Patent ID: 12233188

DETAILED DESCRIPTION

Unless otherwise indicated, all numbers expressing quantities of ingredients, dimensions reaction conditions and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”.

In this application and the claims, the use of the singular includes the plural unless specifically stated otherwise. In addition, use of “or” means “and/or” unless stated otherwise. Moreover, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one unit unless specifically stated otherwise.

Mesenchymal stem cells (MSCs) can be obtained from fractionated bone marrow aspirate. Bone marrow aspirate may be fractionated using a centrifuge-based or similar technique which separates the aspirated fluid into density graded layers. As shown inFIG.1, fractionated bone marrow aspirate100typically includes at least the following layers, ordered from greater to lesser density, a red blood cell (RBC) layer102, a buffy coat layer104, a serum layer106and an adipose supernatant layer108. Conventional techniques for the extraction of MSCs from bone marrow aspirates typically feature the isolation and processing of the buffy coat layer104. Many different methods have been used to isolate the buffy coat. Conventional buffy coat isolation and processing methods discard the adipose layer supernatant108.

C. L. Insausti, M. B. Blanquer, L. M. Olmo, M. C. Lopez-Martinez, X. F. Ruiz, F. J. Lozano, V. C. Perianes, C. Funes, F. J. Nicolas, M. J. Majado, and J. M. Jimenez, ‘Isolation and Characterization of Mesenchymal Stem Cells from the Fat Layer on the Density Gradient Separated Bone Marrow’,Stem Cells Dev,21 (2012), 260-72. (Insausti) first disclosed in 2012 that the adipose layer of fractionated bone marrow aspirate contains MSCs. It was estimated by Insausti that processing the adipose layer along with the buffy coat might increase stem cell yields from a bone marrow draw by as much as approximately 50%. The methods and apparatus disclosed herein may be used to isolate, collect and process the adipose layer108of fractionated bone marrow aspirate, with or without co-processing of the buffy coat. Alternatively, the apparatus and methods disclosed herein may be used to obtain MSCs from other non-marrow sources of adipose tissue. Surprisingly and advantageously, applicants have demonstrated MSC yields from the adipose layer of bone marrow aspirate which are increased in an amount significantly greater than 50% when compared to the MSC yield obtained when processing the buffy coat alone.

As noted above, Insausti estimated that processing the adipose layer along with the buffy coat might increase stem cell yields from a bone marrow draw by as much as approximately 50%. This relatively modest yield was in part caused by difficulty encountered in extracting the MSCs from the surrounding adipose tissue. In particular, applicants believe that the MSCs in the adipose layer supernatant108of fractionated marrow aspirate (or the MSC's in other adipose tissue) may be locked in a fine collagen matrix. For example, abdominal subcutaneous fat has a strong collagen matrix that must be disrupted with chemical digestion before viable stem cells can be obtained. Applicants have determined that mechanical emulsification of the adipose fraction of bone marrow aspirate can greatly increase the MSC yield to values significantly above the 50% increase estimated by Insausti.

Specifically, as detailed below, the novel step of applying mechanical emulsification to adipose layer supernatant resulted in an increased MSC yield by approximately 700%. Applicants believe that the increased MSC yield when compared to Insausti et. al. is due to the mechanical dissociation of stem cells from the finer collagen matrix of this tissue.

Accordingly, the present disclosure provides device embodiments, systems and methods for isolating the stem cell rich adipose layer supernatant108(alternatively referred to herein as the adipose LS108) of whole bone marrow aspirate. Embodiments may optionally include isolating and co-processing the buffy coat layer104. Embodies may also be applied, in certain instances, to other sources of adipose tissue.

One family of system embodiments feature a closed system suitable for use in a physician's office for the withdrawal of marrow from a patient followed by the substantially contemporaneous rapid isolation of the adipose LS108and re-injection or surgical placement of adipose LS108or MSCs isolated therefrom into the patient to enhance tissue repair. In another family of embodiments the system may be open ended or partially open ended such that adipose LS or MSCs isolated therefrom are expanded or otherwise processed before reintroduced into the patient to achieve therapeutic goals.

Device embodiments may be used to isolate adipose LS108alone or in combination with the buffy coat104of a whole marrow aspirate. Device embodiments may also combine the adipose LS108with one or more components of the bone marrow aspirate such as the serum layer106, an isolated fraction of the serum layer and/or buffy coat104and/or RBC layer102such as platelets or white blood cells.

