WO2018031654A1

WO2018031654A1 - Device and methods for harvesting fat derived microvessel segments

Info

Publication number: WO2018031654A1
Application number: PCT/US2017/046094
Authority: WO
Inventors: Christopher Rathbone; Brian Barnes; Samantha REILLY; Patrick Patterson
Original assignee: Arteriocyte Inc
Current assignee: Arteriocyte Inc
Priority date: 2016-08-09
Filing date: 2017-08-09
Publication date: 2018-02-15
Anticipated expiration: 2019-02-09

Abstract

The present disclosure relates to an apparatus for obtaining microvascular fragments (MVFs) or fat derived microvessel segments (FDMS). Tissue samples are loaded into a fluidly connected apparatus, in which they are minced with a paddle blender, digested with enzymes, washed and filtered, resulting in minimally manipulated FDMSs. Methods for processing tissue samples to obtain FDMSs are also disclosed.

Description

DEVICE AND METHODS FOR HARVESTING FAT DERIVED MICROVESSEL

SEGMENTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 62/372,409, filed August 9, 2016, which is fully incorporated by reference herein.

BACKGROUND

[0002] The present disclosure relates to devices and methods for harvesting microvascular fragments (MVFs) or fat derived microvessel segments (FDMSs) that may be used to improve tissue perfusion. Specifically, harvested FDMSs can be used to improve tissue perfusion in areas where compromised blood flow leads to impaired healing. One application of FDMSs could be for the treatment of foot ulcers, such as diabetic foot ulcers (DFUs). Very generally, FDMSs are obtained by mincing, digesting, washing, and filtering tissue.

[0003] The number of people affected by diabetes and the social and economic impact of diabetes are staggering. Nearly 29.1 million Americans, or 9.3% of the population, suffered from diabetes in 2012. Worldwide, approximately 387 million people have diabetes, and this number is expected to rise to 592 million by 2035. In the United States alone, the total yearly costs of diagnosed diabetes have risen to $245 billion, which represents nearly 20% of total healthcare expenditures. As the number of people living with diabetes grows, the disease takes an ever increasing proportion of the national health care budget. The vast majority of these numbers are driven by individuals with type 2 diabetes (T2D), who typically develop the disease in adulthood via insulin resistance and inadequate glucose metabolism. While some people are able to manage their T2D through healthy eating and staying active, type 1 diabetes (T1 D) is normally detected in childhood, lasts a lifetime, and must be tightly controlled by insulin administration. Though T1 D is less prevalent, affecting approximately 3 million Americans, more than 79,000 children were diagnosed worldwide in 2013, and the incidence of T1 D has risen by a startling 23% between 2001 and 2009 for persons under the age of 20. Collectively, the array of complications associated with diabetes, which includes premature cardiovascular mortality, neuropathy, nephropathy, retinopathy and impaired wound healing, requires significant attention in order to improve the impaired qualities of life and shortened lifespans of patients with diabetes, consequentially reducing the enormous societal economic burden.

[0004] One of the most common and severe complications of diabetes is diabetic foot ulcers (DFUs), which is now the leading cause of hospitalization in diabetic patients. Persons with diabetes have a 15% to 25% chance of developing a DFU during their lifetime, and a 50% to 70% recurrence rate over the ensuing 5 years. A significant amount of healthcare resources is spent on the management of DFUs, including emergency room visits, antibacterial medications, amputations, and a multitude of other therapies directed at chronic, non-healing wounds. Various estimates indicate that the cost of DFU treatment consumes 25 to 50% of the total cost of all diabetes treatment. Even more significantly, patients who develop a DFU have a 5% to 8% probability of suffering a major amputation in the first year, and DFUs precede 85% of all lower-limb amputations. Unfortunately, 45% to 55% of these patients die within 5 years of the amputation. Multiple large-scale studies of patient self-reported quality of life have shown that limb loss has a larger negative impact on quality of life than any other complication of diabetes, including end-stage renal disease or blindness. In addition to the loss of mobility and independence, depression and anxiety are more prevalent among people with diabetes who have experienced limb loss.

