WO2020174005A1 - Regenerative combination of plasma and adipose tissue - Google Patents

Abstract

A method of treating a wounded or damaged tissue using adipose tissue and blood plasma. The method comprises steps of retrieving an adipose tissue from a patient or a first donor subject compatible with the patient, retrieving a blood sample from the patient or a second donor subject compatible with the patient, isolating cells comprising stem cells from the adipose tissue, isolating platelet rich plasma from the blood sample,and administering the isolated cells and the platelet rich plasma to the patient.

Description

REGENERATIVE COMBINATION OF PLASMA AND ADIPOSE TISSUE

PROBLEM/BACKGROUND

[0001] Field of Invention

[0002] This invention generally relates to regenerative medicine for reconstruction or reparation of wounded or damaged tissues. More particularly, this invention relates to preparing and administering adult stem cells and platelet rich plasma for reconstruction or reparation of the wounded or damaged tissues.

[0003] Description of the Related Art

[0004] Regenerative medicine, which is based on the use of viable cells for the restoring, maintenance, and enhancement of damaged tissues and organs, has generated great interest in the scientific community. The regenerative medicine exploits the plasticity and regenerative properties of particular types of autologous cells, i.e. the body's own cells such as adult stem cells, that can repair the damaged tissue following the interaction with suitable extracellular matrices and growth factors and/or differentiation factors. In this manner, the autologous cells can be employed to stimulate the reparation of wounded or damaged tissues, restore their functions, or support the regenerative properties of the damaged organs.

[0005] One type of human adult stem cells is mesenchymal stem cells (MSCs) derived from bone marrow, dental pulp, fetal membrane, and term placenta. MSCs may also be derived from human and animal adipose tissues because of their abundance and ease of access. In vitro and in vivo experimental studies have demonstrated that human MSCs may differentiate into several cell lineages, such as mature adipocytes as well as chondrocytes, osteoblasts, myocytes, hepatocytes, neuronal-like and endothelial cells, and other lineages. Human MSCs also secrete a variety of bioactive molecules that act in a paracrine fashion to prime and sustain angiogenic, antifibrotic, antiapoptotic, and immunomodulatory responses in target tissues.

[0006] Human MSCs can also promote vasculogenesis, which is the main mechanism involved in tissue repair, cardiovascular differentiation, and myocardial repair. For example, the stem cells have improved islet graft revascularization in diabetic rats, enhancing engraftment success.

[0007] Cells isolated from adipose tissues, including adult stem cells, have been used in regenerative medicine. U.S. Patent No. 7,901,672 discloses a method of making an enhanced, autologous fat graft by extracting from a subject a first portion of adipose tissue to be used as a fat graft then supplementing said fat graft with a population of concentrated, disaggregated, adipose-derived stem cells and endothelial precursor cells. The stem cells and endothelial precursor cells are prepared by disaggregating and concentrating the stem cells and endothelial precursor cells from a second portion of extracted adipose tissue from the same subject.

[0008] Tremolada et al. (“Adipose Tissue and Mesenchymal Stem Cells: State of the Art and Lipogems® Technology Development,” Curr Stem Cell Rep., 2016; 2(3): 304-312) describes a comprehensive review of the methods for isolating stem cells from adipose tissues. One of the methods is the LIPOGEMS® Technology, which uses a simple closed system designed to harvest, process, and transfer refined adipose tissue and achieves a great regenerative potential and optimal handling ability. The LIPOGEMS® Technology does not use any enzymes or other additives. The fat tissue is microfragmented gently and washed off proinflammatory oil and blood residues. The resulting product contains pericytes retained within an intact stromal vascular niche and is ready to interact with the recipient tissue after transplantation, thereby becoming activated as adult stem cells.

[0009] U.S. Patent Application Publication No. 2013/0123747A1 discloses a method of preparing cells and cell agglomerates from lobular fat extracted from a subject for

transplantation. The extracted fat comprises one or more of oily components, a blood component, or sterile solutions, and a solid component including cell fragments, cells and one or more cell macroagglomerates of heterogeneous size. The method comprises the step of dividing the extracted fat into cell agglomerates smaller than the cell macroagglomerates. The size reduction is accomplished by causing the extracted lobular fat to pass at least once through size reducing means including a series of parallel or intersecting sheets or wires that form at least one size reducing net located in a washing and separating container.

[00010] U.S. Patent Application Publication No. 2015/0030571A1 discloses a method of producing mesenchymal stem cells to be used in cellular therapy, cosmetic treatments, replacing a damaged tissue or an organ, or inducing or accelerating tissue repair or regeneration. The method comprises the following steps of extracting an adipose tissue containing mesenchymal stem cells from a cadaveric donor by liposuction process or by surgical removal of parts of the adipose tissue; mechanically treating the adipose tissue to reduce the size of cell agglomerates by separating and removing the fluid component from the solid component; and generating an emulsion of fluid components by mechanical stirring. [00011] U.S. Patent Application Publication No. 2017/0121666A1 discloses a device for preparing adult stem cells for transplantation from lobular fat extracted by liposuction. The device comprises at least one washing and separating container having a washing chamber for washing the liposuctioned adipose tissue. The washing and separating container has an inlet and an outlet for the liposuctioned adipose tissue to enter the washing chamber through the inlet and for at least part of the tissue such as the fluid component to exit the chamber through the outlet. The washing and separating container is coupled to stirring means for generating an emulsion of the fluid components by mechanical stirring in the washing chamber.

[00012] In addition, platelet rich plasma may also be used in regenerative medicine. Platelet rich plasma is a suite of blood products in which platelets are found at higher concentrations in the plasma than in blood. Once the platelet rich plasma is activated, plasma fibrinogen polymerizes into a three-dimensional transient fibrin scaffold, trapping several growth factors, microparticles, and other biomolecules released from the degranulation of platelets. Growth factors and biomolecules sequestered into the fibrin scaffold are released gradually and in a sustained manner as scaffold fibrinolysis occurs, hence platelet rich plasma is highly suitable for enhancing and accelerating the natural process of tissue repair and ultimately reducing recovery times.

[00013] In particular, the released growth factors trigger biological processes aimed at repairing the wounded or damaged tissue, for instance angiogenesis, chemotaxis, cell migration or proliferation by means of cell membrane signaling. Some types of growth factors are present in plasma, including insulin-like growth factor (IGF) and hepatocyte growth factor (HGF). IGF promotes wound healing, bone formation, myogenesis of striated muscle and keratynocite migration. HGF is involved in wound healing, and stands out for its antifibrotic and anti-inflammatory properties. Other growth factors are stored in platelets and are released when platelet rich plasma is activated, e.g. transforming growth factor bΐ (TGF- bΐ), which presents different effects depending on tissue type where it acts: cell migration, neovascularization or osteogenic differentiation; vascular endothelial growth factor (VEGF), which is a key molecule involved in angiogenesis and organ homeostasis; platelet-derived growth factor (PDGF); basic fibroblast growth factor (FGF-2); or epithelial growth factor (EGF) among others. These growth factors play important roles in healing wounded or damaged tissues.

[00014] In practice, blood can be fractionated under the influence of gravity or centrifugal force, blood spontaneously sediments into three layers at equilibrium. The top low-density layer is a straw-colored clear fluid called plasma. Plasma is a water solution of salts, metabolites, peptides, and many proteins ranging from small (insulin) to very large

(complement components). The bottom, high-density layer is a deep red viscous fluid comprising anuclear red blood cells (erythrocytes) specialized for oxygen transport. The intermediate layer is the smallest, appearing as a thin white band above the erythrocyte layer and below the plasma layer. This is called the buffy coat, which has two major components, nucleated leukocytes (white blood cells) and anuclear smaller bodies called platelets (or thrombocytes).

[00015] Platelets are involved in the hemostatic process and release several initiators of the coagulation cascade. Platelets also release cytokines, stored in alpha granules, involved with initiating wound healing. In response to platelet to platelet aggregation or contact with connective tissue, the cell membrane of the platelet is "activated" to secrete the contents of the alpha granules (including cytokines). The alpha granules release cytokines via active secretion through the platelet cell membrane as histones and carbohydrate side chains are added to the protein backbone to form the complete cytokines.

[00016] Enriched platelet concentrates, i.e. plasma containing more platelets per unit volume compared to the whole blood, are known to possess properties for wound healing and formation of new tissue. The use of platelet rich plasma in surgical sites has been shown to rapidly enhance both hard and soft tissue regeneration and repair. Further, in the field of hair restoration, existing evidence that demonstrates platelet rich plasma is a promising treatment option to promote hair growth.

[00017] U.S. Patent No. 7,708,152 discloses device called a platelet rich plasma separator- concentrator suitable for office use or emergency use for trauma victims. The device comprises a platelet rich plasma separator assembly, and a platelet rich plasma concentrator assembly. The separator assembly comprises a centrifugal drum separator that includes an erythrocyte capture module and a motor having a drive axis connected to the centrifugal drum separator. The centrifugal drum separator has an erythrocyte trap. The concentrator assembly comprises a water-removal module for preparing platelet rich plasma concentrate. The water removal module can be a syringe device with water absorbing beads or it can be a pump- hollow fiber cartridge assembly. The hollow fibers are membranes with pores that allow the flow of water through the fiber membrane while excluding flow of clotting factors useful for sealing and adhering tissue and growth factors helpful for healing while avoiding activation of platelets and disruption of any trace erythrocytes present in the platelet rich plasma. [00018] WO 2013/007308A1 discloses a platelet rich plasma composition for epicutaneous use in a wide range of applications in the cosmetic and dermatological fields and a functional bioactive composition suitable for topical use. The compositions allow complete absorption into the skin, hair or scalp, and which have a regenerating action for relaxing expression wrinkles, a lifting effect for sustaining relaxed skin tissues, for scar reduction and wound healing. The compositions are formulated to allow complete absorption of the platelet rich plasma and guarantees the vital function of platelet growth factors while combining a gradual, more physiological releasing rate, and stimulating the recruiting of stem cells.