Method embodiments may be performed manually or automatically or semi-automatically with appropriate devices. Accordingly, certain automated devices incorporate optical sensors or other detectors to identify the various marrow fractions of interest such as the adipose LS108, serum106, buffy coat104, or RBC layer102.

In one specific device embodiment, as shown inFIG.2A, a centrifuge tube110fabricated to have multiple chambers as described below is centrifuged at 100-500 g for 5-10 minutes. The centrifuge tube110is provided with a plurality of chambers. The first chamber112performs as a typical centrifuge chamber to produce the fractionated bone marrow aspirate with the adipose LS on top108, the serum layer106below the adipose LS, the buffy coat104below the serum layer, and the RBC layer102below the buffy coat. The centrifuge tube110also includes at least a secondary chamber114and a tertiary chamber116which, for example, provide for the serum layer106to be decanted into the secondary chamber114and the adipose LS108to be collected into the tertiary chamber116.

In use, the centrifuge tube110is centrifuged as described above to separate the bone marrow aspirate into layers, as illustrated inFIG.2A. A second centrifuge run is performed to decant the serum layer106into the secondary chamber114as illustrated inFIG.2B. A third centrifuge run performed in an upright position may then eject the adipose LS108and the buffy coat104into the tertiary chamber116.

An alternative device embodiment is illustrated inFIGS.3-4. In this alternative embodiment, certain layers, for example the adipose LS108, serum106, and buffy coat104are collected in the secondary chamber114of a dual chamber centrifuge tube110and then the adipose LS108(with or without the buffy coat) is isolated via the insertion of a tube, catheter, or needle122, into the secondary chamber114such that the serum layer between the adipose LS and buffy coat is collected or drawn into a withdrawal chamber124which can be, but is not limited to a conventional syringe. The serum106can then be expelled from the withdrawal chamber124and the same chamber124can be used to collect the adipose LS108and/or buffy coat104or a fourth chamber can be used for adipose LS or buffy coat collection. As shown inFIGS.3A,3B.3C and4, a density-tuned floating element126may be used to cause the adipose LS (or other layers, depending on the density of the density-tuned floating element126) to collect in the secondary chamber114for efficient withdrawal. The use of density-tuned floating elements126is described in more detail below.

With respect to the embodiment ofFIGS.3A,3B, and3Ca first centrifuge run can be performed to fractionate the bone marrow aspirate as described above. Then, as shown inFIG.3A, a second centrifuge run (performed with or without the addition of a density tuned floating element126to the centrifuge tube110) may separate the buffy coat layer104, serum layer106, and adipose LS108in the secondary chamber114. As shown inFIG.3B, the serum layer106positioned between the adipose LS108and buffy coat layer104may be withdrawn into the withdrawal chamber124. This step may be followed by withdraw of the valuable adipose LS108and buffy coat layer104as shown inFIG.3C.

In an alternative device embodiment, as shown inFIG.5, after the initial centrifuge run as described above, the centrifuge tube128is provided with a novel cap130supporting a tube132inserted to the correct depth to collect the adipose LS108from the centrifuge tube128. Thus, the adipose LS108may be withdrawn into a withdraw chamber124. The withdraw chamber124may be a syringe, later used for direct injection of the adipose LS108for therapeutic purposes, connected to the centrifuge chamber128by a Luer lock or otherwise firmly attached to the tube132. The depth of the tube132may, in certain embodiments, be manually or automatically adjusted to the correct depth to optimize adipose LS108recovery.

In yet another device embodiment as shown inFIGS.6A,6B, and6C, a centrifuge tube134is provided with a plunger136(which may or may not be detachable). As shown inFIGS.6A-6C, the plunger equipped centrifuge tube134may be used to expel the RBC fraction102, the buffy coat104, and serum106from the inferior end of the tube134. This allows the adipose LS108to remain in the chamber134. In certain embodiments the centrifuge tube134may be implemented with a suitably sized syringe that provides for direct clinical injection of the isolated adipose LS108into a patient with or without serum.

In the alternative embodiment ofFIGS.7A and7B, a first chamber138is provided in the form of a disc that fractionates the whole marrow by spinning around a central axis. The adipose LS108, being the least dense fraction, collects in the middle of the disc and may then be drawn off into a second, collection chamber140which may or may not be a syringe which would also provide for direct clinical use of the adipose LS108. In certain embodiments, the collection chamber140may be connected to the first chamber138as shown inFIG.7B. And opening or gate may be provided between the chambers138,140such that the adipose LS108may be forced, expelled or otherwise drawn into the chamber140during the fractionation process. A similar embodiment, illustrated inFIGS.8A and8B, includes a first vertical separation tube142that rotates about its vertical axis causing a fractioning pattern as described above with the additional creation of a fluid meniscus where the adipose LS108in isolated in the center of a depression. The adipose LS may then be isolated and withdrawn using any of the methods described above.