[0005] Only a small handful of advanced wound care technologies have shown some efficacy in accelerating DFU healing in large, prospective, randomized clinical trials. These include the application of becaplermin (Regranex), a topical gel containing recombinant human PDGF-BB; and two living skin equivalents: a bilayered skin substitute (Apligraf) and a human fibroblast-derived dermal substitute (Dermagraft). Disappointingly, these interventions provide only moderate improvement over standard treatments (generally only 15-20%), are considerably more expensive, and are associated with significant safety concerns. For example, becaplermin increased the risk of cancer mortality in patients. Other interventions, including hyperbaric oxygen and negative pressure devices, have shown promise in promoting DFU healing, but have yet to be tested in large clinical trials. [0006] These therapies may be limited by the inability to effectively restore tissue perfusion, a situation that is likely a result of the impaired microenvironment of the wound. For example, although PDGF-BB and negative pressure wound therapy provide strong angiogenic stimulants, a wound microenvironment lacking the essential elements of angiogenesis (e.g., endothelial cells, stem cells) will not be capable of providing the required building blocks for microvessel synthesis. Similarly, although skin equivalents provide the major components of skin, the inability to sustain perfusion is a challenge that prevents full wound healing.

[0007] Therapies to treat DFUs should address insufficient blood perfusion in order to be successful. It is a well-accepted idea that chronic wounds are poorly vascularized and that impaired angiogenesis represents a critical barrier to wound healing. Both micro- and macrovascular beds are affected by diabetes; in fact, the disease is paradoxical in that excessive angiogenesis plays a role in retinopathy and nephropathy, while insufficient angiogenesis contributes to impaired wound healing and chronic DFUs. Insufficient angiogenesis results from deficiencies associated with the wound microenvironment, including, but not limited to, chronic inflammation, impaired cell migration, edema, and fibrosis. Given the poor angiogenesis and failure to form normal granulation tissue in this microenvironment, it has been suggested that therapeutics to enhance angiogenesis may require more than just the addition of growth factors.

[0008] Microvascular fragments (MVFs) orfat derived microvessel segments (FDMSs) are a heterogenous mixture of arterioles, capillaries, and venules isolated from adipose tissue that have been shown to support tissue perfusion in a number of regenerative medicine/tissue engineering applications. Since FDMSs are delivered as intact microvessels, they circumvent the need for angiogenic ingrowth from the host tissue. Several studies have demonstrated the benefits of FDMSs for improving tissue perfusion. FDMSs bypass some of the challenges of delayed/impaired vascular ingrowth because they already consist of intact microvessels that do not require de novo development to be effective. The clinical opportunities for FDMSs have since been recognized and explored, having been used as a means to support tissue perfusion for cardiac and skin tissues and aiding islet implant survival. More recently, it has been demonstrated that FDMSs can be effectively used to improve tissue perfusion in large volumetric muscle loss defects. [0009] Others have attempted to create vascular networks that can be implanted for immediate vascularization. However, in keeping with the theme of simplicity, it is important to point out that these other prevascularization strategies rely on the in vitro creation of microvessels that use several types of cells derived from a variety of sources for vascular development (e.g., fibroblasts, mesenchymal stem cells, human umbilical cord vascular endothelial cells) prior to their implantation in vivo. For example, in one approach, human umbilical cord vascular endothelial cells (HUVECs), human foreskin fibroblast cells, and immortalized C2 skeletal muscle cells were used to create a preformed vasculature for implantation into an abdominal muscle defect. Using another method, HUVECs were co-cultured with mesenchymal stem cells (MSCs) prior to implantation into a subcutaneous defect. Both approaches resulted in improved vascularity within the implants, but the requirement for multiple cells from various tissue sources has a significant patient burden and regulatory hurdles. FDMSs contain all the necessary components for functionality, i.e., endothelial cells, smooth muscle cells, pericytes, and are capable of spontaneous angiogenesis - there is no need to artificially create microvessels.