[00019] The present invention is a novel method for regenerative medicine by administering both platelet rich plasma and stem cells derived from adipose tissue to a subject for healing wounds and repairing tissues.

SUMMARY OF THE INVENTION

[00020] In an aspect, disclosed is a method of treating a wounded or damaged tissue of a patient, comprising steps of:

retrieving an adipose tissue (la) from the patient or a first donor subject compatible with the patient;

retrieving a blood sample (lb) from the patient or a second donor subject compatible with the patient;

isolating cells (2a) comprising stem cells from the adipose tissue;

isolating platelet rich plasma (2b) from the blood sample; and

administering (3) the isolated cells and the platelet rich plasma to the patient.

[00021] In the foregoing embodiment, one or both of the blood sample and the adipose tissue may be retrieved from the patient.

[00022] In each of the foregoing embodiments, the first donor subject and the second donor subject may be the same subject.

[00023] In each of the foregoing embodiments, the step of retrieving the adipose tissue (la) may employ suction-assisted lipoplasty, ultrasound-assisted lipoplasty, excisional lipectomy, or combinations thereof.

[00024] In each of the foregoing embodiments, the step of isolating cells (2a) may comprise washing the retrieved adipose tissue with a washing solution.

[00025] In each of the foregoing embodiments, the washing solution may be an isotonic saline solution. [00026] In each of the foregoing embodiments, the step of isolating cells (2a) may comprise disaggregating the adipose tissue.

[00027] In each of the foregoing embodiments, the disaggregating may employ mechanical cutting and/or enzymatic digestion.

[00028] In each of the foregoing embodiments, the mechanical cutting may employ one or more cutting wire and cutting net.

[00029] In each of the foregoing embodiments, the enzymatic digestion may employ a proteolytic enzyme selected from the group consisting of collagenase, trypsin, lipase, and liberase HI.

[00030] In each of the foregoing embodiments, the step of isolating cells (2a) may further comprise washing the disaggregated adipose tissue.

[00031] In each of the foregoing embodiments, the step of isolating cells (2a) may further comprise emulsifying the disaggregated adipose tissue.

[00032] In each of the foregoing embodiments, the emulsifying may employs mechanical stirring.

[00033] In each of the foregoing embodiments, the step of isolating cells (2a) may further comprise filtering the disaggregated adipose tissue.

[00034] In each of the foregoing embodiments, the isolated cells may comprise individual cells and cell agglomerates.

[00035] In each of the foregoing embodiments, the retrieved blood sample may be mixed with an anticoagulant.

[00036] In each of the foregoing embodiments, the anticoagulant may be selected from the group consisting of heparin, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), acid citrate dextrose solution (ACD).

[00037] In each of the foregoing embodiments, the step of isolating platelet rich plasma (2b) may employ a technique selected from filtration and density fractionation techniques.

[00038] In each of the foregoing embodiments, the density fractionation techniques may comprise centrifugation or continuous-flow centrifugation.

[00039] In each of the foregoing embodiments, the method may further comprise a step of adding a platelet activator to the isolated platelet rich plasma.

[00040] In each of the foregoing embodiments, the platelet activator may be selected from the group consisting of thrombin, calcium chloride, collagen, epinephrine, and adenosine diphosphate. [00041] In each of the foregoing embodiments, the method may further comprise a step of freezing-thawing the isolated platelet rich plasma.

[00042] In each of the foregoing embodiments, the freezing-thawing step may be repeated at least two times, or at least three time, or at least four times, or at least five times.

[00043] In each of the foregoing embodiments, the isolated platelet rich plasma may be in a suspension with a buffer, a diluent, a solvent or a stabilizer.

[00044] In each of the foregoing embodiments, the isolated platelet rich plasma may be suspended in a buffer having a pH between 6.5-8.0, or between 7.0-7.5.

[00045] In each of the foregoing embodiments, the isolated platelet rich plasma may be in a form selected from supernatant, liquid, clot and fibrin membrane.

[00046] In each of the foregoing embodiments, the administering step (3) may comprise mixing the isolated cells and the platelet rich plasma to produce a composition, followed by administering the composition to the patient.

[00047] In each of the foregoing embodiments, the administering step (3) may further comprise, after administering the composition, one or more times of administering the platelet rich plasma to the patient.

[00048] In each of the foregoing embodiments, the administering step (3) may comprise administering the isolated cells, followed by administering the platelet rich plasma.

[00049] In each of the foregoing embodiments, the administering step (3) may comprise administering the platelet rich plasma, followed by administering the isolated cells.

[00050] In each of the foregoing embodiments, the platelet rich plasma and isolated cells in the administering step (3) may have a volume ratio of from about 1 : 10 to about 10: 1, or from about 1 :5 to about 5: 1, or from about 1 :2 to about 2: 1, or about 1 : 1.

[00051] In each of the foregoing embodiments, the platelet rich plasma in the administering step (3) may have a volume in a range of from about 0.5 mL to about 15 mL, or from about 0.7 mL to about 13 mL, or from about 1 mL to about 11 mL, or from 1.5 mL to about 10 mL, or from 2 mL to about 8 mL, or from 3 mL to about 6 mL.

[00052] In each of the foregoing embodiments, the isolated cells in the administering step (3) may have a volume in a range of from about 0.5 mL to about 9 mL, or from about 0.7 mL to about 7 mL, or from about 1 mL to about 5 mL, or from 1.5 mL to about 3mL.

[00053] In each of the foregoing embodiments, the method may further comprise a step, after the administering step (3), examining the patient to assess effectiveness of the treatment. [00054] In each of the foregoing embodiments, the examining step may be performed one or more times at one or more of about 2-8 weeks, about 2-4 months, about 5-7 months and about 11-13 months post the administering step (3).

[00055] In each of the foregoing embodiments, the examining step may be used to determine the necessity of additional administering of the platelet rich plasma.

[00056] In each of the foregoing embodiments, the wounded or damaged tissue may be a result of injury or aging.

[00057] In each of the foregoing embodiments, the injury may be caused by physical activity.

[00058] In each of the foregoing embodiments, the injury may be caused by a disease selected from liver failure, hepatitis B, hepatitis C, myocardial infarctions, renal diseases, kidney damage, retinal necrosis, diabetes, Parkinson's disease, Alzheimer's disease, and stroke.

[00059] In each of the foregoing embodiments, the wounded or damaged tissue may be a result of aging selected from skin wrinkles, hair loss, loose skin, and thinned skin.

[00060] In each of the foregoing embodiments, the administering step (3) may be performed by injecting the isolated cells and the platelet rich plasma directly into the wounded or damaged tissue of the patient.

[00061] In each of the foregoing embodiments, an effective amount of the isolated cells and the platelet rich plasma may be administered to the patient in the administering step (3).

[00062] In each of the foregoing embodiments, the patient may be a human.

[00063] In each of the foregoing embodiments, the patient, the first donor subject and the second donor subject may be humans.

BRIEF DESCRIPTION OF THE DRAWINGS

[00064] FIG. 1 shows a method for treating wounded or damaged tissue.

[00065] FIG. 2 shows a prior art device for isolating cells from an adipose tissue.

DEFINITIONS

[00066] As used herein, the term "stem cells" refers to the heterogeneous population of pluri- and/or multi-potent cells that is found within adipose tissue and/or lipoaspirate, including but not limited to mesenchymal stem cells (MSC), multi-lineage stress- enduring adipose tissue (MuSE-AT), hematopoietic stem cells (HSC), supra-adventitial cells, pericytes and/or others. [00067] As used herein, the term“adipose tissue” refers to fat including the connective tissue that stores the fat. Adipose tissue contains multiple cell types including adipocytes and microvascular cells. Adipose tissue also includes adult stem cells and endothelial precursor cells.

[00068] As used herein, the term "autologous" refers broadly to any material derived from the same subject to which it is later to be administered.

[00069] As used herein, the term "central nervous system" refers broadly to include brain and/or the spinal cord of a mammal. The term may also include the eye and optic nerve in some instances.

[00070] As used herein, the term "effective amount" refers broadly to the amount of platelet rich plasma and/or adipose cells sufficient to provide a beneficial effect to the subject to which the platelet rich plasma and/or adipose cells is administered.

[00071] As used herein, the term "isolated" refers broadly to material removed from its original environment in which it naturally occurs, and thus is altered by the hand of man from its natural environment. Isolated material may be, for example, exogenous nucleic acid included in a vector system, exogenous nucleic acid contained within a host cell, or any material which has been removed from its original environment and thus altered by the hand of man (e.g., "isolated cell").

[00072] As used herein, the term "isolated cells" refers a plurality of cells which has been separated from other components and/or cells which naturally accompany the isolated cell in a tissue or mammal. The cells may be in the form of individual cells and/or in the form of cell agglomerates. Since the cells are isolated from adipose tissues, the isolated cells may also be termed microfat.

