 100 according to certain embodiments of the present disclosure.
FIG. 2 is a partial exploded view of a single-walled bowl according to certain embodiments of the present disclosure.
FIG. 3A is a partial top view of a single-walled bowl according to certain embodiments of the present disclosure.
FIG. 3B is a front view of a single-walled bowl according to certain embodiments of the present disclosure.
FIG. 3C is a partial side view of a single-walled bowl according to certain embodiments of the present disclosure.
FIG. 4A is a partial cross-sectional view of a single-walled bowl taken across the bowl through the pellet concentration areas according to certain embodiments of the present disclosure.
zo .. FIG. 4B is a partial cross-sectional view of a single-walled bowl taken across the bowl perpendicular through the pellet concentration areas according to certain embodiments of the present disclosure.

2 FIG. 5A is a partial exploded view of a of a single walled bowl assembly 102 according to certain embodiments of the present disclosure.
FIG. 5B is a partial isometric view of a single walled bowl assembly 102 according to certain embodiments of the present disclosure.
FIG. 6 is partial exploded view of a mounting plate 504 and a centrifuge assembly 132 according to certain embodiments of the present disclosure.
FIG. 7 is a method of separating endothelial cells from biologic material such as adipose tissue according to certain embodiments of the present disclosure.
SUMMARY
An embodiment of the present disclosure describes a method of separating cells, comprising: agitating, in a digestion area of a cell separation system, a volume of biologic material and a volume digestion media to form a digested volume of biologic material; centrifuging the digested volume of biologic material at a force from about 500 G to about 1000 G from about 5 minutes to about 10 minutes to separate the digested volume into a plurality of concentrated cell volumes and a plurality of waste;
collecting the plurality of concentrated cell volumes in a product reservoir wherein the concentrated cell volume comprises material with a smaller diameter than the pores of a filter such that the material can pass through the filter into a cell concentration area;
isolating the plurality of waste in a waste reservoir wherein the waste reservoir is zo partitioned in the bowl from the digestion area and wherein the plurality of waste comprises waste with a larger diameter than the pores of the filter and is not in contact with the concentrated cell volumes; and removing the concentrated cell volumes from the product reservoir.

3 An embodiment of the present disclosure describes a cell separation system, comprising: a non-transitory storage device comprising a plurality of logic associated with centrifugation programs, wherein execution of a centrifugation program separates a cell volume from a biologic material volume; a heating mechanism electrically coupled to a power supply; a containment mechanism in proximity of the heating mechanism; an assembly removably coupled to the containment mechanism, wherein the assembly comprises: a single-walled bowl comprising a product reservoir, plurality of cell concentration areas contained within the product reservoir, a digestion area, a waste reservoir, and a center column comprising a access tube removably coupled to the product reservoir, a mounting plate, wherein the single-walled bowl is removably coupled to the mounting plate, an alignment mechanism coupled to the center column to restrict movement, wherein, in a first state of a centrifugation program, the assembly is configured to rotate around a central axis in a first direction and in a second direction in an alternating fashion and the heating mechanism is activated, and wherein, in a .. second state of a centrifugation program, the heating mechanism is deactivated and the single-walled bowl is configured to rotate in a single direction around the central axis to separate a plurality of waste from a plurality of cells.
An embodiment of the present disclosure describes a cell separation system comprising: a non-transitory storage device comprising a plurality of logic associated zo with a plurality of different centrifugation programs, wherein, when executed by a processor; agitates in a digestion area a volume of biologic material and a volume of digestion media to form a digested volume of biologic material; separates, via centrifugation, the digested volume of biologic material at a force from about 500 G to