[0010] While other strategies to improve vascularization are relatively complicated and time-consuming, fat derived microvessel segments (FDMSs) can be harvested and implanted on the day of surgery. A recent study has demonstrated the superiority of utilizing freshly isolated FDMSs, pointing out that the use of freshly isolated, non-cultured FDMSs is very conducive to a same-day, one-step procedure. After transplantation, FDMSs rapidly inosculate with surrounding tissue within days providing rapid tissue perfusion. The extraction of FDMSs resembles what has been described for the procurement of stromal vascular fraction (SVF) from lipoaspirate, with the exception that the digestion time required for the extraction of FDMSs is much less (i.e., <10 minutes vs. 30-60 minutes). This is a key distinction because the lesser digestion time allows for the microvessel architecture to remain intact, which includes the resident stem cells associated with them. The possibility that FDMSs are a source of stem cells is intuitive, given recent evidence identifying microvessels as a source of stem cells. The large number of trophic factors secreted by the vessels and associated stem cells are another benefit of using FDMSs. FDMSs, in addition to other cells that comprise microvessels (e.g., endothelial cells), may act as sources of bioactive factors that provide a regenerative stimulus in the surrounding tissue. Furthermore, FDMSs are not completely broken down, therefore, there is no need for de novo vessel growth and reorganization, potentially translating into greatly accelerated and improved healing.

[0011] These qualities of FDMSs suggest that they may be an effective means to improve tissue perfusion to facilitate rapid healing for tissue wounds like DFUs, thereby enabling patients to resume normal daily activities and reduce the risk of amputation. The fact that freshly isolated autologous FDMSs can be utilized supports the idea that they can be rapidly translated to the clinic, thereby affecting a large number of patients suffering from DFUs. Despite all the benefits of FDMSs, to date the clinical potential has not been realized.

[0012] Despite the simplistic nature of the methodology used to isolate FDMSs, it is conceptually different from those currently being explored to isolate cells/stem cells from tissues. The majority of stem cell based therapies use invasive, complicated, and skill- dependent strategies to procure stem cells and subsequent manipulation procedures that are subject to stringent regulatory approval. Current strategies of cell procurement rely on extensive manipulation to purify and subsequently use a single type of cell or stem cell, while the goal of FDMS isolation is to preserve vascular functionality to maintain, as closely as possible, the physiological properties of the microvessels as they existed immediately prior to isolation.

[0013] There is a need to develop a means of obtaining FDMSs naturally, rather than artificially, without disturbing microvessels, resulting in a simpler and more physiologically relevant approach. A minimally manipulative strategy to isolate FDMSs will preserve the functionality of vessels, permitting them to be used in a wide variety of chronic wounds. Virtually any condition in which limited perfusion is an underlying cause of disease/nonrepair could be a potential use for FDMSs, e.g., critical limb ischemia, post-myocardial infarction, and bone non-union.

BRIEF DESCRIPTION

[0014] The present disclosure relates to devices, systems, and apparatuses for the simple production of fat derived microvessel segments (FDMSs) from appropriate tissue. A rapid, point-of-care device capable of isolating FDMSs would provide clinicians with a means to improve perfusion within DFUs. The successful development of an automated device capable of isolating FDMSs also has the potential to drastically shift the current paradigms regarding the approach used to treat chronic wounds. The introduction of this new methodology to the wound healing market is a major shift from the current approaches to treat wounds. Specifically, while the majority of current procedures rely heavily on the wound microenvironment to provide the critical elements for angiogenesis and healing (i.e., migrating vascular cells and growth factors), new approaches that will provide an immediate vascular supply that simply requires host inosculation to be successful are sought after. This would allow for an innovative approach of circumventing the compromised wound environment to be realized.

[0015] Very generally, the apparatuses include systems for performing three different functions, and also include a centrifuge for separation of tissues from each other. Sample tissue is loaded into the apparatus and processed through it to obtain FDMSs. The apparatus includes components for mincing of tissue, digestion of tissue, centrifugation and washing, and filtration / separation of FDMSs from the other tissue. One or more of these functions can be performed in a common vessel, as further discussed herein.

[0016] Disclosed herein in various embodiments are apparatuses for obtaining fat derived microvessel segments (FDMSs), comprising: a first component comprising a paddle blender in a first vessel; a second component fluidly connected to the first component and comprising a second vessel; a centrifuge fluidly connected to the second component; and a third component fluidly connected to the centrifuge and comprising an upper-size filter and a lower-size filter placed within a third vessel.

[0017] The first vessel may have at least one opening for loading sample tissue. A bag containing the sample tissue can be loaded into the first vessel.