[00073] As used herein, the term "platelet rich plasma" refers broadly to plasma having a concentration of platelets greater than the peripheral blood concentration, where the platelets may be suspended in a solution of plasma, or other excipient suitable for administration to a human or non-human animal including, but not limited to isotonic sodium chloride solution, physiological saline, normal saline, dextrose 5% in water, dextrose 10% in water, Ringer solution, lactated Ringer solution, Ringer lactate, or Ringer lactate solution. Platelet counts may range from 500,000 to 7,000,000 per milliliter. Platelet rich plasma is formed from the concentration of platelets from whole blood, and may be obtained using autologous, allogeneic, or pooled sources of platelets and/or plasma. Platelet rich plasma may be formed from a variety of animal sources, including human sources. In preferred embodiments, platelet rich plasma according to the invention is buffered to physiological pH (e.g., pH 7.4). [00074] As used herein, the term“subject” refers to a warm-blooded animal, preferably a mammal, including a human. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human.

[00075] As used herein, the term“stem cell” refers to a multipotent regenerative cell with the potential to differentiate into a variety of other cell types, which perform one or more specific functions and have the ability to self-renew. Some of the stem cells disclosed herein may be multipotent.

[00076] As used herein, the term "wounded or damaged" refers broadly to any tissue damaged including a wound, trauma, lesion, or any tissue degeneration due to aging.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[00077] It must be noted that as used herein and in the appended claims, the singular forms “a”,“an”, and“the” include plural references unless the context clearly dictates otherwise. Furthermore, the terms“a” (or“an”),“one or more,” and“at least one” can be used interchangeably herein. The terms“comprising,”“including,”“having,” and“constructed from” can also be used interchangeably.

[00078] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percent, ratio, 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,” whether or not the term“about” is present. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are

approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[00079] It is to be understood that each component, compound, substituent or parameter disclosed herein is to be interpreted as being disclosed for use alone or in combination with one or more of each and every other component, compound, substituent or parameter disclosed herein. [00080] It is also to be understood that each amount/value or range of amounts/values for each component, compound, substituent or parameter disclosed herein is to be interpreted as also being disclosed in combination with each amount/value or range of amounts/values disclosed for any other component(s), compounds(s), substituent(s) or parameter(s) disclosed herein and that any combination of amounts/values or ranges of amounts/values for two or more component(s), compounds(s), substituent(s) or parameters disclosed herein are thus also disclosed in combination with each other for the purposes of this description.

[00081] It is further understood that each range disclosed herein is to be interpreted as a disclosure of each specific value within the disclosed range that has the same number of significant digits. Thus, a range of from 1-4 is to be interpreted as an express disclosure of the values 1, 2, 3 and 4. It is further understood that each lower limit of each range disclosed herein is to be interpreted as disclosed in combination with each upper limit of each range and each specific value within each range disclosed herein for the same component, compounds, substituent or parameter. Thus, this disclosure to be interpreted as a disclosure of all ranges derived by combining each lower limit of each range with each upper limit of each range or with each specific value within each range, or by combining each upper limit of each range with each specific value within each range.

[00082] Furthermore, specific amounts/values of a component, compound, substituent or parameter disclosed in the description or an example is to be interpreted as a disclosure of either a lower or an upper limit of a range and thus can be combined with any other lower or upper limit of a range or specific amount/value for the same component, compound, substituent or parameter disclosed elsewhere in the application to form a range for that component, compound, substituent or parameter.

[00083] In one aspect, the invention provides a method of treating a wounded or damaged tissue of a patient. The method comprises steps of retrieving an adipose tissue (la) from the patient or a first donor subject compatible with the patient; retrieving a blood sample (lb) from the patient or a second donor subject compatible with the patient; isolating cells (2a) comprising stem cells from the adipose tissue; isolating platelet rich plasma (2b) from the blood sample; and administering (3) the isolated cells and the platelet rich plasma to the patient. In one embodiment, the first donor subject and the second donor subject are the same subject.

[00084] Both the adipose tissue and blood sample may be retrieved from the patient being treated (i.e., autologous sources) or from the donor subjects that are compatible with the patient. The compatibility between the patient and the donor subjects may be identified using standard blood tests, including, for example, matching blood cell surface antigens. The compatibility of the adipose tissue between patient and donor subjects may be determined using standard tissue/organ transplantation matching technology. In one embodiment, one or both of the adipose tissue and blood sample are retrieved from the patient as the autologous source.

[00085] Human adipose tissue is a well-vascularized tissue, and the network of blood vessels that are integrated throughout subcutaneous adipose tissues is surrounded by numerous types of regenerative cells. These regenerative cells include mesenchymal stem cells as well as other cells that are important for tissue healing and regeneration such as monocytes and fibroblasts. In order to obtain adipose tissue, one may harvest fat from the abdomen, the flank, or subcutaneous hip of the patient or donor subject. Adipose tissues make it feasible to easily obtain a greater amount of mesenchymal stem cells than one is able to obtain from bone marrow. This benefit of using adipose tissues may be realized regardless of the age of the person from whom the adipose tissue is obtained.

[00086] Retrieving of an adipose tissue (la) can use any method known to a person of ordinary skill in the art. For example, the adipose tissue may be removed from a patient by suction-assisted lipoplasty, ultrasound-assisted lipoplasty, and excisional lipectomy, or combinations thereof. As the adipose tissue is intended for reintroduction into the patient, the retrieving of the adipose tissue should be performed in a manner that preserves the viability of the cellular component and that minimizes the likelihood of contamination with potentially infectious bacteria and/or viruses, such as in a sterile or aseptic manner. Suction assisted lipoplasty may be desirable to remove the adipose tissue from the patient as it provides a minimally invasive method of collecting tissue with minimal risks of stem cell damage that may be associated with other techniques, such as ultrasound assisted lipoplasty.

[00087] For suction-assisted lipoplastic procedures, the adipose tissue is collected by insertion of a cannula into or near an adipose tissue deposit present in the patient or donor subject followed by aspiration of the adipose into a suction device. In one embodiment, a small cannula may be coupled to a syringe, and the adipose tissue may be aspirated using manual force. Using a syringe or other similar device may be desirable to harvest relatively moderate amounts of adipose tissue (e.g., from 0.1 ml to several hundred milliliters of adipose tissue). Procedures employing these relatively small devices have the advantage that the procedures can be performed with only local anesthesia, as opposed to general anesthesia. Larger volumes of adipose tissue (e.g., greater than several hundred milliliters) may require general anesthesia at the discretion of the patient or donor subject and the person performing the collection procedure. When larger volumes of adipose tissue are desired to be removed, relatively larger cannulas and automated suction devices may be employed in the procedure.

[00088] Excisional lipectomy procedures may also be used, which include, but not limited to, procedures in which adipose tissue is removed as an incidental part of the procedure, where the primary purpose of the surgery is the removal of other tissues (e.g., skin in bariatric or cosmetic surgery) and in which adipose tissue is removed along with these tissues of primary interest.

[00089] The amount of adipose tissue collected will be dependent on a number of factors including, but not limited to, the body mass index of the patient or donor subject, the availability of accessible adipose tissue harvest sites, concomitant and pre-existing medications and conditions (such as anticoagulant therapy), and the clinical purpose for which the adipose tissue is being collected. Experience with transplant of hematopoietic stem cells (bone marrow or umbilical cord blood-derived stem cells used to regenerate the recipient's blood cell-forming capacity) shows that engraftment is cell dose-dependent with threshold effects. Thus, it is likely that the general principle that "more is better" will be applied within the limits set by other factors and that where feasible the harvest will collect as much tissue as possible.

[00090] The step of isolating cells (2a) comprising adult stem cells from the retrieved adipose tissue preferably includes depletion of the mature fat-laden adipocytes, blood and blood cells, and/or free lipids from the adipose tissue. This is typically achieved by a series of washing and disaggregation steps in which the adipose tissue is first rinsed to reduce the presence of free lipids (released from ruptured adipocytes) and peripheral blood elements (released from blood vessels severed during tissue harvest), and then disaggregated to free intact adipocytes and other cell populations from the connective tissue matrix. In certain embodiments, the entire adipocyte component, or non-stem cell component, is separated from the stem cell component of the adipose tissue. In other embodiments, only a portion of the adipocyte component is separated from the stem cells. Thus, the isolated cells may comprise stem cells and adipocytes, as well as endothelial precursor cells.

[00091] Rinsing is an optional, but preferred, technique for washing off free lipid and blood with blood cells with a solution, leaving behind intact adipose tissue fragments. In one embodiment, the washing solution is isotonic saline or other physiologic solution(s) (e.g., PLASMALYE®.), of BAXTER® Inc. or NORMSO®. of Abbott Labs). Intact adipose tissue fragments can be separated from the free lipid and blood by any means known to persons of ordinary skill in the art including, but not limited to, filtration, decantation, sedimentation, and centrifugation.