4 about 1000 G from about 5 minutes to about 10 minutes to separate the digested volume into a plurality of concentrated cell volumes and a plurality of waste;
collects the plurality of concentrated cell volumes in a product reservoir wherein the concentrated cell volume comprises material with a smaller diameter than the pores of a filter such that the material can pass through the filter into a cell concentration area;
isolates the plurality of waste in a waste reservoir wherein the waste reservoir is partitioned in the bowl from the digestion area and wherein the plurality of waste comprises waste with a larger diameter than the pores of the filter and is not in contact with the concentrated cell volumes; and removes the concentrated cell volumes from the product reservoir.
DETAILED DESCRIPTION
Endothelial cells are used to establish non-thrombogenic cell lining within synthetic grafts. Thus, it is desirable to achieve rapid cellular adhesion in or on a permeable matrix, scaffold, or other permeable cell substrate material in a matter of minutes or hours with an instrument that lends itself to the operating room environment, maintains a sterile barrier, is easy to use, and produces consistent graft results.
Currently, there are various approaches for meeting these requirements, but with limited success: (i) the use of decellularized tissue materials; (ii) the use of a self-assembly mechanism, wherein cells are cultured on tissue culture plastic in a medium that induces extracellular matrix (ECM) synthesis; (iii) the use of synthetic biodegradable zo polymers, onto which cells are subsequently seeded and cultured in a simulated physiological environment; and (iv) the use of biopolymers, such as a reconstituted type I collagen gel, which is formed and compacted with tissue cells by the application of mechanical forces to simulate a physiological environment.

5

6 Embodiments of this disclosure describe systems and methods that enable the isolation of large quantities of endothelial cells from fat tissue and the rapid cell sodding of synthetic grafts, and that enable the automation and adhesion of cells in a turn-key, operating room ready instrument for the rapid sodding of the graft.
Embodiments of this disclosure likely have other applications in addition to the lining of grafts for implantation.
The systems and method discussed herein are of a cell separation system that comprises a single-walled bowl that may be formed as a single piece (no seams or welds) or which may be formed as a plurality of pieces. The single-walled bowl ("bowl") may comprise various areas including a center column that houses a single access tube connected to a product reservoir, permitting direct access to the cell concentration areas containing the desired end product. The bowl further comprises a digestion area and a waste reservoir separated by a plurality of internal walls which may be formed integrally with the bowl or which are separable components. The internal walls permit the separation of the digestion area and waste area such that when the bowl is centrifuged, the cell separation process occurs faster and results in a more pure end product. The physical separation of the digestion area and waste area further permits the digestion area to be directly heated, further assisting in the cell separation process.
The single-walled bowl further comprises a plurality of cell concentration areas disposed zo circumferentially around a central axis of the bowl such that, during centrifugation, the gravitational forces separate the cells from a biological material volume.
Waste is collected in the waste reservoir and cells are collected in the cell concentration areas.
The bowl further comprises one or more filters or screens connected to a product reservoir that extends from one end of the bowl to the other end, from one cell concentration area to another. The product reservoir is connected to the access tube in the center column, thereby providing access to the concentrated cells. The product reservoir further comprises one or more screen holders that house screens or filters that prevent waste product from contaminating the concentrated cell product. The bowl couples to a mounting plate which couples to a containment mechanism via a spring locking mechanism. An alignment mechanism is coupled to the bowl such that it engages with the mounting plate on which the assembly of the bowl is disposed.
In one example of cell separation using the cell separation system discussed herein, a plurality of logic is stored in a non-transitory storage device (memory) and comprises a plurality of centrifugation programs. Each program of the plurality of programs comprises instructions that may be based upon a plurality of factors including media type and biological material volume and/or target cell volume or target cell concentration. When executed by a processor, each program initiates cell separation through a plurality of states as discussed herein, resulting in the automated removal of the separated cells and the trapping and/or removal of waste. The programs may differ and/or overlap in various aspects, including temperatures, times, forces, and overall program length (time) from disposal of the biologic material volume and the media until the removal of the separated cell volume.
zo In one example of the cell separation system, a plurality of digestion media and a plurality of biologic material (a volume) are disposed in the bowl, in particular in a mixing and digestion area of the bowl. The bowl is then agitated, rotated in each direction around the central axis to break up the biologic material volume to enable the

