Patent Grant
12018244
Multipotent cells are known to be useful in various medical procedures to assist in the healing of an affected area of a patient, for example by providing enhanced cellular regeneration of a treatment site. The multipotent cells can be sourced from various tissues of the body of a living being for use in a surgical procedure. The multipotent cells may be autologous, where the patient is the donor for the cells that are used to treat the same patient. The term “multipotent cells” includes adipose-derived stem cells, which have also been described as adipose-derived stem/stromal cells, adipose-derived adult stem cells, adipose-derived adult stromal cells, adipose-derived stromal cells, adipose stromal cells, adipose mesenchymal stem cells, lipoblast, pericyte, preadipocyte, and processed lipoaspirate cells.
It is well known that adipose tissue in the human body contains significant numbers of multipotent cells, in fact, far more multipotent cells are stored per unit volume in fat than in bone marrow. Some estimates give factors of 500:1 for the ratio of multipotent cells stored per unit volume in adipose tissue relative to those stored in bone marrow.
In order to retrieve the multipotent cells from fat, a sample of fat is retrieved from the patient by techniques known in the art, generally, for example, surgery or liposuction. It has been known to utilize enzymes, such as collagenase, or trypsin, etc., to breakdown peptide bonds in the collagen network holding the adipose tissue together, and to break down the basement membrane around the individual cells. Once this has been done, the multipotent cells may be separated out, and concentrated using centrifuge, sedimentation or filtration techniques, and the concentrate is washed to remove the enzyme (residuals) used to treat the fat sample. It is thought to be vital to remove the agents that had been added to break down the collagen network, as these enzymes are thought to cause reduced viability of the harvested cells. The washed concentrate is then available for injection back into the patient, for the purpose of accelerated repair of an injury. Unfortunately, this process to prepare a useful sample of multipotent cells, takes several hours (and in some cases up to 14 days), that makes the ad-hoc use of such a procedure difficult or impossible, required multiple processing steps, thereby increasing the potential for contamination, compromised sterility, and the process demands skilled technical knowledge.
It is previously known that in addition to preparing samples of multipotent cells isolated from adipose tissue, the multipotent cells could be isolated from a sample of bone marrow. However, in order to retrieve cells from bone marrow, the patient has to endure a very uncomfortable puncture of the marrow spaces/cavities in bone (e.g., the iliac crest) before bone marrow aspirate (BMA) is drawn. The BMA sample is then spun down in a centrifuge to gain a cellular concentrate that can then be injected into the patient for the repair of some injury. Although the timing of this procedure permits the ad-hoc use in an operatory, the concentrate obtained may have an insufficient dose level for some applications without adopting a culturing method to increase the concentration. The procedure utilizing BMA may be competitive to procedures using multipotent cells from fat, however, the harvesting of tissue for BMA procedures has the disadvantage of requiring a painful access procedure.
Accordingly, a need exists for a rapid multipotent cell collection, isolation and concentration apparatus and procedure that enables the ad-hoc use of harvested cells in a surgical procedure, where the harvested cells can be prepared in a short timeframe (less than 5 minutes), and capable of being performed following a simple protocol with easy steps that do not require extensive technical training. The subject invention addresses that need (and others) by providing a compact, sterile, self-contained, easy-to-use centrifugal separation unit to provide quick and reliable multipotent cell isolation from collected or harvested fatty tissue and methods for quickly and reliably isolating multipotent cells from collected or harvested fatty tissue. The fatty tissue can be collected or harvested by any means known in the art, including, but not limited to, liposuction and surgically harvested fat. In the case of adipose tissue, the biologic mixture consists of the fatty and fibrous tissue, plus a portion of the tumescent fluids used to stabilize the fat for extraction (e.g., saline, epinephrine, lidocaine, etc.), with the multipotent cells residing in the fatty and fibrous tissue. To isolate the multipotent cells for harvesting, the device mechanically breaks down the collagen structure, and separates its fractions by specific gravity, in order to isolate the fraction containing the multipotent cells for collection and use in various types of procedures, be they diagnostic, therapeutic, or surgical.
With regard to fat processing for reimplantation, one may alternatively obtain a sample of harvested fat to be utilized surgically, in a manner that does not require separating out the multipotent stem cells from the tissue structure, as described immediately above. Fat transfer, for example, also referred to as autologous fat grafting, involves the removal and re-implantation of a patient's adipose tissue. The adipose material is typically removed from areas of the body like the abdomen, thighs, or buttocks. Depending on the extraction technique (e.g., surgical removal, liposuction, etc.), it may be necessary to remove the certain portions of the harvested sample (e.g., tumescent solution) from the tissue extract. It may further be necessary, depending on the techniques used to harvest the sample, to size the tissue, in order to create a homogenous product and present a material with appropriate particulate sizes for the purpose intended. Sizing of the tissue is desirable in many clinical applications where there is limited access for re-implanting the sample. For example, where there are aesthetic concerns (e.g., facial cosmetic procedures), in order to minimize scarring from incisions, the procedure may be performed by injecting the material via a small diameter needle. When used as a facial filler, fat grafting can improve the creased and sunken areas of the face, and add fullness to the lips and cheeks. Fat grafting is also commonly used in breast and buttocks augmentation, usually in place of implants.