[00092] The intact adipose tissue fragments are then disaggregated using any conventional techniques, including mechanical force (mincing or shear forces), enzymatic digestion with single or combinatorial proteolytic enzymes, such as collagenase, trypsin, lipase, liberase HI, as disclosed in U.S. Patent No. 5,952,215, and pepsin, or a combination of mechanical and enzymatic methods. For example, the intact adipose tissue fragments may be disaggregated by methods using collagenase-mediated dissociation of adipose tissue, similar to the methods for collecting microvascular endothelial cells in adipose tissue, as disclosed in U.S. Patent No. 5,372,945. Additional techniques using collagenase that may be used in the invention are disclosed in U.S. Patent Nos. 5,830,714 and 5,952,215, and by Williams, et al. "Collagenase lot selection and purification for adipose tissue digestion," Cell Transplant , 4(3): 281-9 (1995). Similarly, a neutral protease may be used instead of collagenase, as disclosed in Twentyman and Yuhas, "Use of bacterial neutral protease for disaggregation of mouse tumors and multicellular tumor spheroids," Cancer Lett 9(3): 225-8 (1980). Furthermore, a combination of enzymes may be employed, such as a combination of collagenase and trypsin, as disclosed in Russell et al. "Inflammatory cells in solid murine neoplasms. I. Tumor disaggregation and identification of constituent inflammatory cells," Int J Cancer, 18(3): 322- 30 (1976); or a combination of an enzyme, such as trypsin, and mechanical dissociation, as disclosed in Engelholm et al. "Disaggregation of human solid tumors by combined mechanical and enzymatic methods," Br J Cancer, 51(1): 93-8 (1985).

[00093] In one particular embodiment, the adipose tissue is washed with sterile buffered isotonic saline and incubated with collagenase at a temperature and time sufficient to provide adequate disaggregation. The collagenase may be approved for human use by the relevant authority (e.g., the U.S. Food and Drug Administration). Suitable collagenase preparations include recombinant and non-recombinant collagenase. Non-recombinant collagenase may be obtained from F. Hoffmann-La Roche Ltd, Indianapolis, Ind. and/or Advance Biofactures Corp., Lynbrook, N.Y. Recombinant collagenase may also be obtained as disclosed in U.S. Patent No. 6,475,764.

[00094] In one embodiment, the collagenase is at concentrations from about 10 pg/ml to about 50 pg/ml and are incubated at from about 30° C to about 38° C for from about 20 minutes to about 60 minutes. These conditions may vary according to the source of the collagenase enzyme, optimized by empirical studies, in order to validate that the system is effective at extracting the desired cell populations in the used time frame. A particular preferred concentration, time and temperature is 20 pg/ml collagenase BLENDZYME® (Roche) incubated for 45 minutes, at about 37° C.

[00095] Following collagenase disaggregation, the cell population may be washed/rinsed to remove additives and/or by-products of the disaggregation process (e.g., collagenase and newly-released free lipid). The cell population could then be concentrated by centrifugation or other methods known to persons of ordinary skill in the art. These post-processing wash/concentration steps may be applied separately or simultaneously.

[00096] In one embodiment, the cells are concentrated and the collagenase removed by passing the cell population through a continuous flow spinning membrane system or the like, such as, for example, the system disclosed in U.S. Patent Nos. 5,034,135; and 5,234,608.

[00097] Besides or in addition to enzymatic digestion, a mechanical device/component may be used to disaggregate the adipose tissue, which include cutting the adipose tissue fragments into cells or cell agglomerates that have smaller size. Cutting will also expose lipid fat and blood inside of the tissue fragments, thus permit washing off these components.

[00098] In some embodiments, the disaggregated adipose tissue fragments through enzymatic or mechanical technique, which comprise stem cells, may be directly administered (3) to the patient.

[00099] Optionally, mature adipocytes may be further removed from the disaggregated adipose tissue fragments. Specifically, the suspension of the disaggregated adipose tissue fragments is passed to a cell collection container. The suspension may flow through one or more conduits to the cell collection container by using a pump. Other embodiments may employ the use of gravity or a vacuum while maintaining a closed system. Separation of the cells in the suspension may be achieved by buoyant density sedimentation, centrifugation, elutriation, differential adherence to and elution from solid phase moieties, antibody- mediated selection, differences in electrical charge, immunomagnetic beads, flourescence activated cell sorting (FACS), or other means. Examples various techniques and devices may be found in Hemstreet, et al. "Tissue disaggregation of human renal cell carcinoma with further isopyknic and isokinetic gradient purification," Cancer Res, 40(4): 1043-9 (1980); Schweitzer, "Isolation and culture of human bone marrow endothelial cells," Exp Hematol, 23(1): 41-8 (1995); Gryn, et al. "Factors affecting purification of CD34(+) peripheral blood stem cells using the BAXTER® ISOLEX™ 300i." J Hematother Stem Cell Res , 11(4): 719- 30 (2002); Prince, et al. "ISOLEX™ 300i CD34-selected cells to support multiple cycles of high-dose therapy,” Cytotherapy, 4(2): 137-45 (2002); Watts, et al. "Variable product purity and functional capacity after CD34 selection: a direct comparison of the CLINIMACS® (v2.1) and ISOLEX™ 300i (v2.5) clinical scale devices," Br J Haematol, 118(1): 117-23 (2002); Mainwaring and Rowley "Separation of leucocytes in the dogfish (Scyliorhinus caniculd) using density gradient centrifugation and differential adhesion to glass coverslips," Cell Tissue Res, 241(2): 283-90 (1985); Greenberg and Hammer "Cell separation mediated by differential rolling adhesion." Biotechnol Bioeng, 73(2): 111-24 (2001); and U.S. Patent Nos. 6,277,060; 6,221,315; 6,043,066; 6,451,207; 5,641,622; and 6,251,295.

[000100] In an exemplary embodiment, the cells or cell agglomerates in the suspension are separated from the acellular component in the suspension using a spinning membrane filter.

In other embodiments, the cells or cell agglomerates in the suspension are separated from the acellular component using a centrifuge. In one yet another embodiment, the cell collection container may be a flexible bag that is structured to be placed in a centrifuge (e.g., manually or by robotics). After centrifugation, the cellular component forms a pellet, which may then be resuspended with a buffered solution to form the cells ready to be administered to the patient.

[000101] In another embodiment, the cell suspension may optionally layered over (or under) a fluid material formed into a continuous or discontinuous density gradient and placed in a centrifuge for separation of different cell populations on the basis of cell density. Examples of media suitable for formation of such gradients include PERCOLL® and FICOLL- PAQUE™ (Qian, et al.,“PERCOLL® Density Gradient-Enriched Populations of Rat Pituitary Cells: Interleukin 6 Secretion, Proliferative Activity, and Nitric Oxide Synthase Expression ,” Endocr Pathol, 1998. 9(1): p. 339-346; Lehner, et al.,“Endotoxin-free purification of monocytes for dendritic cell generation via discontinuous density gradient centrifugation based on diluted FICOLL-PAQUE™ Plus,” Int Arch Allergy Immunol, 2002. 128(1): p. 73-6). This embodiment would be capable of separating out certain residual blood cell populations and immature adipocytes (pre-adipocytes) from the stem cell population.

[000102] The isolated cells, in the form of cell suspension, may be administered (3) directly into the patient. In other words, the cells and cell agglomerates (e.g., the stem cells and/or endothelial precursor cells) are administered to the patient without being removed from a closed separation system or exposed to the external environment of the system before being administered to the patient. Providing a closed system reduces the possibility of

contamination of the cells being administered to the patient. Thus, processing the adipose tissue in a closed system provides advantages over existing methods because the isolated cells are more likely to be sterile. In such an embodiment, the only time the stem cells and/or endothelial precursor cells are exposed to the external environment, or removed from the system, is when the cells are being withdrawn into an application device (such as syringe) and being administered to the patient. In one embodiment, the application device can also be part of the closed system thus even this risk may be eliminated (FIG. 2).

[000103] One device to be used in the invention for isolating cells (2a) from the retrieved adipose tissue is described in U.S. Patent Application Publication No. 2013/0123747A1, which allows preparation of cells in the form of cell agglomerates with only mechanical stirring, without using chemicals or physico-chemical treatments, while eliminating most of the oily component from the adipose tissue and with minimized or no contact with the outside environment to reduce the risks of contaminations. The isolated cells so obtained may be administered to the patient. This device is shown in FIG. 2.

[000104] The device comprises at least one washing and separating container having a washing chamber for washing the retrieved adipose tissue. The container has an inlet and an outlet for the retrieved adipose tissue to enter the washing chamber through the inlet and for at least part of unwanted material, particularly the fluid component, to exit the washing chamber through the outlet. The washing chamber including a stirring mechanism for mechanically forming an emulsion of fluid components, on which the cells will float, being separated from the liquid component.

[000105] The stirring mechanism may be of any type, particularly of active or passive type. An active stirring mechanism is motorized, driven by a motor or a motive force to provide the stirring movement. A passive stirring mechanism exerts its action upon stirring of the container, and hence operate, for example, by inertia. Preferably, the mechanical stirring action of the container consists in a rotation of the container about an axis, e.g. a longitudinal axis, perpendicular to the end surfaces of the container, and either external or internal to the container. Other kinds of stirring may be also provided, such as shaking of the container or the like.

[000106] The stirring mechanism for forming an emulsion by simple manual or possibly mechanical stirring of the washing and separating container, without using chemicals or enzymes that might lead to disintegration of the cell material, afford separation of the solid component from the liquid component in the washing chamber, and particularly allow the solid component comprising cells and cell agglomerates to float on an emulsion of liquid substances that include blood, sterile solutions and oil yielded from broken fat cells. The floated cells and cell agglomerates may then be collected.