FIGS.9A,9B, and10illustrate alternative types of centrifuge tube suitable for use with certain embodiments described above. For example the centrifuge tube ofFIG.9Ais implemented as a specialized tubular chamber144including a superior portion146which is restricted in diameter relative to the inferior portion148. This configuration allows the superior adipose LS layer108to be elongated after fractionation for easier manual or automatic removal. This system may be part of a closed system providing for direct therapeutic use of the adipose LS108, once isolated or part of an open system where the adipose LS108is for the process before therapeutic use.

In yet another centrifuge tube embodiment (FIG.9B), a specially fabricated density-tuned floating disc126is provided having a selected specific gravity that causes the disc126to float just below the adipose LS108and above the serum106after fractionation, allowing for easier manual or automatic removal of the adipose LS108. In a similar embodiment illustrated inFIG.10, a two-chambered centrifuge tube110utilizes the specific gravity or density-tuned disc126to act as a stopper that can be affixed to the side walls of the primary chamber112of the centrifuge tube110after the disc126floats just above the serum layer106and below the adipose LS108. The adipose LS108can then be manually or automatically decanted into a secondary chamber114for isolation.

In an alternative device embodiment illustrated inFIG.11, the centrifuge tube is implemented as a superiorly tapered tube154with an inferior plunger156. The superiorly tapered tube154is attached directly, or via a tube158, to a withdraw chamber124. The superior tapered portion160of the tube154provides for the adipose LS108to be elongated and selectively pushed via the inferior plunger156or drawn through suction into the withdraw chamber124for isolation. Each tube or chamber154,124may in certain embodiments be implemented as a syringe providing for the direct closed loop clinical use of the adipose LS108.

In an alternative device embodiment illustrated inFIG.12, the centrifuge tube110is implemented with a cap130on the superior end and a highly lipophilic porous membrane168positioned at or near the superior end of the130. In use, a port and withdrawal tube170may be connected to a withdraw chamber, possibly implemented as a syringe or a vacuum line such that the adipose LS108may be lifted out of the fractionated solution opposite the porous highly lipophilic membrane168.

Optionally, as shown inFIG.13saline172or another biologically inert fluid may be added to the centrifuge tube110to float the adipose LS108and lift the adipose LS108into contact with the lipophilic membrane168. Then, as shown inFIG.14, the lipophilic membrane168can then be washed to displace the adipose LS108into an empty tube110where the adipose LS108may be collected by withdraw into a withdraw chamber124which may be implemented with a syringe providing for the direct clinical use of the adipose LS108.

In another device embodiment illustrated inFIG.15, the adipose LS108(which can optionally be intermixed slightly with the fibrinogen rich serum106) is polymerized via the addition of thrombin, CaCl2, or another clotting agent174. Then the polymerized adipose LS176is either manually or automatically removed from the top of the serum layer or drawn though a tube into a withdrawal chamber124.

In yet another device embodiment illustrated inFIG.16, the centrifuge tube110is provided with a side port178or multiple side ports that can be attached to or be punctured by a needle122or other conduit that connects to a withdraw chamber124which may be implemented with a syringe. The side port178or side ports are located at or just below the adipose LS108boundary such that the adipose LS can be drawn into the withdraw chamber124for isolation after fractionation.

In any of the above described embodiments the device may also contain an integrated or separate well system that allows the isolated adipose LS to be processed such that the stem cells and other cellular components are separated from the fine collagen matrix present in the adipose tissue. Emulsification may be accomplished by mechanical or chemical means. For example, as shown inFIG.17A, an emulsification system180may be provided as one or more additional chambers associated with the device or may be located in a separate apparatus such as a syringe providing for clinical use with a patient. In one representative, but non-limiting embodiment, the emulsification system108may use two or more chambers or a single chamber to accomplish emulsification processing. For example, emulsification may occur as the adipose LS108is forced through a small aperture182between a first emulsification chamber184and the second emulsification chamber186. The adipose LS108may be forced repeatedly through the aperture182to accomplish the desired degree of emulsification.