Current fat grafting is performed by harvesting the adipose material, using a variety of techniques and surgical tools. Consequently, the product that is harvested may be quite different in cell viability, texture (e.g., particle size) and composition (e.g., fatty tissue, blood, tumescent solution, oil, saline, water), as a result of the technique utilized for harvesting. This results in variability in the material that may beneficially be accounted for during the processing of the fat sample prior to re-implantation. Furthermore, the preparation techniques and instruments applied to the fat sample for re-implantation may also vary, potentially resulting in a product prepared for re-implanting that may be sized to a particle size that is too small for the intended use of the material, resulting lower cellular viability attributable to the excessive processing, increasing the potential for washout of the implanted material and/or volume loss in the implanted site. Alternatively, a sample that is sized to particle size that is too large for the intended use may result in challenges upon implantation, such as uneven texture, blockages of the narrow gauge needles utilized for re-implantation, and difficulty in the revascularization of the large particle size graft which may negatively affect viability.
What is needed is a device that is able to size the material to a useful consistency, and is able to provide a reliable composition of the material for implantation, regardless of the original collection technique, in order to avoid the above mentioned problems.
What is needed further needed is a unitary device that can quickly process, in a sterile, closed system, the fat harvested for fat grafting, into a homogenous material, having a reliably uniform particle size. The ideal device would consistently size the material in a manner that is independent of the manner of initial harvesting of the fat sample. Additionally, what is needed is a device capable of removing at least a substantial portion of unwanted components from the harvested sample, and preserving the components to be implanted, such as by removing from the sample one or more of: blood, water, saline, oil, tumescent solution. Additionally, the ideal device would minimize the potential for damage to the cellular components and tissue structure within the sample, in order to maximize the viability of cells to be implanted.
In accordance with an aspect of this invention, a centrifuge for processing a biologic mixture, e.g., adipose tissue, and selectively concentrating its constituents is provided. Those constituents have differing specific gravities and are stratifiable in a centrifugal field produced by the centrifuge. The centrifuge comprises a processing assembly and a rotation source. The processing assembly comprises an inner chamber, an outer chamber, at least one cutting element and an annular screen. The inner chamber is arranged to contain a biologic mixture, and has a central longitudinal axis about which the inner chamber is arranged to be rotated and comprises a conical member, a base and at least one extrusion hole at a first location along the central longitudinal axis and extending radially through the inner chamber. The outer chamber is arranged to receive a biologic mixture from the inner chamber and is arranged coaxially upon the central longitudinal axis of the inner chamber and around the inner chamber. The outer chamber is arranged to rotate about the central longitudinal axis and comprises an outer chamber wall and a dish. The at least one cutting element is positioned between a portion of the inner chamber and the outer chamber and is arranged to remain stationary relative to the rotation of the inner and outer chambers. The annular screen is positioned between the cutting element and the outer chamber. The screen provides a series of openings therein and is arranged to rotate about the central longitudinal axis. The rotation source is coupled to the inner and outer chambers.
In accordance with another aspect of this invention a centrifuge for selectively concentrating at least one constituent of a biologic mixture, e.g., adipose tissue, is provided. The constituents have differing specific gravities and are stratifiable in a centrifugal field produced by the centrifuge. The centrifuge comprises an inner chamber arranged to receive the biologic mixture and has a central longitudinal axis about which the inner chamber is arranged to be rotated. The inner chamber comprises a sidewall having a tapered inner surface, a base, an annular screen, and optionally, a trap and at least one roller. If present, the trap is located in the inner chamber adjacent the inner surface of the sidewall. The annular screen has an inner surface and is located at a first radial distance from the central longitudinal axis. The annular screen projects away from the base. The at least one roller is arranged to effectively roll around the inner surface of the screen to propel at least a portion of the biologic mixture through the screen and away from the central longitudinal axis and towards the tapered sidewall.
In accordance with another aspect of this invention a method of for processing a biologic mixture, e.g., adipose tissue, and selectively concentrating constituents of the biologic mixture is provided. The constituents have differing specific gravities and are stratifiable in a centrifugal field. The method basically entails providing the biologic mixture into an inner chamber of a centrifuge. The inner chamber has at least one extrusion hole. The centrifuge additionally comprises an outer chamber disposed about the inner chamber. The inner chamber is rotated about an axis to extrude a portion of the biologic mixture through the extrusion hole. Portions of the biologic mixture from the extrusion hole are cut off to produce a morselized biologic mixture. The morselized biologic mixture is introduced into the outer chamber and the outer chamber is rotated about an axis to cause the morselized biologic mixture to stratify in the outer chamber into at least two concentric stratified constituent layers (e.g., one of which being multipotent cells).
In accordance with another aspect of this invention a method of for processing a biologic mixture, e.g., adipose tissue, and selectively concentrating constituents of the biologic mixture is provided. The constituents have differing specific gravities and are stratifiable in a centrifugal field. The method basically entails providing the biologic mixture into an inner chamber of a centrifuge, and while rotating the chamber about a longitudinal axis, causing at least a portion of the biologic mixture in the chamber to be sized by passing through a rotating screen element having small openings therein. Continued rotation of the chamber will cause the sized biologic mixture to stratify in the outer chamber into at least two concentric stratified constituent layers.
In the various exemplary embodiments described herein, there is provided a motor or drive unit, which serves as a rotation source for the processing unit. Preferably, the motor unit is separable from the processing unit, such that the motor unit may be reused, while the processing unit is preferably a single-use component, though it is contemplated that the processing unit may be cleaned and sterilized, such that it may be reused as well. The processing unit is an assembly, made up of an inner chamber and an outer chamber. The inner chamber is constructed of a sidewall and a base. The sidewall has a tapered inner surface. The inner chamber includes one or more extrusion holes extending radially through the sidewall of the inner chamber at its widest diameter. The inner and outer chambers are arranged to rotate and be driven by the rotation source.