[000107] In a preferred embodiment, the solid component is washed in the washing chamber by introducing a sterile washing solution, e.g. a sterile saline, once or multiple times into the washing and separating container through the inlet. With stirring of the container, this washing solution allows the cells and cell agglomerates to be cleaned of any waste liquid, such as blood and oil yielded from broken fat cells.

[000108] The device further includes size reducing container, which may be in the washing and separating container or outside of, but adapted to be fluid-tightly connected to the washing and separating container. The size reducing container has a mechanism for reducing the size of the solid component of the adipose tissue, particularly cell macroagglomerates, to averagely equal smaller cell agglomerates. The mechanism can comprise at least one series of parallel or intersecting sheets or cutting wires, to form at least one size reducing net, through which the adipose tissue is passed.

[000109] In a preferred embodiment, the size reduction of the adipose tissue occurs before washing, with a first size reduction net, through which the adipose tissue is forced before entering the washing chamber of the washing and separating container, and a second size reduction occurs with a second size reducing net, which size reduction is performed after at least one washing step in the washing and separating container. Preferably, the second size reducing net has narrower meshes than the first size reducing net.

[000110] At least one filter may be provided in the washing chamber of the washing and separating container proximate to the outlet which allows the passage of at least part of the liquid phase, consisting of an oily component, a blood component, sterile solutions such as anesthetic liquid and/or saline and retains the solid cell phase, consisting of cells and cell agglomerates, thereby allowing further separation of the liquid phase of the emulsion from the solid phase.

[000111] The filter may consist of a net of fine meshes, which is smooth, i.e. with no projecting parts or irregular or sharp surfaces that might damage cell walls, whose meshes or through interstices are smaller than the cell agglomerates contained in the washing chamber of the washing and separating container. The meshes of the net that forms the selectively permeable membrane may have a size ranging from 1 pm to 50 pm.

[000112] In another embodiment, the device described above may be used in conjunction with chemical and/or physical treatment of the solid component obtained by the above device, as disclosed in U.S. Patent Application Publication No. 2015/0030571A1. The chemical and/or physical treatment step may allow to have a cell preparation rich in or only composed of mesenchymal stem cells, and/or to induce the development of mesenchymal stem cells and/or differentiation of mesenchymal stem cells into a population of cells of interest depending on the tissue or organ to be regenerated or repaired. [000113] The chemical and/or physical development can be carried out before administering to the patient, or it can be carried out in situ , that is after the administering the isolated cells to the patient, by stimulating the development and/or differentiation of the stem cells directly in the tissue or organ targeted by the treatment.

[000114] The physical treatment can be a treatment of the cells by sound energy, light energy or the like. For example, it can be placed in radiofrequency fields of Wi-Fi range (Radio Electric Conveyed Fields) conveyed by suitable apparatus (REAC Radio Electric

Asymmetric Conveyer). These physical stimuli initiate the differentiation of the stem cells into one or more types of cells of interest.

[000115] Another type of physical treatment is subjecting the cells to stress such as freezing, hypoxia and/or centrifugation, preferably pusher centrifugation. These physical treatments, which can be used alone or in combination, cause the adipocytes to break, with the formation of oil that can be easily removed for example by the device described above, but not the stem cells that therefore remain in the solid component and they allow a cells preparation to be obtained substantially composed only of mesenchymal stem cells.

[000116] Another type of chemical and/or physical treatment is for promoting the stem cells differentiation, in vitro or in situ , into types of interest depending on the tissue or organ to be treated. For example it is possible to differentiate the stem cells to cardiovascular cells by a treatment with esters or mixtures of hyaluronic acid with butyric acids, or other histone deacetylase inhibitors, and retinoic acids.

[000117] The cells comprising stem cells treated chemically and/or physically can be directly administered to the patient for performing tissue repairing or regeneration. The cells may also be used for enriching the so-called scaffolds i.e. biocompatible supports, currently available on the market and for generating an integrated system of scaffolds/stem cells from

preconditioned adipose tissue before the implantation or conditioned in situ , by chemical and/or physical stimuli. According to a further variant from the chemically treated cells it is possible to select only the stem cells, without arriving to a cell expansion, to be used for transplantation, with or without integration on biocompatible supports.

[000118] The selection of stem cells can be performed by the digestion of the solid cell component for forming a cell suspension having the stem cells and cells of non-interest (e.g., adipocytes) that can be eliminated by washing or chemical treatment. For example, the physical treatment can eliminate the adipocytes and a cell preparation enriched with mesenchymal stem cells to be obtained. [000119] The device used to isolate cells from the adipose tissue can be integrated into a closed system such that the adipose tissue from the donor to the administering to the patient has no or minimal contact with outside environment, as described in U.S. patent application publication 2017/0121666A1. This integrated system may be used in the present invention.

[000120] The washing and separating container has an inlet, via a two-way connection, to a saline vessel and vessel containing the retrieved adipose tissue, respectively. The outlet of the washing and separating container is connected via a two-way connection to a waste product vessel and a collection vessel for the isolated cells respectively. Preferably the two-way connections are equipped with self-closing valves. The two-way connections allow two different inlet or outlet ports to be integrated into a single connection.

[000121] In one embodiment, a syringe containing the retrieved adipose tissue is connected to the inlet. The self-closing valve allows the adipose tissue to be introduced into the washing and separating container, but as soon as the syringe is disconnected, the self-closing valve closes to prevent exit of the adipose tissue and to seal the inner environment of the washing and separating container.

[000122] The washing and separating container and the connections with their self-closing valves may be connected by screw, interlocking, welding or chemical bonding arrangements, or by complex connections, as needed, provided that they can withstand with appropriate safety margins the mechanical and pressure stresses exerted thereon during treatment and extraction of the adipose tissue being processed. In one embodiment, the washing and separating container is connected to the saline vessel via pumping mechanism for controlling the washing flow.

[000123] Thus, the integrated system, through the operation of the self-closing valves, allows introduction of adipose tissue into the washing and separating container from the inlet and removal thereof from the outlet. The washing and separating container is connected to the waste fluid collection vessel through a solenoid-operated pinch valve. The solenoid-operated pinch valve may be electronically controlled to open and close the connecting tube according to the process steps, and be synchronized and cooperate with the other actuators of the apparatus, with time intervals decided by the user or resulting from the processing operation of the process control unit.

[000124] Optionally, a pumping mechanism may be advantageously used in combination with the solenoid-operated pinch valve, such that the discharge passage is closed and the only available passage for the isolated cells is the connector with the collecting syringe.

[000125] The operation of this closed system includes the steps of: a) filling the washing chamber of the washing and separating container with the saline contained in the vessel. The saline fills the entire washing chamber, thereby completely removing air through the inlet and the two-way connection.

b) introducing the adipose tissue into the washing chamber. A syringe is introduced into one of the two ports of the two-way connection and the adipose tissue in the syringe is pushed into the washing chamber through the inlet. In some cases, a plurality of syringes may be used to introduce a larger amount of adipose tissue.

c) stirring the washing and separating container to facilitate emulsion of fluid components, particularly the oily component with the sterile fluid substances, by the provision of a stirring mechanism for generating an emulsion of the fluid components. d) placing the washing and separating container in such a position as to obtain a stratification of the solid components on the liquid emulsion. Particularly, the container is in such a position that the container has its longitudinal axis parallel to a vertical axis.

Thus, the washing chamber has a solid component composed of cells and one or more cell agglomerates floating on an emulsion of the fluid components in the lower portion of the washing chamber in contact with the outlet of the washing and separating container.

e) injecting saline and withdrawing the solid component for administering to the patient.

[000126] Step c) is carried out in an automated fashion, the washing and separating container being coupled to stirring mechanism, as described above. Steps a), c), d) and e) are preferably also automated.

[000127] The closed system, such as the LIPOGEMS® technology, improves and optimizes the natural properties of adipose tissue. Without the use of enzymes, additives, or separation centrifugation and relying instead on mild mechanical forces, the system yields a

microfragmented autologous adipose tissue that acts as a large-scale tool to supply damaged tissues with a regenerative environment. The availability of minimally manipulated products having adequate adult stem cells allows for shorter procedure times, avoids regulatory constraints, and enables autologous grafting in a one-step intervention.

[000128] The isolated cells may be administered (3) to the patient immediately or

administered to the patient without any significant delay. In one embodiment, at least a portion of the isolated cells may be stored for later administering. The cells may be divided into multiple aliquots or units such that part of the population of stem cells and/or endothelial precursor cells is retained for later administering while part is administered immediately to the patient. Moderate to long-term storage of all or part of the isolated cells in a cell bank is also within the scope of this invention.

[000129] In another aspect, the platelet rich plasma is prepared from whole blood, which is retrieved (lb) from the patient (autologous source) or a subject acting as blood donor. The blood may be from a single source (patient or the subject), or a pooled source of multiple donor subjects. The blood sample may be retrieved (lb) using a blood collection syringe. The amount of blood collected may depend on a number of factors, including, for example, the amount of platelet rich plasma desired, the health of the patient, the severity or location of the tissue damage, and the availability of pre-prepared platelet rich plasma.

[000130] Platelets are non-nucleated blood cells that are found in bone marrow and peripheral blood. They have several important functions such as controlling bleeding and tissue healing because their ability to produce many growth factors including platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), fibroblast growth factor (FGF), insulin like growth factor-1 (IGF-1), connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF).