[0018] The second vessel may also include an enzymatic inlet for introducing enzymes. The centrifuge may have a wash/rinse outlet through which fluid can exit. The third vessel may have an outlet through which FDMSs are collected.

[0019] Also disclosed herein are apparatuses for obtaining fat derived microvessel segments (FDMSs), comprising: a mincing vessel, a digestion vessel, a washing centrifuge, and a filtration vessel. The mincing vessel receives sample tissue and minces the sample tissue. The digestion vessel receives enzymes and the sample tissue to make digested tissue. The washing centrifuge washes the sample tissue. The sample tissue passes through filters in the filtration vessel to separate the washed tissue into tissue of improper size, and tissue of a desired size (i.e. the microvascular fragments).

[0020] The mincing vessel may comprise a paddle blender, an opening for loading sample tissue, and an outlet through which minced tissue exits the mincing vessel.

[0021] The filtration vessel may comprise an inlet, an upper size filter and a lower size filter through which tissue is passed, a waste outlet for removal of tissue of improper size, and an FDMS outlet for collecting FDMSs of a desired size.

[0022] The digestion vessel may comprise an inlet for receiving tissue, an enzymatic inlet, and an outlet.

[0023] The washing centrifuge may comprise an inlet for receiving tissue, a wash/rinse inlet for introducing fluid into the washing centrifuge, a wash/rinse outlet through which the fluid can exit the washing centrifuge, and an outlet.

[0024] In some embodiments, the mincing vessel and the digestion vessel are a common vessel in which the mincing and digesting occur. The sample tissue can subsequently pass from the common vessel to the centrifuge, and subsequently to the filtration vessel.

[0025] In other embodiments, the mincing vessel and the filtration vessel are a common vessel in which the mincing and filtering occur. The sample tissue then passes from the common vessel to the digestion vessel, and subsequently to the centrifuge.

[0026] In still other embodiments, the mincing vessel, the digestion vessel, and the filtration vessel are a common vessel. The sample tissue subsequently passes from the common vessel to the centrifuge.

[0027] Also disclosed are processes for obtaining fat derived microvessel segments (FDMSs), comprising: (a) obtaining sample tissue; (b) loading the sample tissue in an apparatus; (c) processing the sample tissue in the apparatus to separate FDMSs from undesired tissue; and (d) extracting FDMSs from the apparatus.

[0028] The sample tissue may be loaded into a sterile bag which is loaded into the apparatus. The apparatus may include a paddle blender, a digestion tank, a centrifuge, and a filtration tank for processing the sample tissue. [0029] The paddle blender compresses and minces sample tissue to create tissue fragments. Enzymes are used to digest the tissue fragments. About 1 mg to about 5 mg of enzymes can be used per ml_ of sample tissue. The enzymes may digest the tissue fragments for a period of from about 5 minutes to about 15 minutes.

[0030] The digested tissue fragments may be centrifuged at a force of about 100 g's to about 600 g's. The digested tissue fragments may be centrifuged for a period of from about 1 minute to about 10 minutes.

[0031] The digested tissue fragments may be filtered through a first filter and a second filter to obtain fragments of a desired size. The first filter may be an upper size filter that, for example, has a size of 400 micrometers. The second filter may be a lower size filter that, for example, has a size of 40 micrometers.

[0032] These and other non-limiting characteristics of the disclosure are more particularly disclosed below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following is a brief description of the drawings, which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same.

[0034] FIG. 1 is a schematic diagram of a first exemplary embodiment of the present disclosure. Sample tissue is loaded into a mincing tank and processed/passed through a digestion tank, a washing centrifuge, and a filtration tank, from which FDMSs are extracted.

[0035] FIG. 2 is a schematic diagram of a second exemplary embodiment of the present disclosure. Mincing and digestion first occur in a common vessel, followed by centrifugation and filtration.

[0036] FIG. 3 is a schematic diagram of a third exemplary embodiment of the present disclosure. Mincing and filtration take place first in a common vessel, followed by digestion in a second component and subsequent centrifugation.

[0037] FIG. 4 is a schematic diagram of a fourth exemplary embodiment, in which mincing, digestion and filtration occur in a common vessel followed by centrifugation. DETAILED DESCRIPTION

[0038] A more complete understanding of the compositions and methods disclosed herein can be obtained by reference to the accompanying drawings. These figures are merely schematic representations based on convenience and the ease of demonstrating the present disclosure, and are, therefore, not intended to define or limit the scope of the exemplary embodiments.