7 separation during centrifugation. The bowl may be heated prior to and/or during the agitation via a heating mechanism, the heat generated by the heating mechanism causes the air in a gap between the bowl and the mounting plate and/or the containment mechanism to heat up and circulate. The bowl may be heated from about 25 C to about 45 C, and the heating mechanism may be shut off subsequent to completion of agitation such that the remainder of the cell separation occurs at room temperature (from about 20 C to about 25 C).
Subsequent to the agitation, centrifugation is performed. During centrifugation, the bowl rotates freely with respect to the containment mechanism, an alignment mechanism coupled to the bowl and to the mounting plate, and in some embodiments further coupled to the containment mechanism, prevents a portion of the bowl from rotating.
The centrifugation may be performed at a force from about 500G to about 1000G
for from 5 minutes to 20 minutes, or from 1 minute to 10 minutes, or other ranges of time in various embodiments. Cells are separated from the mixture in the mixing and digestion area and are gravitationally forced into a plurality of cell concentration areas of the bowl.
After the centrifugation, the speed of the bowl is reduced and waste materials collect in the waste reservoir. As the speed of the bowl is reduced, a vacuum is created in the product reservoir through the access tube and the separated cells are collected during operation. Cells and media that are of a smaller diameter than the holes in the zo filters/screens pass through the screens and into the cell concentration areas while waste and other materials with a diameter larger than the filter/screen holes are prevented from passing through the screens. This waste is maintained in the waste reservoir and does not contact the separated cell volume.

8 In an embodiment, after centrifugation, the rotation may be stopped, and the collected cells remain in the product reservoir. The cells are removed via the access tube using a vacuum. By using a single-walled bowl, the heat transfer may be more effective, thus increasing the efficiency of the system. A single walled bowl system reduces the cost associated with cell separation. Direct access to the cell concentration areas permits the capture of a more pure and more concentrated cell product. The access to the product reservoir further permits the cell product to be captured while the assembly is still rotating.
FIG. 1 is a partial isometric view of a cell separation system 100 according to certain embodiments of the present disclosure. The cell separation system 100 comprises a centrifuge assembly 132 and an assembly 102 of a single-walled bowl 104 coupled to a mounting plate 106 via a spring locking mechanism. The mounting plate 106 is coupled to the drive shaft of a centrifuge (not shown). The assembly 102 rotates freely while the alignment rod 110 (and single access tube 108) remain stationary. During agitation and centrifugation, the assembly 102 spins in either direction 130A or 130B around a central axis 124, shown in a coordinate system in FIG. 1 and referenced throughout.
The coordinate system further comprises a second axis 126 that is perpendicular to the central axis 124, and a third axis 128 perpendicular to both 124 and 128.
In an embodiment, the assembly 102 is agitated by rotating less than 360 degrees in zo each direction 130A and 130B around central axis 124. The bowl 104 may be heated during agitation and/or during centrifugation. During agitation and centrifugation, the biological material is separated and the cells are concentrated in the product reservoir 206 and waste is collected in the waste reservoir 202.

9 In an embodiment, the mounting plate 106 is seated in and removably coupled to a containment mechanism 112, which acts to direct the heat and hot air generated by the activation of the heating unit 114. Containment mechanism 112 is also removably coupled to a centrifuge that operates to rotate the assembly 102. The coupling of the containment mechanism 112 to the assembly 102 creates an air gap between the two through which hot air may be circulated via a plurality of apertures (not shown) on the bottom of the mounting plate 106. In an embodiment the single walled bowl assembly 102 and associated tubing are disposable.
In an embodiment, the heating unit 114 is employed to elevate a temperature of the io system 100 during at least agitation. The heating unit 114 is coupled to a base 118 comprising a plurality of feet 116 configured to prohibit movement of the base 118 and system 100 during execution of a plurality of centrifugation programs. The heating unit 114 may be powered by a direct wired connection via 122, or may comprise a portable power source, such as a rechargeable battery (or batteries). The heating unit further comprises a plurality of heating elements configured to elevate a temperature of the containment mechanism 112 and thus the assembly 102 coupled to the mechanism 112. The containment mechanism 112 may also be coupled or removably coupled to the base 118 as well.
In an embodiment, the cell separation assembly 102 is able to spin relative to the zo containment mechanism 112 because the alignment rod 110 is coupled to the center column 402 and the bowl 104 is secured to the mounting plate 106 by a spring locking mechanism. The assembly is removably coupled to containment mechanism 112, enabling free rotation of the assembly 102.