Alternatively, as shown inFIGS.17B and17Ca fine emulsification grate188may be located in one chamber and physically passed through the isolated adipose LS108(FIG.17B). Alternatively, the adipose LS tissue may be passed from one chamber to another through an emulsification grate188. Alternatively, the grate may be fixed with the adipose LS108passing from one part of a single chamber to another part of the same chamber.

In alternative device embodiments, the adipose LS can be processed in any one of the above described chambers or an out board vessel with a digestion agent such as collagenase or lecithin to dissociate the cells from the collagen matrix of the adipose LS108. In other embodiments, the adipose LS can be processed using sonic energy or vibration to dissociate the cellular components.

In other alternative device embodiments, the dissociated cells plus the remaining adipose LS structural tissue (collagen and oils) can be further centrifuged to isolate a cell pellet that can then be washed. This pellet can then be added to the isolated bone marrow serum, platelets, RBCs, buffy coat, mesenchymal stem cells, other adult stem cells, or a nucleated cell mixture and/or isolated nucleated cell types for clinical use.

Alternative embodiments disclosed herein include methods of processing bone marrow aspirates and/or methods of collecting, preparing or reintroducing mesenchymal stem cells into an animal or human patient. Method embodiments include collecting bone marrow aspirate and fractionating the bone marrow aspirate to cause the formation of at least an adipose layer supernatant108. The adipose layer supernatant may then be isolated utilizing one or more of the devices described above or similar devices suitable for isolating the adipose layer supernatant. For example, the bone marrow aspirate may be centrifuged to cause fractionation and the adipose layer supernatant withdrawn or decanted according to the techniques described above, or other suitable techniques.

The methods may further include processing the adipose layer to collect MSCs. For example, the adipose layer may be emulsified, mechanically emulsified, chemically digested, polymerized, subjected to sonic or vibrational energy, centrifuged or otherwise treated to aid with the extraction or collection of MSC's from the adipose layer tissue or fluid.

Upon collection, the adipose layer supernatant108or MSCs collected therefrom may be reintroduced into an animal or human patient to achieve therapeutic goals. In certain embodiments, bone marrow may be drawn; an adipose layer supernatant108collected and MSCs may be extracted therefrom and reintroduced into the patient in a single closed-loop treatment session. Alternatively, MSCs or adipose layer supernatant may be collected and stored or processed for subsequent use. For example MSCs collected and isolated as described herein may be expanded in culture prior to reintroduction into a patient for therapeutic purposes.

EXAMPLES

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. As noted above, applicants have been able to collect surprisingly high quantities of MSCs from bone marrow-derived adipose tissue when compared to the quantity of MSC's collected from similarly obtained buffy coat tissue. The results of preliminary laboratory investigations are described below and graphically represented inFIGS.18-23.

Example 1

10 cc of bone marrow aspirate was withdrawn from several patients. Following a brief centrifugation step of the whole bone marrow aspirate in a sterile conical tube at 200×g, the buoyant adipose layer was collected manually via serological pipette along with a portion of bone marrow aspirate serum. In an initial plating of this bone marrow fraction, a ‘dirty’ culture consisting of cell debris and ‘oily’ substances in the native lipid layer was observed. These components were difficult to remove in later media changes. Further, subsequent re-plating of the media containing lipid suspension resulted in the establishment of large numbers of fibroblast-like morphologies in cells believed to be MSCs. This indicated that the initial plating was sub-optimal and potentially resulted in discarding target cells, if not re-plated, thereby consuming additional resources and time.

Example 2

10 cc of bone marrow aspirate was withdrawn from seven patients. The adipose-plasma solution was passed through a small gauge emulsifier several times to dissociate adipose cells from the associated MSCs. This preparation was used for cell counting, flow cytometric analysis and in vitro plating for cell expansion.

Emulsification was employed in an effort to distort the lipid layer matrix to increase initial plating efficiency. Emulsification and plating resulted in an apparent increase of adherent cells compared to those not emulsified derived from the same lipid sample (see Example 1). In addition, re-plating of the supernatant following 2 days in culture did not result in the establishment of cells of the appropriate morphology and the initial culture was easily cleaned of the features described in the native layer. Therefore, mechanical disruption of the lipid layer via emulsification is believed to be optimal for initial in vitro plating of the lipid layer, potentially by exposing suspected MSCs to the environment and allowing for adhesion.