[0039] Although specific terms are used in the following description for the sake of clarity, these terms are intended to refer only to the particular structure of the embodiments selected for illustration in the drawings, and are not intended to define or limit the scope of the disclosure. In the drawings and the following description below, it is to be understood that like numeric designations refer to components of like function.

[0040] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0041] As used in the specification and in the claims, the term "comprising" may include the embodiments "consisting of" and "consisting essentially of." The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. However, such description should be construed as also describing compositions or processes as "consisting of" and "consisting essentially of" the enumerated ingredients/steps, which allows the presence of only the named ingredients/steps, along with any impurities that might result therefrom, and excludes other ingredients/steps.

[0042] Numerical values in the specification and claims of this application should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0043] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of "from 2 to 10" is inclusive of the endpoints, 2 and 10, and all the intermediate values). [0044] The term "about" can be used to include any numerical value that can vary without changing the basic function of that value. When used with a range, "about" also discloses the range defined by the absolute values of the two endpoints, e.g. "about 2 to about 4" also discloses the range "from 2 to 4." The term "about" may refer to plus or minus 10% of the indicated number.

[0045] The abbreviation FDMS refers to fat derived microvessel segments. This term is generally used herein to refer to segments obtained from sample tissue containing vascular parts such as blood vessels.

[0046] The present disclosure relates to devices and apparatuses that take as input large biological tissue samples and output minimally manipulated FDMSs that can be used for clinical treatment of conditions such as diabetic foot ulcers (DFUs). The apparatuses perform four different functions, which are (a) tissue mincing; (b) tissue digestion; (c) centrifugation and washing of the digested tissue; and (d) filtration to separate FDMSs from the other tissue fragments.

[0047] A first exemplary embodiment is shown in the schematic diagram of FIG. 1. Initially, it is contemplated that a sample of biological tissue is used as the source for the FDMSs. Such tissue may include adipose tissue or lipoaspirate. This tissue is generally relatively large compared to the desired size for the FDMSs. Sample tissue is loaded into a first component 110. The first component may include a paddle blender within a first vessel. The blender minces the sample tissue into smaller pieces. The minced tissue is then passed to the second component 120, which may include a second vessel. Enzymes are added to the second vessel to digest the minced tissue. It is particularly contemplated that the enzymes may be added through an enzymatic inlet that is separate from the inlet through which the minced tissue enters the second component. The digested tissue then passes from the second component to the centrifuge 130 where it is rinsed and washed. The washed and digested tissue then passes to the third component 140, where filtering of the tissue occurs to obtain the desired FDMSs of the desired size. The third component may include a third vessel that uses an upper-size filter and a lower-size filter to obtain the desired FDMSs.

[0048] The first component may include a paddle blender within a first vessel, an opening, for accessing the first vessel, and an outlet for the minced tissue to exit. This component may also be referred to as a mincing vessel. The tissue sample to be processed can be added loose, or in a bag, through the opening. The bag can be clamped into position. Paddle blenders use paddles to crush and knead, compressing the tissue sample and alternately press on the outer surface of the bag, thereby kneading the bag's contents to achieve a grinding action that minces the tissue sample into minced tissue. The minced tissue fragments then travel out of the first vessel through the first component outlet, which is fluidly connected to an inlet of the second component.

[0049] The second component is generally contemplated to include a second component inlet, a second vessel, and a second component outlet. The second component may also have an enzymatic inlet which is separate from the second component inlet. Minced tissue fragments enter the second vessel through the second component inlet. Enzymes are then added to the minced tissue fragments to digest the tissue. Such enzymes may include collagenase, trypsin, elastase, and protease type XIV. It is contemplated the amount of enzyme used may range from about 0.01 mg to about 5 mg of enzyme per milliliter (ml_) of sample tissue. The tissue is digested for a period of about 1 minute to about 15 minutes. This allows the microvessel architecture of the tissue to remain intact. Following digestion, the tissue fragments may pass through the second component outlet and into the washing centrifuge, these two components being fluidly connected to each other. This component may also be referred to as a digestion tank.