In some of the exemplary embodiments described herein, there may be a static element positioned between the rotating inner and outer chambers. The static element has at least one cutting element which, in cooperation with the one or more extrusion holes of the rotating inner chamber, serves to morselize the tissue into smaller fragments. In these embodiments, as the inner chamber is rotated, the centrifugal force drives the biologic mixture through an extrusion hole, and upon encountering the cutting element of the static element, the ejected material is cut into smaller units, becoming morselized. Furthermore, some of these embodiments may also have a screen arranged between the static element and the outer chamber. As the morselized tissue encounters the screen, continued centrifugal force will urge the material through the screen, thereby capturing the fibrous material on the screen, and passing the non-fibrous material to the outer chamber. This screen may also serve to further reduce the particle size of the material as it passes through the openings.
Once the morselized material is in the outer rotating chamber, the larger diameter of the outer chamber will subject the morselized material to greater centrifugal forces, relative to those in the inner chamber, if the rotational speed is kept constant. Alternatively, should one want to maintain the level of G forces at a constant level, the rate of rotation could be reduced once the majority of the tissue material is in the outer chamber. While in the rotating outer chamber, the morselized material will stratify into annular layers, based upon the specific gravity of the constituents of the biologic mixture. It is understood that the rotation rate may be varied during the processing and separation, such as rotating at a first velocity while the material is within the inner chamber and while passing through the extrusion hole and past the static cutting element; then rotating at a second velocity while the material is within the outer chamber in order to achieve the separation of the constituents by their specific gravities.
In various other exemplary embodiments of the device, the processing unit is an inner chamber, with an internal screen element. The biologic mixture is added to the interior of the chamber, and as the device is rotated, the material will encounter the screen. Continued rotation will urge the material through the screen, which will morselize the material as it passes through the opening. Furthermore, the screen may capture much of the fibrous elements in the material, and passing the non-fibrous elements through the openings to the chamber wall, where the morselized material can separate by specific gravity. In some of these exemplary embodiments having a screen, an optional roller may be provided to further urge the material through the screen. In such an embodiment, as the material spreads out along the inside surface of the rotating screen, the material will encounter a roller arranged parallel to the screen, essentially rolling in place against the rotating screen, thus the material will be pushed through the openings in the screen as the material encounters the roller.
In various exemplary embodiments described herein, the chamber wall, and the base of the inner chamber may form a trap in order to capture the highest density fraction of the fluid in the chamber, as the constituents are separated by specific gravity due to the rotation of the centrifuge about the central longitudinal axis. This trap is arranged so that upon cessation of rotation of the chambers of the centrifuge device, the effects of gravity overcome the centrifugal force acting on the material within the device, the constituent fraction within the trap will remain within the trap, and not mix with the remaining material within the chamber, as that lighter fraction pools due to gravity in the center of the inner chamber. The fraction remaining within the trap may then be harvested by various techniques and applied to tissue to aid in repair.
Alternatively, in other exemplary embodiments where the cells are being retained within the native structure of the tissue material, a substantial portion of the liquids will be removed from the tissue and accumulate in the trap, however, a substantial portion of the desired cells will remain within the inner chamber in fat for collection and use in surgical procedures where a scaffold material may be useful.
In accordance with another aspect of this invention, a centrifuge for processing a biologic mixture, e.g., adipose tissue, by sizing the material, and selectively concentrating its constituents is provided. The centrifuge comprises a processing assembly, and a rotation source. The processing assembly comprises a rotatable chamber arranged to receive the biologic mixture, and a rotatable tube housing a rotatable sizing helix therein. The rotatable chamber comprises a sidewall with a tapered inner surface, and optionally, a trap. The rotatable chamber and the rotatable sizing helix are arranged to be driven by the rotation source. As the sample material is introduced into the chamber through the delivery tube, the rotation of the helix will reduce the particle size of the material. The chamber may be rotated about its longitudinal axis to separate the components of the biologic mixture by specific gravity.
The isolated fraction containing the multipotent cells may be harvested and stored for later use, or immediately directed into a patient for treatment in a medical procedure.
Referring now to the various figures of the drawing, wherein like reference characters refer to like parts, there is shown in
While it has previously been known that the fibrous network in fatty tissue can be broken down by using enzymatic agents, it is currently sought to break down the fibrous network in the harvested adipose tissue by using solely mechanical means, so as to allow, in some embodiments, the release of the multipotent cells contained within the fibrous network. This mechanical breaking down of the fibrous network should avoid the need to wash out an enzymatic agent, and may be accomplished using the various embodiments of the centrifuge devices described herein. For clarity, the term morselized is used to describe the process of mechanically reducing a tissue having an initial fragment size into fragments of a smaller size by the centrifuges of this invention, also known as sizing of the tissue. The terms “morselize” and “size” are used interchangeable herein.