In an embodiment, the system 100 may comprise at least one storage device (not shown) comprising a plurality of centrifugation programs and a processor, as well as a plurality of controls (not shown) activated by the execution of a centrifugation program via the processor. The storage device and/or the plurality of controls that may be located on the system 100 or located remotely and accessed via a tablet, mobile phone, wearable technology, kiosk, laptop computer, or desktop computer. Each centrifugation program may comprise a plurality of parameters employed in the centrifugation cycle, and may be selected manually or dynamically and in an automated fashion based upon inputs such as the biologic material volume used and/or the volume and/or type of .. digestion enzyme(s) employed.
FIG. 2 is a partial exploded view 200 of a single-walled bowl 104 according to certain embodiments of the present disclosure. The single-walled bowl 104 may be fabricated as a single, seamless piece or as multiple pieces assembled into the bowl 104.
The "single-wall" of the bowl is in contrast to a bowl that has at least two nested walls.
In an embodiment, the bowl 104 comprises one or more cell concentration areas removably coupled to a product reservoir 206. Cell concentration areas 212 comprise smooth internal geometries and act to isolate and concentrate, via the removal of fluid and solids, the cells separated during centrifugation. A center portion of the bowl 104 houses the alignment rod 110 and a single access tube 108. The alignment rod 110 is zo coupled to the bowl 104 and further to the product reservoir 206 by way of rotary seals 214. The bowl 104 further comprises a waste reservoir 202 that is separated from a mixing and digestion area 204 by a plurality of internal walls which may be formed integrally with the bowl 104 or which are separable components.

In an embodiment, the bowl 104 further comprises one or more filter holders removably coupled to product reservoir 206 and which hold one or more filters 210. The one or more filters 210 prevent waste product, including undigested material, from entering into the product reservoir 206 and contaminating the concentrated cell product.
The one or more filters 210 may be as large as a 500 micron filter size. The one or more filters may be of a size between 250 microns and 500 microns. The one or more filters may be of a size between 100 microns and 250 microns. The one or more filters 201 may be less than 100 microns in size.
The one or more filters 201 may be configured in a serial assembly with a range of larger filter sizes to smaller filter sizes.
In an embodiment, the bowl 104 further comprises one or more feet 216 that couple to a spring loaded locking mechanism (not shown) and act to secure the bowl 104 in the mounting plate 106 during operation. Once secured, the bowl 104 can rotate freely with respect to the containment mechanism 112, which does not rotate and acts at least in part to direct and circulate heated air towards the bowl 104 during agitation (and breakdown) of the biologic material volume.
FIGS. 3A-C are various views of a single-walled bowl 104 according to certain embodiments of the disclosure. FIG. 3A is a partial top view 300 of a single-walled bowl 104. Bowl 104 may comprise an at least one seam 306 along a centerline of the bowl 104. The cell concentration areas 212 and single access tube 108 are also shown. FIG.
zo 3B is a partial front view 302 of a single-walled bowl 104 according to certain embodiments of the disclosure. The seam 306, cell concentration areas 212, single access tube 108, and alignment rod 110 are also shown. FIG. 3C is a partial side view 304 of a single-walled bowl according to certain embodiments of the disclosure. The cell concentration areas 212, single access tube 108, and feet 216 are shown.