[000131]Blood sample(s) from the patient or donor subject can be retrieved (lb) as needed or can be obtained hours, days, or several weeks in advance of the treatment, based on blood storage and preservation methods as generally practiced in the art.

[000132] The blood sample may be mixed with an anticoagulant prior to preparation of the platelet rich plasma or at one or more points during preparation. Suitable anticoagulants include those known in the art, such as heparin, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), acid citrate dextrose solution (ACD), and mixtures thereof. The anticoagulant may include a chelating agent (e.g., citrate, EDTA). For example, the anticoagulant may be placed in a syringe used for drawing blood from the subject, or may be mixed with the blood after it is drawn.

[000133] Platelet rich plasma can be isolated (2b) from the blood sample by one or more techniques including filtration, and density fractionation methods such as centrifugation of whole blood, centrifugation of blood in multiple stages, and continuous-flow centrifugation. For example, platelet rich plasma may be prepared by the methods as described in Whitman, et al. (1997) J Oral Maxillofac Surg 55: 1294-1299 and Marx, et al. (1998) Oral Surg Oral Med Pathol Oral Radiol 85: 638-646. Several simplified protocols for the preparation of platelet rich plasma are also known. See, Landesberg, et al. (2000) J Oral Maxillofac Surg 58: 297; Efeoglu, et al. (2004) J Oral Maxillofac Surg 61(11): 1403-7; and Nagata, et al. (2010) European Journal of Dentistry 4: 395-402. Further, neutrophil-depleted platelet rich plasma may be obtained using the methods described in U.S. Patent No. 8,142,993; European Patent Application EP1547606; U.S. Patent Application Publication No. 2005/0170327; and U.S. Patent No. 8,105,495.

[000134] In one embodiment, the blood sample may be centrifuged using a gravitational platelet system, such as the Cell Factor Technologies GPS System® centrifuge. The blood- filled syringe containing between about 20-150 mL of blood (e.g., about 55 mL of blood) and about 5 mL citrate dextrose may be slowly transferred to a disposable separation tube which may be loaded into a port on the GPS centrifuge. The blood sample may be capped and placed into the centrifuge. The blood sample is spun in the centrifuge to separate platelets from blood and plasma. The blood sample may be spun at about 2000-5000 rpm for about 5- 30 minutes. For example, centrifugation may be performed at 3200 rpm for extraction from a side of the separation tube and then isolated platelet rich plasma may be extracted from a side port using, for example, a 10 mL syringe or pipette. From about 55 mL of blood, about 5 mL of platelet rich plasma may be obtained.

[000135] Generally, at least 60% or at least 80% of the available platelets within the blood sample can be captured using centrifuge. These platelets may be resuspended in a volume that may be about 3-20% or about 5-10% of the blood sample volume.

[000136] Another exemplary device that may be used in isolating platelet rich plasma (2b) by density fractionation includes a centrifugal drum separator and an erythorocyte capture trap. In one embodiment, the walls of the centrifugal drum separator are coated with a depth filter having pores and passageways that are sized to receive and entrap erythrocytes. Blood is placed in the centrifugal drum, and the drum is spun along its axis at sufficient speed so as to force erythrocytes from the blood into the depth filter. After spinning, the erythrocytes remain in the filter and the remaining platelet rich plasma is extracted. The platelet rich plasma may be concentrated by desiccation. Embodiments of such devices include the Vortech™ Concentration System (Biomet Biologies, Inc., Warsaw, Ind.), and are disclosed in U.S. Patent Application Publication Nos. 2006/0175244A1 and 2006/0175242A1.

[000137] In some embodiments, to minimize the possibility of contamination, the retrieving (lb) of blood sample and isolating (2b) of platelet rich plasma may be formed using a closed circuit.

[000138] The concentration of platelets within the platelet rich plasma may vary. For example, in some embodiments, the platelet concentration in the platelet rich plasma can be from about 3 -fold to about 10-fold greater than the platelet concentration in the blood sample (which is whole blood). Furthermore, the platelet rich plasma can contain cytokines, growth factors, and other proteins and molecules in addition to those contained within the platelets.

[000139] In some embodiments, a platelet activator may be added to the platelet rich plasma. The platelet activator serves to release the growth factors within the platelets in the platelet rich plasma. Platelet activators may include thrombin, calcium chloride, collagen, epinephrine, adenosine diphosphate and mixtures thereof. Activation of the platelets in platelet rich plasma by the platelet activator can occur just prior to administration of the platelet rich plasma to the patient, concomitant with administration the platelet rich plasma to the patient, or following administration of the platelet rich plasma to the patient.

[000140] The activated platelets release cytokines, such as IL-IB, IL-6, TNF-A, chemokines, optionally ENA-78 (CXCL5), IL-8 (CXCL8), MCP-3 (CCL7), MAP- 1 A (CCL3), NAP-2 (CXCL7), PF4 (CXCL4), RANTES (CCL5), inflammatory mediators, optionally PGE2, and growth factors, optionally Angiopoitin-1, bFGF, EGF, FGF, HGF, IGF -I, IGF -II, PDAF, PDEGF, PF-4, PDGF AA and BB, TGF-beta 1, 2, and 3, and VEGF.

[000141] However, exogenous or extra activators may not need to be administered to the patient, because collagen, a major component of connective tissues, is a strong activator of platelets. Thus, when the platelet rich plasma is introduced into and/or around a connective tissue, platelets in the platelet rich plasma may bind to the collagen and be activated. This reduces or eliminates the need for administering an exogenous activator (e.g., thrombin). Exogenous activators may still be employed if a physician determines that they are medically necessary or desirable.

[000142] In some embodiments, an angiogenic factor may be added to the platelet rich plasma. Angiogenic factors can include angiogenin, angiopoietin-1, del-1 protein, fibroblast growth factors such as acidic FGF (also known as aFGF or FGF-1) and basic FGF (also known as bFGF or FGF-2), follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), interleukin-8 (IL-8), leptin, midkine, placental growth factor, platelet-derived endothelial growth factor (PD-ECGF), platelet-derived growth factor (PDGF), pleiotrophin (PTN), progranulin, proliferin, transforming growth factor alpha (TGF- a), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a), vascular endothelial growth factor (VEGF), and vascular permeability factor (VPF). In various embodiments, isolated, recombinant, and/or synthetic angiogenic factors may be used.

Angiogenic factors can be applied to the site just prior to the administration of the platelet rich plasma, concomitant with administration the platelet rich plasma, or following administration of the platelet rich plasma to the patient. [000143] In another embodiment, one or more cytokines may be added to the platelet rich plasma. Cytokines may be selected platelet-derived growth factor, transforming growth factor beta, fibroblast growth factor, insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin 8 (IL-8), keratinocyte growth factor (KGF), and connective tissue growth factor.

[000144] In one embodiment, the platelet rich plasma is subjected to a freeze-thawing treatment. For example, the platelet rich plasma is frozen at a temperature of at least -10° C, specifically at least -25° C, specifically at least -30° C, specifically at least -35° C, preferably in the absence of any cryoprotectant, for a sufficient time period needed for platelet lysis, specifically for at least 15 minutes, specifically at least 20 minutes, specifically at least 30 minutes, specifically at least 45 minutes, specifically 1 hour, specifically for at least 1 .5 hours, specifically for at least 2 hours. Specifically, the freezing temperature is higher than - 120° C, specifically higher than -85° C, specifically higher than -80° C, specifically it is up to -80° C, -70° C, -60° C, -50° C, -40° C, -35° C, or -30° C.

[000145] Freezing is followed by a thawing process at room temperature, specifically at 25°

C, or about 27.5° C, or about 30° C, or about 35° C, or about 37.5° C.

[000146] Optionally, the platelet rich plasma may be repeatedly treated by freeze-thawing cycles, specifically said cycles can be repeated two times, three-times, four-times, five-times or more until the desired lysis of platelets is reached.

[000147] The platelet rich plasma may be admixed with a solution, buffer, diluent, solvent, or stabilizer to form a composition. Examples of the additional agents include, but are not limited to, thrombin, epinephrine, collagen, calcium salts, pH adjusting agents, materials to promote degranulation or preserve platelets, additional growth factors or growth factor inhibitors, NSAIDS, steroids, anti- infective agents, and mixtures of the foregoing.

[000148] The platelet rich plasma may be buffered using an alkaline buffering agent to a physiological pH. The buffering agent may be a biocompatible buffer such as HEPES, TRIS, monobasic phosphate, monobasic bicarbonate, or any suitable combination thereof that may be capable of adjusting the platelet rich plasma to physiological pH between about 6.5-8.0. The physiological pH is from about 7.3-7.5 (e.g., about 7.4). For example, the buffering agent may be an 8.4% sodium bicarbonate solution. For each mL of platelet rich plasma isolated from whole blood, 0.05 mL of 8.4% sodium bicarbonate may be added. The syringe may be gently shaken to mix the platelet rich plasma and bicarbonate.

[000149] In some embodiments, an excipient may be added to the platelet rich plasma.