The exemplary processing assembly or unit 100A of
It is preferred that the base unit 20 be reusable so that it can be used consecutively with multiple processing assemblies. It is, however, contemplated that the base unit can be disposable, if desired. The processing unit is, however, preferably disposable, but that is not mandatory providing that it can be sufficiently cleaned and sanitized for reuse. In the embodiment where the drive unit is reusable, the cost for the user can be kept lower than would be the case where the drive unit is disposed along with the rotatable separation unit. It is contemplated that the act of joining of the engageable components (i.e., the drive unit 20 and processing unit 100A) may trigger an automatic start-up reaction in the drive unit, in order to begin processing of the fibrous material. For example, by incorporating magnetic switches in the drive unit, the act of inserting the processing unit into the drive unit may wake up and optionally start the drive unit. Alternatively, the drive unit may include manually operated controls, to allow the operator to have complete control over some or all of the processing steps.
The processing unit 100A also includes an outer housing 101 in which the outer chamber 102, the inner chamber 103 and a stationary sleeve 117 are disposed. The inner chamber 103, stationary sleeve 117 and the outer chamber 103 will be described in detail later. The inner chamber is a hollow, tapered (e.g., conically shaped) member having a sidewall and a base. The outer chamber 103 is arranged to have the tissue to be processed introduced into its interior via an injection port 110. To that end, the inner chamber is arranged to be rotated about the central axis 125 whereupon the centrifugal force produced by the rotation causes the introduced tissue to be extruded through one or more extrusion holes 114 in the inner chamber. The stationary sleeve 117 is disposed between the inner chamber 103 and the outer chamber 102 and includes at least one outlet hole 115, which is arranged to receive the tissue extruded through the extrusion hole(s) 114 as each is brought into alignment with the outlet hole as the inner chamber rotates with respect to the sleeve 117. This action serves to cut or otherwise shear off the tissue extruded through the extrusion hole, thereby morselizing that tissue. The morselized tissue then enters into the interior of the outer chamber 102 as a slurry. The outer chamber is a hollow, tapered (e.g., conically shaped) member having a sidewall and a dish. As mentioned above, the outer chamber is also arranged to rotate about the central axis 125 by the operation of the motor of the base unit. That action causes the slurry material to stratify, with the higher specific gravity migrating away from the central longitudinal axis. The outer chamber includes an annular trap 136 located at the furthest radial distance from the central longitudinal axis. The trap is arranged to receive the portion of the slurry having the highest specific gravity, e.g., the concentration of multipotent cells when the centrifuge is used to process adipose tissue to enable those cells to be readily recovered from the trap, as will be described later.
The inner chamber 103 basically comprises a base 118 and a conical member 134, both being driven via a shaft 129, that is integrally fastened to the base 118. This inner revolving assembly is mounted in a sleeve bearing 119 and a large bearing 104. A stationery sleeve 117, and the sleeve extension 124 is placed around the inner rotating base 118, with the clearance between the sleeve 117 larger end and the base 118 set to a precise value, typically the tolerance is set in the range of 0.001 inch to 0.02 inch, and preferably 0.001 inch to 0.005 inch. The outer chamber 102 is mounted over the inner chamber 103 and is secured thereto at an upper joint 135. The outer chamber basically comprises a dish 120 secured to the sidewall of the outer chamber at lower joint 130. The dish 120 thus forms the larger end of the outer chamber, and is supported for rotation on a dish end bearing 121. The extension 124 of the sleeve 117 is pressed fit into a bottom plate 123, which is stationarily mounted with respect to the housing 101. Thus, in this embodiment, all three of components 123, 124 and 117 are stationary, in that they do not rotate when the centrifuge is activated. At least one extrusion hole 114 is provided in the base 118 of the inner chamber 103. The extrusion hole may be formed by inserting (e.g., pressing) a small plug 113 into a hole in the wall of the base 118, with the plug having an extrusion hole (or extrusion nozzle) 114 on its centerline and with a lead or chamfer 116 formed on the inner end of the hole. Although, only one plug is shown, it is contemplated that more than one plug may be provided, such as by being distributed at intervals around the circumference of the base 118. Alternatively, the opening of the extrusion hole 114 may be integrally formed in the sidewall of the inner chamber, e.g., the sidewall of base 118, rather than requiring a distinct plug or multiple plugs to be inserted into the opening(s). The entrance chamfer 116 can be of any angle or can be a radius, so as to prevent fiber agglomeration at the entrance chamfer. The extrusion hole 114 is shown adjacent to a conical outlet hole 115 in the sleeve 117. One or more outlet holes 115 may be provided in the sleeve 117, and as shown in cross-section in
At the small diameter end of inner chamber 103 a spring 108, a stepped washer 107 and an end-cap 106 are located. The end cap includes threads 111 and is arranged to be threadedly secured on opposing counter-threads provided on the upper neck 105 of the outer housing 101. These engaging threads allow the end cap 106 to be rotated, thus providing for compression of the spring 108, which when compressed, serves to preload the large bearing 104 via the stepped washer 107. The preload is transmitted via the inner chamber 103 to a sleeve bearing 119. The sleeve bearing 119 is located between the base 118 of the inner chamber 103 and the stationary sleeve 117. Thus, the preload is provided to the sleeve extension 124, from thence to the plate 123 and from thence to the outer housing 101. A small bearing 109 is mounted in the small diameter end of revolving inner chamber 103 in order to allow the passage of a non-rotating needle or cannula (or other tubular member) into the revolving chamber through the injection port 110, as the inner chamber 103 is rotating.
Although the sleeve 117 has been described as stationary or non-rotating, it is contemplated that in an alternative embodiment the sleeve may also rotate. However, in such a case there must be difference in the rotation rates of the inner chamber and the sleeve. In particular, in order to achieve the goal of severing portions of material exiting through the extrusion hole 114 to form the morselized material, there need be some difference between the rate of rotation of the inner chamber and that of a rotating sleeve. The rotation of the sleeve may be either in the same, or opposite, direction of rotation as that of the inner chamber. For this embodiment, so long as there is momentary alignment of the extrusion hole 114 and the conical outlet hole 115 of the sleeve, then the exiting (extruded) material may be severed into smaller particles (morselized).