FIG. 4A is a partial cross-sectional view 400 of a single-walled bowl according to certain embodiments of the present disclosure. The view 400 of the bowl 104 is taken across the bowl through the pellet concentration areas 212. The product reservoir 206 extends across the bowl 104 from one pellet concentration area 212 to a diametrically opposite pellet concentration area 212. Filters 210 in filter holders 208 are shown removably coupled to product reservoir 206.
FIG. 4A further discloses a center column or cavity 402 that contains single access tube io 108. Single access tube 108 is configured to permit the introduction of sterile media, to permit access to concentrated cell product, and to permit the removal of the concentrated cell product including during operation. Single tube 108 remains stationary during rotation, thus enabling access to the concentrated cell product during rotation of the assembly 102. This configuration also reduces contamination of the separated cell product. FIG. 4A further comprises an access port 404 which is configured to permit access to the interior of the bowl only and permits the introduction of raw material into the mixing and digestion area 204 of bowl 104 while stationary. The material may be biological material, media, digestion enzymes, or other material necessary to assist in the cell separation.
zo .. FIG. 4B a partial cross-sectional view 406 of a single-walled bowl 104 according to certain embodiments of the present disclosure. View 406 is taken across the bowl 104 perpendicular through the cell concentration areas 212. FIG. 4B shows features illustrated in FIGS. 2-3, including waste reservoir 202 and the mixing and digestion area 204. Also shown in FIG. 4B are internal walls 408 that form a partial barrier between the mixing and digestion area 204 and the waste reservoir 202. During the digestion and mixing process, the assembly 102 rotates in each direction 130A and 130B
around central axis 124. During centrifugation, the assembly 102 spins in either direction 130A
or 130B around a central axis 124. The cells are separated and collected in the product reservoir 206. As the bowl is agitated or centrifuged the waste material is spun over the internal walls 408 and collected in the waste reservoir 202.
In an embodiment, the waste reservoir 202 is configured to hold and isolate large and undesired solids as well as used media and other liquids. By collecting in the waste io .. reservoir, these particles are prohibited from clogging the system. An additional measure to prevent clogging are the filters 210 (held in filter holders 208) which are removably coupled to the outer edges of the product reservoir 206 (seen in FIG. 4A).
FIG. 5A is a partial exploded view of a of a single walled bowl assembly 102 according to certain embodiments of the present disclosure. FIG. 5A shows that the single-walled bowl 104 fits into the spring loaded locking mechanism of mounting plate 504 by way of feet 216 being inserted into apertures 506 and locking into place. FIG. 5B
depicts the assembly 508 when the locking mechanism is engaged and bowl 104 is coupled to mounting plate 504.
FIG. 6 is partial exploded view 600 of mounting plate 504 and centrifuge assembly 132 zo according to certain embodiments of the present disclosure. FIG. 6 shows where the mounting plate 504 is located within and removably coupled to the containment mechanism 112 of the centrifuge assembly 132.