[0050] The centrifuge may include a centrifuge inlet and a centrifuge outlet. The centrifuge may further comprise a wash/rinse inlet and a wash/rinse outlet, through which the water or another aqueous rinse solution may be added to the centrifuge separately from the minced / digested tissue fragments. Generally, the digested tissue is mixed with a wash/rinse solution, then centrifuged, and the supernatant removed.

[0051] The centrifuge may be operated at a speed sufficient to create a force of about 100 gravities (g's) to about 600 g's, including a speed of about 300 g's to about 600 g's. Each round of centrifugation may occur for a period of about 1 minute to about 10 minutes, including from about 1 minute to about 5 minutes. This wash/rinse cycle can be repeated as many times as needed. Following centrifugation, the washed tissue fragments may pass through the centrifuge outlet into the third component. After centrifugation, the tissue fragments may be in the form of a pellet. [0052] The third component may also be referred to as a filtration tank. The third component may include a third component inlet, an upper size filter and a lower size filter within a third vessel, and a third component outlet. The third component may further include a suspension liquid inlet, through which liquid (for example phosphate buffered saline) may be added to re-suspend the tissue fragments if desired. The washed tissue fragments then may enter the third component through the third component inlet. After entering the third component, the tissue fragments are filtered through an upper-size filter and a lower-size filter to separate the tissue fragments into undesired tissue (i.e. of improper size) and the desired fat derived microvessel segments (FDMSs).

[0053] In particular embodiments, the upper-size filter is between 200 micrometers (pm) and 600 micrometers. In specific embodiments, the upper-size filter is 400 micrometers. In particular embodiments, the lower-size filter is between 20 micrometers and 80 micrometers. In specific embodiments, the lower-size filter is 40 micrometers.

[0054] The FDMSs may be obtained from the third vessel through the third component outlet. It is particularly contemplated that the FDMSs can be removed from the third vessel by a syringe. The desired physical form of the FDMSs can depend on the application. For example, if it is intended that the FDMSs be injected, they can be suspended in a liquid. If the FDMSs are to be applied topically, they may be in a gel formulation. The FDMSs can be combined with other ingredients to form the liquid or gel, either in the third vessel or after being removed therefrom, as desired.

[0055] It is contemplated that these functions can be combined and performed in a single vessel. FIG. 2 is an example of one such embodiment, in which mincing and digestion happen in a common vessel 210, followed by centrifugation 220 and filtration 230. FIG. 3 is an example of another embodiment, in which mincing and filtration take place in a common vessel 310, followed by digestion 320 and centrifugation 330. FIG. 4 is an example of a third embodiment, in which mincing, digestion and filtration occur in a common vessel 410, followed by centrifugation 420.

[0056] As seen here, it is also contemplated that the order of the process steps can be varied. Generally, smaller tissue fragments must first be made, so the mincing occurs first. Digestion or filtration can occur next, depending on the extent of the mincing. The centrifugation / washing then occurs. Filtration can also occur last. As a result, FDMSs are obtained. Desirably, the apparatus will be of a size sufficiently small / portable for clinical use.

[0057] FDMSs generally contain arterioles; capillaries; venules; endothelial cells; stem cells; smooth muscle cells; and pericytes. As best understood, arterioles and venules should make up the majority of the FDMS extract, potentially as high as 90% or more of the extract, with capillaries being the next most common ingredient. Desirably, the washing and digestion procedure has reduced the amount of adipocytes, collagenase, red blood cells, white blood cells, and other stem progenitors to a minimal amount in the FDMSs. FDMS functionality is defined by the ability to undergo spontaneous angiogenesis. This can be tested in vitro by suspending the FDMSs in a collagen hydrogel and evaluating their ability to form sprouts.

[0058] The present disclosure has been described with reference to exemplary embodiments. Obviously, modifications and alterations will occur to others upon reading and understanding the preceding detailed description. It is intended that the present disclosure be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

CLAIMS:

1 . An apparatus for obtaining fat derived microvessel segments (FDMSs), comprising:

a first component comprising a paddle blender in a first vessel; a second component fluidly connected to the first component and comprising a second vessel;

a centrifuge fluidly connected to the second component; and a third component fluidly connected to the centrifuge and comprising an upper-size filter and a lower-size filter placed within a third vessel.