In operation of the various exemplary embodiments described herein, adipose tissue can be obtained from a patient by known techniques, including liposuction or surgical excision. In the case of tissue obtained by liposuction, the fatty tissue and tumescent solution mixture are likely to be in about a 1:1 ratio and will have passed through suction cannula orifices that will have reduced the fat fragments to a size of about 2 mm. This biologic mixture can be fed straight into the various embodiments of a centrifuge device described herein, via the injection port 110 or stationary tube 235, as appropriate. Alternatively, In the case of fatty tissue obtained via surgically excision, the fat will typically be removed from the patient as a semi-coherent mass, in contrast to the tissue collected as particles through liposuction. In the case of surgically excised fat, the fat should be broken up into smaller pieces, and then is to be mixed with portions of liquid, typically with saline or tumescent solution, up to two times the volume of fat, though it is contemplated that other proportions may be suitable as well. The mixing of the harvested fat and mixing liquid may be performed by passing the mixture to and fro between syringes having nozzles of about 2 mm before placing in the centrifuge device.
In operation of any of the various exemplary embodiments described herein, the adipose tissue harvested may optionally be treated with an additive, such as a biologically active agent. It is contemplated that one may wish treat the adipose tissue with, for example, drugs, antibiotics, cellular modifiers, pH modifiers, enzymes, blood products (e.g., whole blood, platelet rich plasma (PRP), red blood cells, platelet poor plasma (PPP), bone marrow aspirate (BMA) or bone marrow aspirate concentrate (BMAC)), prior to, or during the processing of the adipose tissue in the various exemplary embodiments described herein. Alternatively, one or more preservatives or anti-coagulants (e.g. heparin, coumarin, ethylene diamine tetra acetic acid (EDTA), citrates (e.g., Anticoagulant Citrate Dextrose A (ACDA), oxalate) may be added alone, or other additives, to the adipose tissue prior to, or as it is being processed in the various exemplary embodiments of the devices described herein. It is contemplated that additives may beneficially aid in separation of cells during centrifugation, may alter the behavior of the cells in the harvested sample for processing as described herein, or enable the storage of the harvested tissue sample for subsequent processing as described herein. For example, the addition of ACDA may prevent coagulation, allowing storage of the solution containing red blood cells or platelets, or additionally, the ACDA may serve to alter the morphology of stem cells and platelet cells. For example, Applicants believe that adding ACDA to the charge of biologic mixture may be beneficial, in the case of platelet cells typically having a plate-like morphology, may convert to a more spherical morphology, thereby beneficially affecting the ability of the platelet cells to separate by specific gravity, as the more spherical shape of the cell may maneuver more easily through the other constituents of the biologic mixture, e.g., adipose tissue particles.
As mentioned earlier, the centrifuge device of
During this centrifugation process, the fatty constituent of the material tends to migrate toward the central longitudinal axis 125, and the heavier cells and aqueous solution tend to move toward the outer walls of the outer chamber 102. The heaviest density fluid (having the highest specific gravity), containing the highest concentrations of multipotent cells, moves to the outermost diameter, to the annular trap 136. That trap basically comprises an angularly extending channel, though the size and shape of the trap may be modified to capture different fractions of the biologic mixture. For example, the trap may not be angled as shown, but rather may be a channel that is arranged parallel to the axis of rotation 125. In any event, when the rotation of the centrifuge chambers is stopped, the fraction of the biologic mixture not within the trap 136, and within the chamber 102, then drops into a cavity 151 in dish 120 by gravity, and the more viscous fat material collapses onto this liquid. Thus the multipotent cell containing liquid can be isolated in the trap 136 for harvesting. The liquid containing the majority of multipotent cells residing in trap 136 may then be removed by syringe and a shaped cannula, via ports 138 and 137 in the housing 101 and the outer chamber 102, respectively.
For greater ease of manufacture, the inner chamber 103 and outer chamber 102 are arranged to rotate together, in a synchronous fashion, however, in this and in the other embodiments described herein, it is contemplated that the centrifuge could be arranged so that the inner chamber 103 and outer chamber 102 rotate in an asynchronous manner. Thus, the inner chamber 103 may rotate at a first speed, so long as that rotation creates a centrifugal field which will generate sufficient pressure upon the charge of tissue, so as to cause the ejection of the tissue material through the extrusion hole 114; while the outer chamber 102 rotates at a second speed, whether in the same or different direction of rotation, so long as the rotation creates a centrifugal field, so as to effect the stratification of the morselized slurry material.