FIG. 7 is a method of separating endothelial cells from biologic material according to certain embodiments of the present disclosure. The method 700 is an automated mechanism for the washing, separation, concentration, and removal/harvesting of viable cells. The harvested cells may be employed for grafts or otherwise employed for use in healthcare (e.g., FDA approved implantation or further processing) and/or healthcare research (trials to approve cell separation methods and cell-derivative products for use).
In an embodiment, at block 702 of the method 700, a biologic material volume is disposed in a mixing and digestion area 204 (FIG. 2) of a single-walled bowl 104 of a cell separation system such as the system 100 in FIG 1. At block 704, a plurality of media and a collagenase enzyme or enzymes may be introduced into the bowl 104 in particular into the mixing and digestion area 204 via access port 404.
Examples of media may be Lactated Ringers (LR), Hartmans, Water For Injection (WFI) or any other similar media. In some examples, the mixing and digestion area 204 may be pre-heated to from about 30 C to about 40 C prior to disposal of the biologic material volume and/or media in the area. In some examples, block 702 may occur prior to block 704, and in other examples, block 704 may occur prior to block 702. In still other examples, blocks 702 and 704 may occur near-simultaneously such that the biologic material volume and media are disposed in the mixing and digestion area 204 at approximately the same time.
zo In an example where the mixing and digestion area 204 is heated, it may be heated to and maintained at a temperature from about 30 C to about 40 C prior to block 702, block 704, or after either or both of the biologic material volume and/or media are disposed in the mixing and digestion area 204. This heating may be referred to as a pre-heating, which may take from about 10 minutes to about 45 minutes, or less than 10 minutes depending upon a target temperature or temperature range.
Further in the method 700 at block 706, the biologic material volume disposed at block 702 is completely or partially digested via the enzyme media disposed at block 704.
The digestion at block 706 of the biologic material volume disposed at block 702 may occur via heating of the mixing and digestion area 204, or via the agitation of the single-walled bowl 104, or by a combination of both. This digestion may occur over various time periods from 5 minutes to 1 hour, from 15 minutes to 45 minutes, or other periods of time depending upon a type of enzyme(s) used, a number of enzyme(s) used, and/or a volume of each enzyme used at block 704, and the volume of biologic material disposed at block 702.
Further in method 700 at block 706, the agitation at block 706 may occur by the partial or complete rotation of the bowl in alternating directions around a central axis, and is not the same as, nor does it generate the force of, the centrifugation discussed herein. The agitation employed to promote digestion at block 706 may comprise whole or partial rotations in different directions around a central axis 124 (FIG. 1), and may occur for a predetermined period of time. The digestion at block 706 may be referred to as a first state of the apparatus, wherein centrifugal forces are not applied and the media and biologic material volume are agitated in the chamber for a predetermined period of time zo or for a predetermined number of agitation cycles. In one example, the temperature of the mixing and digestion area 204 is maintained at about 37 C during the digestion at block 706, this first state is maintained for a predetermined time period based upon a type of enzyme(s) used, a number of enzyme(s) used, and/or a volume of each enzyme used at block 704, and the volume of biologic material disposed at block 702.
The systems discussed herein may comprise a plurality of stored programs comprising parameters for the digestion at block 706 as well as for other states, blocks, and phases discussed herein.
Further in method 700 at block 708, in response to and subsequent to the completion of digestion at block 706, centrifugation occurs. As discussed herein, the "completion" of the digestion at block 706 refers to when the digestion has progressed to a point where the cells are still viable but the biologic volume has been broken down such that it is capable of centrifugation at block 708. This centrifugation at block 708 may be characterized by the separation of cells from the digested volume formed at block 606.
In an embodiment, the configuration at block 708 comprises a g-force from 600 G to 1000 G for a time period from about 5 minutes to about 10 minutes. The g-forces or "G"
referred to herein is the force of gravity applied to a body, in this case, the force applied to the cells collected in the cell concentration areas which continue to have fluid .. removed (thus becoming a more concentrated cell volume with the reduced fluid/waste) and remain isolated from the mixing and digestion area 204 during at least the centrifugation at block 708.
The centrifugation at block 708 may be referred to as a second state of the cell separation system. The centrifugation at block 708 may comprise programs of various zo RPM speeds and times for cycles. These cycles may increase in RPM and/or in duration until cessation at block 712 as discussed below. In an embodiment, a single centrifugation cycle may be employed from 500 G to 1000 G for a time period from about 5 minutes to about 10 minutes and in still other examples, multiple centrifugation cycles may be employed that first increase in the G-force applied and then decrease the force applied, leading into the slowed rotation discussed in detail in block 710 below.
In an embodiment, the digestion at block 706 separates a plurality of cells from adipose tissue and fluids, the centrifugation cycle(s) at block 708 acts to force the separation of the cells and the movement of those separated cells into the cell concentration areas 212.