2. The apparatus of claim 1 , wherein the first vessel has at least one opening for loading sample tissue.

3. The apparatus of claim 2, wherein a bag containing the sample tissue is loaded into the first vessel.

4. The apparatus of claim 1 , wherein the second vessel also includes an enzymatic inlet for introducing enzymes; or

wherein the centrifuge has a wash/rinse outlet through which fluid can exit; or

wherein the third vessel has an outlet through which FDMSs are collected.

5. An apparatus for obtaining fat derived microvessel segments (FDMSs), comprising:

a mincing vessel, a digestion vessel, a washing centrifuge, and a filtration vessel;

wherein the mincing vessel receives sample tissue and minces the sample tissue;

wherein the digestion vessel receives enzymes and the sample tissue to make digested tissue;

wherein the washing centrifuge washes the sample tissue; and wherein the sample tissue passes through filters in the filtration vessel to separate the washed tissue into tissue of improper size, and tissue of a desired size.

6. The apparatus of claim 5, wherein the mincing vessel comprises a paddle blender, an opening for loading sample tissue, and an outlet through which minced tissue exits the mincing vessel; or

wherein the digestion vessel comprises an inlet for receiving tissue, an enzymatic inlet, and an outlet; or

wherein the washing centrifuge comprises an inlet for receiving tissue, a wash/rinse inlet for introducing fluid into the washing centrifuge, a wash/rinse outlet through which the fluid can exit the washing centrifuge, and an outlet; or

wherein the filtration vessel comprises an inlet, an upper size filter and a lower size filter through which tissue is passed, a waste outlet for removal of tissue of improper size, and an FDMS outlet for collecting FDMSs of a desired size.

7. The apparatus of claim 5, wherein the mincing vessel and the digestion vessel are a common vessel in which the mincing and digesting occur.

8. The apparatus of claim 7, wherein the sample tissue passes from the common vessel to the centrifuge, and subsequently to the filtration vessel.

9. The apparatus of claim 5, wherein the mincing vessel and the filtration vessel are a common vessel in which the mincing and filtering occur.

10. The apparatus of claim 9, wherein the sample tissue passes from the common vessel to the digestion vessel, and subsequently to the centrifuge.

1 1 . The apparatus of claim 5, wherein the mincing vessel, the digestion vessel, and the filtration vessel are a common vessel.

12. The apparatus of claim 1 1 , wherein the sample tissue subsequently passes from the common vessel to the centrifuge.

13. A process for obtaining fat derived microvessel segments (FDMSs), comprising:

(a) obtaining sample tissue;

(b) loading the sample tissue in an apparatus;

(c) processing the sample tissue in the apparatus to separate FDMSs from undesired tissue; and

(d) extracting FDMSs from the apparatus.

14. The process of claim 13, wherein the sample tissue is loaded into a sterile bag which is loaded into the apparatus.

15. The process of claim 13, wherein the apparatus includes a paddle blender, a digestion tank, a centrifuge, and a filtration tank for processing the sample tissue.

16. The process of claim 15, wherein the paddle blender compresses and minces sample tissue to create tissue fragments.

17. The process of claim 13, wherein enzymes are used to digest the tissue fragments.

18. The process of claim 17, wherein from about 0.01 mg to about 5 mg of enzymes are used per ml_ of sample tissue; or

wherein the enzymes digest the tissue fragments for a period of from about 5 minutes to about 15 minutes; or

wherein the digested tissue fragments are centrifuged at a force of about 100 g's to about 600 g's; or

wherein the digested tissue fragments are centrifuged for a period of from about 1 minute to about 10 minutes.

19. The process of claim 17, wherein the digested tissue fragments are filtered through a first filter and a second filter.

20. The process of claim 19, wherein the first filter has a size of 400 micrometers, or wherein the second filter has a size of 40 micrometers.

21 . A process for treating chronic wounds, comprising applying fat derived microvessel segments (FDMSs) thereto.

22. The process of claim 21 , wherein the chronic wounds are due to diabetic foot ulcers; venous ulcers; pressure ulcers; critical limb ischemia; myocardial infarction; or bone non-union.