It has been observed that fat of different composition behaves differently in the centrifuge device. Whereas a portion of the liquid having the highest specific gravity does indeed move to the outer diameter during centrifugation and a portion of that highest specific gravity fluid fills the trap 136, the residual fat may, or may not, emulsify into a stable creamy paste. In those instances where the residual fat is in the form of a stable paste, the paste material will be self-supporting, at least for a few minutes, rather than flowing, as would a paste that is not self-supporting. If the paste is relatively stable, upon ejection through the extrusion hole 114, the paste may coat the inner wall of the outer chamber 102 at smaller diameters, nearer the cone apex, with the paste remaining in place against the wall, even after the rotation of the centrifuge has been stopped. Alternatively the fat can remain as small granules, that do not adhere to either the outer chamber 102 wall or to each other, rather these granules remain free to move relative to each other, in contrast to a material having a self-supporting paste consistency. In these instances, when the chamber stops spinning, the fat granules tend to fall toward the chamber's large diameter end and may disturb the higher specific gravity fluid that has been collected in the trap 136, potentially reducing the concentration of that fraction. To minimize the possibility of the granules of fat interfering with the liquid collected in the trap, the centrifuge may include an alternative processing unit 100B as shown in
Referring to
With this embodiment of the device, and when processing porcine deep adipose tissue, it is possible to retrieve up to 90% of the viable mononuclear cells from the fat samples.
It is contemplated that there may be a benefit to utilize a roller element 210 which is provided with a freedom of movement, such that it can articulate, as it rotates about the static axle 220. Examples of possible articulation mechanisms are shown in
Referring again to
In operation of the embodiment depicted in
In any of the various embodiments described herein, wherein there is a chamber comprising one or more of: a wedge element 265, a first port 275, or a second port 280, the ejection of one or more portions of the biologic mixture within the chamber may be accomplished as follows. The biologic mixture, having been sized by any of the methods described herein, is then rotated within the rotatable chamber to cause the contents to separate by specific gravity. Thus, an outer band of high density fluid (having a higher specific gravity) will form, upon rotation of the chamber, at the outermost surface of the chamber (farthest away from the longitudinal axis 125. An inner band of low density fluid (having a lower specific gravity) will form in the liquid closest to the center of the chamber (closest to the longitudinal axis 125). In between, the outermost and innermost layers, will be at least intermediate layer comprising at least one fraction having a specific gravity between that of the innermost and outermost layers. It is contemplated that the rotation of the chamber and its contents will form an air core, where there is no fluid at the longitudinal axis, so long as the volume of fluid in the chamber is less than the volume of the chamber itself. In those embodiments, where there is a need to eject out of the chamber the heaviest fraction of the biologic mixture, for example, where the fraction having the highest specific gravity contains almost no multipotent cells, this outermost fraction may be discharged through selectively openable first port 275 having an inlet within the chamber at the greatest distance from the longitudinal axis, such that when the valving for the first port is opened, the rotation of the chamber will create a centrifugal force urging the liquid with the highest specific gravity to exit the chamber through the first port 275. The first port is to remain open to allow at least a portion of the highest specific gravity fraction to exit the chamber, whereupon the first port may be closed, whether by action of the operator monitoring the location of an interface, on the tapered surface of the chamber, or operation of an automatic valve. For example, the operator may monitor a color interface that occurs between red blood cells and the multi-potent stem cell fraction, which can be detected through a transparent sidewall of the centrifuge devices described herein. Furthermore, in those embodiments where there is a need to eject the lightest fraction of the biologic mixture, for example, where the fraction having the lowest specific gravity contains almost no multipotent cells, this innermost fraction may be discharged through selectively openable second port 280, having an inlet located within the chamber at a radial distance that is less than that of the radial distance for the inlet of the first port 275, such that when the valving for the second port is opened, the rotation of the chamber will create a centrifugal force urging the fraction of the liquid with the lowest specific gravity to exit the chamber through the second port 280. The second port may remain open to allow at least a portion of the lowest specific gravity fraction to exit the chamber, whereupon the second port may be closed, whether by action of the operator or operation of an automatic valve. In many instances, the second port may be allowed to remain open until the air core, expanding as fluid exits chamber, reaches the entrance to the second port 280, thereby cutting off the flow of fluid out of the second port. In this manner, the inner band of lower density fluid (having a lower specific gravity) and optionally, fat, can be discharged through the second port 280 and into the container 272, leaving the desired concentrate fraction within the dished area 250, at the center of the inner chamber 103, once the rotation ceases. The at least one fraction, having a specific gravity intermediate that of the 2 ejected fractions, will remain within the chamber, and may then be collected by insertion of a cannula into the chamber.
In this or the other processing unit embodiments having a screen element 215, there may be included an optional secondary screen element 216. In such a case, the morselized tissue that has been directed through the screen element 215, will encounter the secondary screen element 216, as the material is directed outwards by the force of the rotation. The secondary screen element 216 is similar to the screen element 215, except that it has a smaller average opening size. While the secondary screen 216 may serve to further morselize the tissue, it is primarily intended to capture the fibrous material that does not readily pass through the openings, while passing the liquid and non-fibrous material therethrough. Use of this arrangement may benefit from reducing the rotational velocity while the processed material is encountering the secondary screen, so as to avoid having excessive centrifugal forces propel the material through the screen, where a slower rotation would aid in capturing the fibrous material against the screen while the liquid is urged through the openings.
As should be appreciated by those skilled in the art by reference to
In the various embodiments described herein, the angle of the inner chamber and wedge, relative to the axis of rotation, will affect how forcefully, and thus how quickly, the stratification of the various components will occur. For example, in an embodiment where the angle of the inner chamber and wedge is shallow, the separation of the constituents will require an increased period of time of rotation, or alternatively higher rotation speeds may be required to drive the separation. By contrast, in an embodiment where the inner chamber and wedge are at a steep angle, off the axis of rotation 125, this steep angle will tend to produce a more forceful and rapid separation of the components. The angle required may be tailored to the viscosity of the fluid being processed. For example, where the charge of tissue is of a high viscosity, it is believed that a steep angle will allow more effective movement of the heaviest components through the fluid. Alternatively, where the charge of fluid is less viscous, it may be possible to employ a shallow angle, and still achieve adequate separation of the constituents. The goal of achieving rapid separation of the constituents is vital, as it is believed that extended duration of the exposure of living cells to elevated G forces during separation may negatively affect the viability of the cells. Thus, it is believed that minimizing the period of time in which the cells are rotated at high speed will lead to better viability of the processed cellular material. In practice of the various embodiments described herein, it is anticipated that the angle of the inner chamber and wedge will likely be between 5 degrees and 30 degrees, but angles of up to 45 or 60 may also work adequately.