In an embodiment, the heating mechanism used to preheat the mixing and digestion area 204 during digestion at block 706 is shut off subsequent to block 706 and prior to the initiation of block 708, such that the centrifugation at block 708 may proceed between room temperature (from about 20 C to about 25 C) and the temperature employed at block 706. Further at block 708, during separation of the cells into the product reservoir 206 and cell concentration areas 212, after a predetermined time period of centrifugation at block 708, clean media may be introduced into the chamber of the single walled bowl 104 via single access tube 108 (block 716). This media further displaces a plurality of materials including fat and other tissues and liquids from the mixing and digestion area 204 such that those materials are removed from the mixing and digestion area 204 as the volume increases and captured into the waste reservoir 202.
In some examples, a speed of from 500 G to 800 G may be used at block 708 in order zo to separate cell volume in the concentration areas 212, and in other examples, a speed from 700 G to 900 G may be used for centrifugation at block 708. The clean media of block 716 may be added at block 708 to increase the total volume inside the bowl 104 to promote the expulsion of waste product into the waste reservoir 202. The introduction of media at block 716 may occur depending upon the rotation speeds, centrifugation programs (cycles), and geometry of the system and collection regions.
In an embodiment where wash media is introduced at block 716, the amount employed may double or triple the total volume of all fluids thus far introduced into the bowl 102.
Block 710 is a slowed rotation phase and may also be referred to as the third state of the cell separation apparatus. During this phase, which may be from 3 minutes to 45 minutes, a lower centrifugal force is applied at block 710. In one example, at block 710, the rotation of the single-walled bowl 104 is slowed to a predetermined speed or range of speeds, and a temperature of the bowl may be from room temperature to the maximum temperature employed at block 706. The parameters such as speed (force generated) and temperature may be employed at block 710 to enable the separated cell volume to remain in the cell concentration areas 212 while the remaining fluid from the media and biological volume drains down the interior walls of the bowl to the waste reservoir 202.
Waste may be collected in waste reservoir 202 in whole or in part while the cell volumes are retained in the cell concentration areas 212 by the centrifugal force applied at block 710. Some waste may remain trapped in the mixing and digestion area 204, where it is isolated from the separated cells. In some examples, the slowed spin of block 710 may be iterative, for example, a first slowed rotation at block 710 may be at 90%
of an zo average speed of block 708, a second, subsequent slowed rotation at block 710 may be at 80% of an average speed of block 708, and subsequent slowed rotations may be at lesser and lesser speeds until a predetermined period has expired or until a predetermined amount of fluid and/or solids has been removed to the waste reservoir 202, as determined by volume and/or optic sensors. During block 710, material that is too large to pass through filters 210 is retained and isolated in waste reservoir 202 such that the separated cell volume is not contacted by this material.
A vacuum is employed to draw cells in from the cell concentration area 212 into the center of the product reservoir 206. Thus, the cells collected in 212 move from those collection areas to an area of the product reservoir 206 wherein, at block 714, the concentrated cells (separated cell volume) may be removed using vacuum and via the single access tube 108. In some embodiments, this removal is automated and occurs in response to completion of blocks 710 and 712. In some embodiments, this removal is io automated and occurs before block 712. Blocks 712 and 714 may be collectively referred to as a fourth state of the cell separation apparatus when the bowl is no longer rotating relative to the containment mechanism 112.
The blocks discussed in the method 700 are associated with an automated, dynamic method of cell separation and collection, such that the loading of the bowl 104 at blocks 702 and 704 proceeds through the removal and collection at block 712 without manual intervention. In one example, a non-transitory memory stored on a storage device and coupled to the cell separation apparatus comprises a plurality of code executable by a processor. This plurality of code comprises centrifugation programs for samples of varying properties, each program may comprise a flow rate for blocks 702 and/or 704, zo as well as cell volume and/or concentration targets, flow rates, times, and ranges for forces generated (rotation rate/RPM) at blocks 706, 708, 710, 714, and 716 as appropriate for the actions occurring at each block. Each program may be associated with an overall time to completion from the deposition of the media and biologic material volume to the removal of the separated cells.
In another example, the plurality of code contains programs that automatically detect when the system has clogged and shuts down the separation process. The program then initiates either media flush of the system or agitates the assembly 102 such that the clog is dispersed and the separation procedure is then reinitiated.
While preferred embodiments have been shown and described, modifications thereof can be made by one skilled in the art without departing from the scope or teachings herein. The embodiments described herein are exemplary only and are not limiting.
Many variations and modifications of the systems, apparatus, and processes described herein are possible and are within the scope of the invention. For example, the relative dimensions of various parts, the materials from which the various parts are made, and other parameters can be varied. Accordingly, the scope of protection is not limited to the embodiments described herein, but is only limited by the claims that follow, the scope of which shall include all equivalents of the subject matter of the claims.
Unless expressly stated otherwise, the steps in a method claim may be performed in any order.
The recitation of identifiers such as (a), (b), (c) or (1), (2), (3) before steps in a method claim are not intended to and do not specify a particular order to the steps, but rather are used to simplify subsequent reference to such steps.