Suitable excipients include isotonic sodium chloride solution, physiological saline, phosphate buffered saline, potassium buffered saline, normal saline, dextrose 5% in water, dextrose 10% in water, Ringer solution, lactated Ringer solution, Ringer lactate, or Ringer lactate solution. Other compounds may be added to the platelet rich plasma, for example, medically inert ingredients (e.g., solid and liquid diluent), such as lactose, dextrosesaccharose, cellulose, starch, calcium phosphate, olive oil, ethyl oleate, water, or vegetable oil; lubricating agents such as silica, talc, stearic acid, magnesium or calcium stearate and/or polyethylene glycols; gelling agents such as colloidal clays; thickening agents such as gum tragacanth or sodium alginate; and other therapeutically acceptable accessory ingredients, such as humectants, preservatives, buffers and antioxidants.

[000150] The platelet rich plasma can be stored in cryostorage and be used for the patient at a later date. For example, the platelet rich plasma may be stored and be readily available to treat the patient for a multitude of injuries (e.g., orthopedic, post myocardiacal infarction).

The platelet rich plasma may be stored at +4° C, -20° C, or -70° C.

[000151] The platelet rich plasma may have four forms that are all ready for administering to the patient: supernatant, liquid, clot and fibrin membrane for different applications. For example, supernatant and liquid forms may be used in fields such as ophthalmology. The liquid form may also be applied to wet surfaces, infiltrations in traumatology or dermatology. The clot may be used with biological scaffold to form autologous and heterologous grafts.

The elastic fibrin membrane may be embedded with growth factors.

[000152] The isolated cells from the adipose tissue and platelet rich plasma from the blood sample can be administered (3) to the patient at the location where the wounded or damaged tissue is to be healed. In one embodiment, the isolated cells and platelet rich plasma are mixed and then administered (3) to the patient as one composition. In another embodiment, the isolated cells are administered (3) to the patient at the location, followed by administering (3) the platelet rich plasma to the same location. In yet another embodiment, the platelet rich plasma is administered (3) to the patient at the location, followed by administering (3) the isolated cells to the same location. In yet another embodiment, the isolated cells and platelet rich plasma are mixed and administered (3) to the patient, followed by one or more administering (3) of the platelet rich plasma to the patient.

[000153] The volume ratio between the isolated cells and platelet rich plasma being administered to the patient may be from about 1 : 10 to about 10: 1, or from about 1 :5 to about 5: 1, or from about 1 :2 to about 2: 1, or about 1 : 1.

[000154] The amount of isolated cells administered to the patient may be in the range of from about 0.5 mL to about 9 mL, or from about 0.7 mL to about 7 mL, or from about 1 mL to about 5 mL, or from 1.5 mL to about 3mL. This amount is dependent on the type of wound or injury being treated, as well as the location of the wound or injury.

[000155] The amount of platelet rich plasma administered to the patient may be in the range of from about 0.5 mL to about 15 mL, or from about 0.7 mL to about 13 mL, or from about 1 mL to about 11 mL, or from 1.5 mL to about 10 mL, or from 2 mL to about 8 mL, or from 3 mL to about 6 mL. This amount is dependent on the type of wound or injury being treated, as well as the location of the wound or injury.

[000156] In one embodiment, the isolated cells and platelet rich plasma are used for pain management by being administered (3) in either joints or soft tissue in the painful area of the patient. In some cases, the area may not be painful but may be injured or have osteoarthritis and other symptoms besides pain. The cause of the pain may be arthritis, inflammation, tendinopathy of tendons, or partial or full thickness tears of ligaments, tendons or other soft tissue damages.

[000157] In one embodiment, the patient receives a first initial treatment of combined injection of the isolated cells and platelet rich plasma. The patient may be examined after the initial treatment at approximately 6 weeks (2-8 weeks), 3 months (2-4 months), 6 months (5-7 months) and at 12 months (11-13 months) post-treatment or within 4 weeks of this timeframe. The patient may not require any further injections. But if the examination determine that further injections are necessary, the patient may receive up to three further injections of just platelet rich plasma at a variable interval throughout the 12 months post initial injection. Depending on the type of wound or injury, as well as the location of the wound or injury, injections may be given 1 to 2 weeks apart or spread over the course of the 12 months.

[000158] The examinations or consultations are for assessment of the effectiveness of the treatment. The effectiveness may be quantified by using a variety of scoring systems known in the art, such as VAS scoring for pain, OXFORD knee scoring for pain. The effectiveness of the treatment may also be assessed by objective measures including Visual Analog Scale joint specific outcome measure and quality of life measures. The examinations or

consultations of the patient which will occur either face-to-face or remotely via voice or video calls between the clinician and the patient.

[000159] The isolated cells and platelet rich plasma may be administered to the patient by injection using a syringe or catheter. The isolated cells and platelet rich plasma may also be delivered via other delivery device such as a dermal patch, a spray device, sutures, stents, screws, plates, or some other implantable medical device such as bioresorbable tissue patch. [000160] The isolated cells and platelet rich plasma may be administered in any of the following routes: epicutaneous, infusion, intraarterial, intracardial, intradermal,

intramuscular, intraperitoneal, intrathecal, intravenous, oral, parenteral, pulmonary, rectally via an enema or suppository, subcutaneous, subdermal, sublingual, topically, via

microneedles, and transdermal. The administration can be local, where the isolated cells and platelet rich plasma are administered directly, close to, in the locality, near, at, about, or in the vicinity of, the site of disease or injury. Administration can be topical with a local effect, where the isolated cells and platelet rich plasma are applied directly to the site of disease or injury.

[000161] In some embodiments, the isolated cells and platelet rich plasma may be used for treatment of numerous diseases or symptoms thereof, including, and not limited to, bone- related disorders, diseases, or injuries, including slow/non-union fractures, osteoporosis (age- related or chemotherapy -induced), inherited diseases of bone (osteogenesis imperfecta); adipose related disorders or diseases; liver related diseases, disorders, or injuries, including liver failure, hepatitis B, and hepatitis C; myocardial infarctions, including heart attack or chronic heart failures; renal diseases or kidney damage; retinal diseases or damage or necrosis; wound healing (e.g., from surgery or diabetic ulcers); skeletal muscle disorders both traumatic and inherited; cartilage and joint repair both traumatic and autoimmune; lung injuries; diabetes; intestinal disorders; nervous system disorders, diseases, or injuries, such as central nervous systems disorders, diseases, or injuries, including spinal cord injuries, Parkinson's disease, Alzheimer's disease, and stroke.

[000162] In one embodiment, the isolated cells and platelet rich plasma may be used for treatment of tissue injury. The injury may be a shoulder injury, optionally rotator cuff tendinitis or tear, rotator cuff impingement syndrome or bursitis, bicipital tendinitis, labrum tears, arthritis, and instability. In another embodiment, the injury may be a wrist hand injury optionally de quervain's tenosynovitis, arthritis, other wrist or finger tendinitis, ligament tears, or dysfunction of the fingers. In another embodiment, the injury may be an elbow injury, optionally medial and lateral epicondylitis (tennis & golfers elbow). In another embodiment, the injury may be hip injury, optionally iliotibial band tendinitis (ITB

Syndrome), psoas tendinitis and bursitis, greater trochanteric bursitis, hip labrum tears, piriformis syndrome, sacroiliac joint dysfunction, and arthritis. In another embodiment, the injury may be a knee injury, optionally patellar tendinitis, patellar femoral syndrome, chondromalacia patella, partially torn or strained major ligaments of knee (ACL/LCL/MCL), meniscus tears, arthritis, and patellar instability. In another embodiment, the injury may be an ankle/foot injury, optionally Achilles tendinitis, peroneal tendinitis, arthritis, recurrent ankle sprains, other foot or ankle tendinitis. In another embodiment, the injury may be a neck injury optionally whiplash injuries, headaches related to the neck, and arthritis. In another embodiment, the injury may be a back injury, optionally facet joint arthritis, rib injury, or pain associated with scoliosis. In another embodiment, the injury may be the result of gum recession, loss of bone, including the jaw, bone fractures, dermal treatment for burns and non-healing wounds, post-laser treatment bums, enterocutaneous fistula, gingival gum regeneration, hair loss (in both men and women), orthopedic problems, osteoarthritis, plantar fasciitis, recto-vaginal fistula, rotator cuff injuries, sports medicine injuries, optionally tears and sprains of the ligaments and tendons, tennis elbow, ulcers, and non-healing wounds.

[000163] In one embodiment, the isolated cells and platelet rich plasma may be used for treating tissue damage as result of gastrointestinal and general surgery, such as Fistulas surgery, Pilonidal Fistulas surgery and surgery for Fecal incontinence; or for wound healing such as healing of complex lesions, chronic ulcers and diabetic foot ulcers; or for GYN diseases such as lichen and vaginal atrophy; or for orthopaedics such as osteoarthritis, knee, hip, shoulder, ankle chondral lesions and tendinopathies; or for pain management such as chronic back pain, chronic pelvic pain and chronic joint pain; or for injury as result of plastic and reconstructive surgery such as scarring, burns and mandibular reconstruction.

[000164] In some embodiments, the isolated cells and platelet rich plasma may be used for cosmetic purposes, such as skin repair, skin regeneration, skin protection, preventing hair loss, for promoting the physiological growth of hair, reducing photoaging, reducing skin oxidative stress, regenerating the hair bulb cells and scalp. In one embodiment, the isolated cells and platelet rich plasma may be used for reducing wrinkles, improving rosacea, increasing skin thickness, increasing skin tome, improving skin texture, and tightening skin.