In the various embodiments described herein, there may also be a benefit in aiding in the separation of the multipotent cells from the fibrous collagen network in the biologic mixture, such as by adding a volume of saline or other fluids (e.g., blood, bone marrow aspirate, or other body fluids, buffered solutions, cell culture media, detergent solutions, therapeutic solutions such as antibiotic, or anti-coagulants, etc.), as has been discussed previously. This additional fluid added to the harvested fatty tissue may serve to decrease the overall viscosity of the biologic mixture, which will in turn provide for more effective movement of the constituents of the mixture into stratified layers upon exposure to rotational forces. Additionally, the added fluid may enhance the separation of the desired cellular concentration from the other portions of the tissue sample. For example, the addition of whole blood or bone marrow aspirate, when separated by density, will result in the platelet-rich buffy coat comingling with the multipotent stem cells of the adipose tissue sample, as they would have similar specific gravities. The red blood cells, due to their highest specific gravity in the combined sample, would tend to accumulate at the outside layer within the rotating chamber. The plasma of the whole blood will form a separation layer between the multipotent cells and the fatty tissue. The platelets will likely form a layer adjacent to and/or intermingle with the multipotent cells. Furthermore, the addition of whole blood or bone marrow aspirate would also provide a visual indicator by color. Radial stratification would occur with layers forming, in order from the outermost to the innermost, with the red blood cells outermost, the multipotent cells and platelets next, clear plasma next, and the fatty tissue radially innermost, with the red blood cell boundary marking the edge of the fraction with the desired cellular constituents. Additionally, the addition of a liquid to the adipose tissue would likely serve to dilute out the epinephrine and lidocaine that may have been added for the collection of the fat sample.
Furthermore, it is contemplated that there may be a benefit to the various embodiments described herein by providing an agitation step after the morselization step, wherein the centrifuge device is operated in a manner that would impart a gentle, mixing movement to the biologic mixture, so as to ensure the cells are further separated from the fibrous network. The gentle mixing would thereby serve to avoid subjecting the cells to the potentially harmful effects of extended high G forces to achieve separation of the cells from the fibrous network, as it is believed that extensive periods of rotation at high speed may be detrimental to cell viability. This gentle mixing action may be achieved by random orbital movement, such as rocking off-axis, or alternately starting and stopping the rotation of the device, or varying the rate of rotation of the device. For example, the device may be rotated in an oscillating manner, at low frequency (e.g., less than 10 Hz, preferably around 1 Hz) and subjecting the cells to low G forces, in order to free the multipotent cells from the fatty and residual fiber network, or mix in additional fluid into the charge of tissue. The effect of the mixing may be enhanced by including projections, such as fingers, ribs or radial fins, extending into the rotating chamber. Such projections can be arranged as vertical elements, spiral elements, or combinations thereof, on the surface of at least one of the wedge 265, the outer surface of the mesh of the screen element 215, the inner surface of the conical sidewall 134, and the base 118, so long as a mixing feature is extended into the dished area 250. The oscillating motion would be quite similar in operation to that of a conventional clothes washing machine, where the alternating start-stop, and optionally, oscillating movement, all at much lower speeds than would be required to achieve centrifugal separation, should not result in significant reduction of cell viability, all the while providing the benefit of aiding in mechanically disassociating the cells from the fatty and residual fibrous material and other constituents of the biologic mixture.
Another alternative embodiment of a processing unit 100F constructed in accordance with this invention is shown in
As should be appreciated by those skilled in the art the embodiment of
Another alternative embodiment of a processing unit 100G constructed in accordance with this invention is shown in
Another alternative embodiment of a processing unit 100H constructed in accordance with this invention is shown in
Due to the unique methods of morselizing the tissue, as described herein, whether by operation of the helix within the stationary tube, or by passing the tissue material through a mesh screen element, the tissue material that is processed is anticipated to be reduced to a suitable particle size for re-implantation, but is not anticipated to cause damage to cellular components and the tissue structure, such as may occur by over-processing the tissue to a particle size that is too small. It is anticipated that by providing a tissue that is processed to an appropriate particle size, the material will have preserved cellular viability, while maintaining adequate tissue structure so as to not be susceptible to washout or significant volume loss once implanted.
In all of the embodiments having a sizing helix 305, it is contemplated that the drive unit 20, as shown in
Referring back to
For all of the embodiments having a sizing helix 305, while in use, the biologic mixture is to be introduced into the device while the chamber 103, the sizing helix 305 and rotatable tube 315 are rotating in a first direction. The biologic mixture passes through the stationary tube 235, while the sizing helix is rotating about the core wire 236, within the stationary tube, and thus serves to whisk the biologic mixture, and sizes the biologic mixture to a desirable particle size that is smaller than the initial average particle size of the biologic mixture, prior to being placed in the device. Once the entire sample of the biologic mixture to be processed is within the chamber 103, the direction of rotation may then be reversed, thereby halting the rotation of the sizing helix 305, and the chamber 103 can then rotated to effect the separation of the biologic mixture by specific gravity, as has been discussed previously.