A very significant difference in the number and percentage of cells that stained positive for the stem cell markers CD44, CD73, CD90 and CD105 was observed when comparing isolations from the buffy coat with the adipose layer. For example,FIG.18illustrates the results of a manual cell count for 7 patients and indicates a lower mononucleated cell count (MNC) in the adipose layer (graph bars200) compared to the buffy coat layer (graph bars202). However, when compared to the contaminating cell background, as shown inFIG.19, the adipose layer after emulsification demonstrated MSCs to comprise approximately 5% of the total cell population (graph bars204). On the contrary, the buffy coat includes only 0.01-0.001% MSCs as determined by flow cytometry analysis (graph bars206).

Further, as shown inFIG.20, the gross MSC count obtained from the buffy coat (graph bars208) ranged from approximately 50-300 cells per 10 cc of bone marrow; while the gross MSC count obtained from the adipose layer collected and processed as described above (graph bars210) was determined to range from approximately 500-4000 cells per 10 cc of bone marrow.

Accordingly, the number of non-MSC ‘contaminating cells’ in the buffy coat layer of bone marrow is significant higher than in the adipose layer; the percentage of MSCs in the buffy coat typically ranges from 0.01-0.001% as compared to the adipose layer where the range appears to be between 3%-15%. Based upon the data represented inFIGS.19and20, it is believed that the number of MSCs in the adipose layer far exceeds that of the buffy coat layer due to the large difference in the percentage of MSCs that exist in the respective regions.

As shown inFIGS.21-23, in vitro studies confirmed that elevated levels of MSCs can be obtained from the bone marrow adipose layer after processing bone marrow aspirate with the devices and methods described herein versus comparable levels of buffy coat derived MSCs.FIG.21depicts the number of MSCs expanded ex vivo from 10 cc. of bone marrow derived adipose tissue (graph bars212) versus the number of MSCs expanded ex vivo from 10 cc. of bone marrow derived buffy coat layer (graph bars214). This data supports the foregoing flow cytometric data ofFIGS.19and20) and it is clear that a significantly larger number of MSCs were expanded from the adipose layer compared to the buffy coat layer using the methods described.

As shown inFIG.23, additional passages revealed that adipose derived MSCs (graph bars222) are also characterized by a lower doubling time resulting from increased rate of division when compared to huffy coat derived MSCs seeded at the same cell density (graph bars220). This indicates that the innate rate of division differs between the adipose derived and huffy coat derived MSCs, suggesting therapeutic advantages from the adipose derived MSCs.

As shown inFIG.23, additional passages revealed that adipose derived MSCs (graph bars220) are also characterized by a lower doubling time resulting from increased rate of division when compared to buffy coat derived MSCs seeded at the same cell density (graph bars222). This indicates that the innate rate of division differs between the adipose derived and buffy coat derived MSCs, suggesting therapeutic advantages from the adipose derived MSCs.

Example 3

Bone marrow aspirate samples were withdrawn from three patients and divided into equal volume subsamples to investigate the effect of emulsification. One subsample from each patient was emulsified as described herein. A 2nd subsample was not emulsified. The cells were plated in a T-25 flask and grown in a 10% FBS/90% DMEM growth medium for 6 days. As shown inFIGS.24-25, for each patient, the cells from the emulsified culture (graph bars224) initiate and proliferate faster when compared to cells grown from the non-emulsified culture. In addition, the cell cultures prepared from emulsified samples were observed to be cleaner and more easily cleared of debris with passage. Thus, the non-emulsified samples appeared “dirty” for longer periods of time.

Various embodiments of the disclosure could also include permutations of the various elements recited in the claims as if each dependent claim was a multiple dependent claim incorporating the limitations of each of the preceding dependent claims as well as the independent claims. Such permutations are expressly within the scope of this disclosure.

The description of the various embodiments has been presented for purposes of illustration and description, but is not intended to be exhaustive or limiting of the embodiments to the form disclosed. The scope of the present disclosure is limited only by the scope of the following claims. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments described and shown in the figures were chosen and described in order to best explain the principles of the disclosed embodiments, the practical application, and to enable others of ordinary skill in the art to understand the various embodiments with various modifications as are suited to the particular use contemplated.

The description of the various embodiments has been presented for purposes of illustration and description, but is not intended to be exhaustive or limiting of the embodiments to the form disclosed. The scope of the present disclosure is limited only by the scope of the following claims. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments described and shown in the figures was chosen and described in order to best explain the principles of the disclosed embodiments, the practical application, and to enable others of ordinary skill in the art to understand the various embodiments with various modifications as are suited to the particular use contemplated.