[000165] In some embodiments, the isolated cells and platelet rich plasma may be used in combination with a device, implant, or biocompatible polymer. For example, the isolated cells and platelet rich plasma may be used as part of medical devices, including implantable and non-implantable medical devices.

[000166] The isolated cells and platelet rich plasma may be placed into the patient surrounded by a resorbable plastic sheath such as that manufactured by MacroPore Biosurgery, Inc. U.S. Patent Nos. 6,269,716 and 5,919,234. In this setting the sheath would prevent prolapse of muscle and other soft tissue into the area of a bone fracture thereby allowing the emplaced isolated cells and platelet rich plasma to promote repair of the fracture. [000167] Biocompatible polymers include but are not limited to, homopolymers, copolymers, block polymers, cross-linkable or crosslinked polymers, photoinitiated polymers, chemically initiated polymers, biodegradable polymers, and nonbiodegradable polymers. In some embodiments, the biocompatible polymers form a polymer matrix that is nonpolymerized, to allow it to be combined with a tissue, organ, or engineered tissue in a liquid or semi-liquid state, for example, by injection. In other embodiments, the biocompatible polymers form a liquid matrix that may polymerize or substantially polymerize in situ. In still other embodiments, the isolated cells and platelet rich plasma may be admixed with the polymer, polymerized or substantially polymerized prior to the administration.

[000168] The polymerized or nonpolymerized matrix may comprise collagen, including but not limited to contracted and non-contracted collagen gels, hydrogels comprising, for example, but not limited to, fibrin, alginate, agarose, gelatin, hyaluronate, polyethylene glycol (PEG), dextrans, including dextrans that are suitable for chemical crosslinking,

photocrosslinking, or both, albumin, polyacrylamide, polyglycolyic acid, polyvinyl chloride, polyvinyl alcohol, poly(n-vinyl-2-pyrollidone), poly(2 -hydroxy ethyl methacrylate), hydrophilic polyurethanes, acrylic derivatives, or pluronics (e.g., polypropylene oxide and polyethylene oxide copolymer.) The fibrin or collagen is autologous or allogeneic with respect to the intended recipient. The skilled artisan will appreciate that the matrix may comprise non-degradable materials, for example, but not limited to, expanded

polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), polyethyleneterephthalate (PET), polyurethane, polyethylene, polycabonate, polystyrene, silicone, or selectively degradable materials (e.g., poly (lactic-co-glycolic acid; PLGA), PLA, or PGA).

[000169] It is to be understood, however, that even though numerous characteristics and advantages of the present invention have been set forth in the foregoing description, together with details of the structure and function of the invention, the disclosure is illustrative only, and changes may be made in detail, especially in matters of shape, size and arrangement of parts within the principles of the invention to the full extent indicated by the broad general meanings of the terms in which the appended claims are expressed.

[000170] The applicant(s) do not intend to dedicate any disclosed embodiments to the public, and to the extent any disclosed modifications or alterations may not literally fall within the scope of the claims, they are considered to be part hereof under the doctrine of equivalents.

Claims

CLAIMS:

1. A method of treating a wounded or damaged tissue of a patient, comprising steps of:

retrieving an adipose tissue (la) from the patient or a first donor subject compatible with the patient;

retrieving a blood sample (lb) from the patient or a second donor subject compatible with the patient;

isolating cells (2a) comprising stem cells from the adipose tissue;

isolating platelet rich plasma (2b) from the blood sample; and administering (3) the isolated cells and the platelet rich plasma to the patient.

2. The method of claim 1, wherein one or both of the blood sample and the adipose tissue are retrieved from the patient.

3. The method of claim 1, wherein the first donor subject and the second donor subject are the same subject.

4. The method of any one of claims 1-3, wherein the step of retrieving the adipose tissue (la) employs suction-assisted lipoplasty, ultrasound-assisted lipoplasty, excisional lipectomy, or combinations thereof.

5. The method of any one of claims 1-3, wherein the step of isolating cells (2a) comprises washing the retrieved adipose tissue with a washing solution.

6. The method of claim 5, wherein the washing solution is an isotonic saline solution.

7. The method of any one of claims 1-3, wherein the step of isolating cells (2a) comprises disaggregating the adipose tissue.

8. The method of claim 7, wherein the disaggregating employs mechanical cutting and/or enzymatic digestion.

9. The method of claim 8, wherein the mechanical cutting employs one or more cutting wire and cutting net.

10. The method of claim 8, wherein the enzymatic digestion employs a proteolytic enzyme selected from the group consisting of collagenase, trypsin, lipase, and liberase HI.

11. The method of any one of claims 7-10, wherein the step of isolating cells (2a) further comprises washing the disaggregated adipose tissue.

12. The method of any one of claims 7-11, wherein the step of isolating cells (2a) further comprises emulsifying the disaggregated adipose tissue.

13. The method of claim 12, wherein the emulsifying employs mechanical stirring.

14. The method of any one of claims 7-13, wherein the step of isolating cells (2a) further comprises filtering the disaggregated adipose tissue.

15. The method of any one of claims 1-14, wherein the isolated cells comprise individual cells and cell agglomerates.

16. The method of any one of claims 1-14, wherein the retrieved blood sample is mixed with an anticoagulant.

17. The method of claim 16, wherein the anticoagulant is selected from the group consisting of heparin, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), acid citrate dextrose solution (ACD).

18. The method of any one of claims 1-17, wherein the step of isolating platelet rich plasma (2b) employs a technique selected from a filtration and a density fractionation technique.

19. The method of claim 18, wherein the density fractionation technique comprises centrifugation or continuous-flow centrifugation.

20. The method of any one of claims 1-19, further comprising a step of adding a platelet activator to the isolated platelet rich plasma.

21. The method of claim 20, wherein the platelet activator is selected from the group consisting of thrombin, calcium chloride, collagen, epinephrine, and adenosine diphosphate.

22. The method of any one of claims 1-21, further comprising a step of freezing- thawing the isolated platelet rich plasma.

23. The method of claim 22, wherein the freezing-thawing step is repeated at least two times, or at least three time, or at least four times, or at least five times.

24. The method of any one of claims 1-23, wherein the isolated platelet rich plasma is in a suspension with a buffer, a diluent, a solvent or a stabilizer.

25. The method of claim 24, wherein the isolated platelet rich plasma is suspended in a buffer having a pH between 6.5-8.0, or between 7.0-7.5.

26. The method of any one of claims 1-25, wherein the isolated platelet rich plasma is in a form selected from supernatant, liquid, clot and fibrin membrane.

27. The method of any one of claims 1-26, wherein the administering step (3) comprises mixing the isolated cells and the platelet rich plasma to produce a composition, followed by administering the composition to the patient.

28. The method of claim 27, wherein the administering step (3) further comprises, after administering the composition, one or more times of administering the platelet rich plasma to the patient.

29. The method of any one of claims 1-26, wherein the administering step (3) comprises administering the isolated cells, followed by administering the platelet rich plasma.

30. The method of any one of claims 1-26, wherein the administering step (3) comprises administering the platelet rich plasma, followed by administering the isolated cells.

31. The method of any one of claims 1-30, wherein the platelet rich plasma and isolated cells in the administering step (3) have a volume ratio of from about 1 : 10 to about 10: 1, or from about 1 :5 to about 5: 1, or from about 1 :2 to about 2: 1, or about 1 : 1.

32. The method of any one of claims 1-31, wherein the platelet rich plasma in the administering step (3) has a volume in a range of from about 0.5 mL to about 15 mL, or from about 0.7 mL to about 13 mL, or from about 1 mL to about 11 mL, or from 1.5 mL to about 10 mL, or from 2 mL to about 8 mL, or from 3 mL to about 6 mL.

33. The method of any one of claims 1-31, wherein the isolated cells in the administering step (3) has a volume in a range of from about 0.5 mL to about 9 mL, or from about 0.7 mL to about 7 mL, or from about 1 mL to about 5 mL, or from 1.5 mL to about 3mL.

34. The method of any one of claims 1-33, further comprises a step, after the administering step (3), examining the patient to assess effectiveness of the treatment.

35. The method of claim 34, wherein the examining step is performed multiple times at about 2-8 weeks, about 2-4 months, about 5-7 months and about 11-13 months post the administering step (3).

36. The method of claim 35, wherein the examining step is used to determine the necessity of additional administering of the platelet rich plasma.

37. The method of any one of claims 1-36, wherein the wounded or damaged tissue is a result of injury or aging.

38. The method of claim 37, wherein the injury is caused by physical activity.

39. The method of claim 37, wherein the injury is caused by a disease selected from liver failure, hepatitis B, hepatitis C, myocardial infarctions, renal diseases, kidney damage, retinal necrosis, diabetes, Parkinson's disease, Alzheimer's disease, and stroke.

40. The method of claim 37, wherein the wounded or damaged tissue is a result of aging selected from skin wrinkles, hair loss, loose skin, and thinned skin.

41. The method of any one of claims 1-40, wherein the administering step (3) is performed by injecting the isolated cells and the platelet rich plasma directly into the wounded or damaged tissue of the patient.

42. The method of any one of claims 1-41, wherein an effective amount of the isolated cells and the platelet rich plasma are administered to the patient in the administering step (3).

43. The method of any one of claims 1-42, wherein the patient is a human.

44. The method of any one of claims 1-43, wherein the patient, the first donor subject and the second donor subject are humans.