As can be seen in the exemplary embodiment of
Another alternative embodiment of a processing unit 100M constructed in accordance with this invention is shown in
As can be seen in the exemplary embodiment of
Another alternative embodiment of a processing unit 100J constructed in accordance with this invention is shown in
As can be seen in the exemplary embodiment of
Another alternative embodiment of a processing unit 100K constructed in accordance with this invention is shown in
As can be seen in the exemplary embodiment of
Another alternative embodiment of a processing unit 100L constructed in accordance with this invention is shown in
As can be seen in the exemplary embodiment of
It should be pointed out at this juncture that any of the above described exemplary embodiments (or any other embodiments constructed in accordance with the teachings of this invention) will produce a concentrated cell fraction that may be usefully combined with (e.g., hydrated into, mixed with, kneaded into, provided as a depot within, or layered onto) a synthetic or natural scaffold or structure which may be implanted into a treatment site of a living being. Such combining of the cell fraction with the scaffold may be accomplished in various manners, for example, by hydrating the scaffold with the cellular fraction, mixing the cell fraction with scaffold material, kneading the scaffold material and cell fraction together, providing the cell fraction as a depot contained within the scaffold material, coating the scaffold with the cell fraction, applying the cell fraction as a layer alongside a scaffold material, sequentially adding the cell fraction to a target site followed by placement of a scaffold material to the target site, or vice versa. Various other procedures for combining a scaffold with a cell fraction may be well known to those skilled in the art and may be suitable for use with the cell fraction created as described herein.
Moreover, while the previously described embodiments have focused on the concentration of multi-potent cells, in any of the embodiments, it is recognized that various cells along with, or instead of, the multipotent cells may be concentrated, which may include adipocytes, as well as the stromal vascular fraction (SVF) of cells including preadipocytes, fibroblasts, vascular endothelial cells and a variety of immune cells (e.g., adipose tissue macrophages, etc.). It is contemplated that by manipulating the location of the outlet ports 275 and 280, the range of specific gravities to be collected can be controlled, such that all of the sample, or just a select portion of the cellular components in the sample can be isolated through the use of the various embodiments described herein.
The above described embodiments may be made available in kit form, including the centrifuge device and accessories needed for operation of the device, including instructions for use and packaging suitable for storage and preserving sterility. In some instances, the kit may provide instructions along with the centrifuge device (either as a single unit, or separable components), and optionally including accessories such as any or all of needles, syringes, cannulas, lidocaine, epinephrine, tumescent solution, liposuction kits and instructions for use.
As should be appreciated by those skilled in the art from the foregoing the apparatus and methods of this invention can be used to provide an injectable concentrate having a larger quantity of multipotent cells that is comparable or better than bone marrow concentrated aspirate of the same volume without requiring the need for a painful iliac crest puncture to harvest cells therefrom. In addition, the subject invention enables one to reduce the time of the procurement process of a usable multipotent cell sample, to a few minutes, so as to allow the use of the equipment in the operatory ad-hoc, if so required. Further still the subject invention eliminates the need for the use of enzymes or chemicals to be added to the sample for processing, yet which would need to be washed from the sample, prior to being injected back into the patient. Thus, the subject invention overcomes the inefficiencies of enzymatic treatments, which typically lead to lower cellular yields.
For any of the above described embodiments, it is contemplated to optionally include a heating source, in order to maintain the biologic mixture at a temperature above ambient temperature. This may be useful where the biologic mixture includes adipose tissue, and the increase in temperature, preferably to body temperature (37 C) would serve to reduce the viscosity of the adipose tissue. In this manner, when the tissue is processed, cell viability may be improved as the cells, e.g., multipotent stem cells, would be exposed to lower levels of shear stress during processing. In contrast, where the processing is performed at a lower temperature, the viscosity of the adipose tissue would increase, and potentially harming cell viability due to the increase in shear stress that would occur when processed by any of the embodiments described herein.
Thus since the inventive process and inventions disclosed herein may be embodied by additional steps or other specific forms without departing from the spirit of general characteristics thereof, some of which steps and forms have been indicated, the embodiments described herein are to be considered in all respects illustrative and not restrictive. The scope of the invention is to be indicated by the appended claims, rather than the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.
This application is a continuation of U.S. application Ser. No. 16/893,464, filed 5 Jun. 2020, now U.S. patent Ser. No. 11/549,094, which is a continuation of U.S. application Ser. No. 15/949,714, filed 10 Apr. 2018, now U.S. Pat. No. 10,711,239, which is a divisional application of U.S. application Ser. No. 14/610,613, filed 30 Jan. 2015, now U.S. Pat. No. 10,125,345, which claims the benefit of U.S. Provisional Application No. 61/934,069, filed 31 Jan. 2014, the entire contents of each of which are hereby incorporated by reference.
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| Number | Date | Country | |
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| 20230159881 A1 | May 2023 | US |
| Number | Date | Country | |
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| 61934069 | Jan 2014 | US |
| Number | Date | Country | |
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| Parent | 14610613 | Jan 2015 | US |
| Child | 15949714 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16893464 | Jun 2020 | US |
| Child | 18094446 | US | |
| Parent | 15949714 | Apr 2018 | US |
| Child | 16893464 | US |