Claims

PCT/US2018/065405A method of separating cells, comprising:
(a) agitating, in a digestion area of a cell separation system, a volume of biologic material and a volume digestion media to form a digested volume of biologic material;
(b) centrifuging the digested volume of biologic material at a force from about 500 G to about 1000 G from about 5 minutes to about 10 minutes to separate the digested volume into a plurality of concentrated cell volumes and a plurality of waste;
(C) collecting the plurality of concentrated cell volumes in a product reservoir wherein the concentrated cell volume comprises material with a smaller diameter than the pores of a filter such that the material can pass through the filter into a cell concentration area;
(d) isolating the plurality of waste in a waste reservoir wherein the waste reservoir is partitioned in the bowl from the digestion area and wherein the plurality of waste comprises waste with a larger diameter than the pores of the filter and is not in contact with the concentrated cell volumes;
(e) removing the concentrated cell volumes from the product reservoir.
zo The method of claim 1, wherein the (a) comprises rotating the single-walled bowl in each direction around a central axis in an alternating fashion for a predetermined period of time.

The method of claim 2, further comprising heating the single-walled bowl from about 30 C to about 40 C prior to (a) the volume of biologic material and the plurality of digestion media into the reservoir.
The method of claim 2, further comprising heating the single-walled bowl from about 30 C to about 40 C during (a).
The method of claim 1, wherein (b), (c), (d), and (e) occur from about 20 C
to about 25 io C.
The method of claim 1, further comprising initiating (a) by execution of a centrifugation program via a processor.
The method of claim 6, wherein the centrifugation program is complete subsequent to (e).
The method of claim 1, wherein a plurality of centrifugation programs are stored in a non-transitory storage device of the cell separation system.

A cell separation system, comprising:
a non-transitory storage device comprising a plurality of logic associated with centrifugation programs, wherein execution of a centrifugation program separates a cell volume from a biologic material volume;
a heating mechanism electrically coupled to a power supply;
a containment mechanism in proximity of the heating mechanism;
an assembly removably coupled to the containment mechanism, wherein the assembly comprises:
a single-walled bowl comprising a product reservoir, plurality of cell concentration areas, a digestion area, a waste reservoir, and a center column comprising a access tube removably coupled to the product reservoir, a mounting plate, wherein the single-walled bowl is removably coupled to the mounting plate, an alignment mechanism coupled to the center column to restrict movement of the access tube when coupled to the containment mechanism, wherein, in a first state of a centrifugation program, the assembly is configured to rotate around a central axis in a first direction and in a second direction in an alternating fashion and the heating mechanism is activated, and wherein, in a second state of a centrifugation program, the heating mechanism is deactivated and the single-walled bowl is configured to rotate in a single direction around the central axis to separate a plurality of waste from a plurality of cells.
The system of claim 9, wherein the separated cells are separated in the second state of a centrifugation program into a plurality of cell concentration areas in the product reservoir of the single-walled bowl.
The system of claim 9, further comprising a third state, wherein, when the system is configured in the third state, the system is configured to collect waste in a waste reservoir formed in the single-walled bowl.
The system of claim 9, wherein the product reservoir comprises one or more filters that only permit the passage of material less than 200 microns in diameter to pass into the cell concentration areas.
The system of claim 9, wherein, when configured in a fourth state, the single-walled bowl is stationary.
The system of claim 9, wherein the access tube permits the removal of concentrated zo cell product from the product reservoir during centrifugation.

A cell separation system comprising:
a non-transitory storage device comprising a plurality of logic associated with a plurality of different centrifugation programs, wherein, when executed by a processor;
agitates in a digestion area a volume of biologic material and a volume of digestion media to form a digested volume of biologic material;
separates, via centrifugation, the digested volume of biologic material at a force from about 500 G to about 1000 G from about 5 minutes to about 10 minutes to separate the digested volume into a plurality of concentrated cell volumes and a plurality of waste;
collects the plurality of concentrated cell volumes in a product reservoir wherein the concentrated cell volume comprises material with a smaller diameter than the pores of a filter such that the material can pass through the filter into a cell concentration area;
isolates the plurality of waste in a waste reservoir wherein the waste reservoir is partitioned in the bowl from the digestion area and wherein the plurality of waste comprises waste with a larger diameter than the pores of the filter and is not in contact with the concentrated cell volumes; and removes the concentrated cell volumes from the product reservoir.

The system of claim 15, wherein the system further comprises a first assembly comprising a single-walled bowl removably coupled to a mounting plate to enable the assembly to spin relative to a containment mechanism during at least the agitation and centrifugation.
The system of claim 16, wherein the first assembly is removably coupled to a containment mechanism and wherein a heating mechanism is located in proximity to the containment mechanism.
The system of claim 15, wherein at least some centrifugation programs of the plurality of centrifugation programs are further configured to, when executed by the processor, heat the bowl at least one of prior to or during the agitation from about 30 C to about 40 C.
.. The system of claim 16, wherein cell product is collected by the transfer of cell product from the plurality of concentrated cell volumes in the plurality of cell concentration areas of the single-walled bowl into the product reservoir.
The system of claim 16, wherein the first assembly is disposable.