MXPA05013967A - Methods of using adipose tissue-derived cells in augmenting autologous fat transfer - Google Patents

Abstract

Methods of treating patients for conditions such asbreast augmentation, soft tissue defects, and urinary incontinence, are described. The methods include removing adipose tissue from a patient, processing a portion of the adipose tissue to obtain a substantially isolated population of regenerative cells, mixing the regenerative cells with another portion of adipose tissue to form a composition, and administering the composition to the patient from which the adipose tissue was removed.

Description

METHODS OF EMPLOYMENT OF ADIPOSE TISSUE-DERIVED CELLS TO INCREASE AUTOMATIC GREASE TRANSFER RELATED APPLICATIONS This application corresponds to a continuation-in-part of the US patent application. Serial No. 10 / 316,127, filed on mber 9, 2002, entitled "SYSTEMS AND METHODS FOR TREATING PATIENTS WITH PROCESSED LIPOASPIRATE CELLS" (SYSTEMS AND METHODS TO TREAT PATIENTS WITH PROCESSED LIPOASPIRATED CELLS), which claims the benefit of the provisional application of US patent Serial No. 60 / 338,856, filed mber 7, 2001. This application also claims priority of the provisional US patent application. Serial No. 60 / 479,418, titled METHODS OF USING ADIPOSE TISSUE DERIVED CELLS IN AUGMENTING AUTOLOGOUS FAT TRANSFER (METHODS FOR USING ADIPOSE TISSUE CELLS TO INCREASE AUTOMATIC FAT TRANSFER), filed on June 18, 2003. The contents of all the aforementioned applications are expressly incorporated herein by this reference. BACKGROUND OF THE INVENTION 1. Field of the Invention This invention generally relates to cells derived from adipose tissue, and more particularly to adipose-derived regenerative cells (e.g., stem and / or progenitor cells), to methods for using derived regenerative cells. of adipose, to compositions containing adipose-derived regenerative cells and systems for preparing and utilizing adipose-derived regenerative cells that are used to increase fat transfer. 2. Description of Related Art Fat transfer is a relatively common cosmetic, therapeutic and structural procedure, involving the collection of adipose tissue (fat) of one site and reimplantation in another site (Coleman 1995; Coleman 2001). While it is used substantially for repair of small cosmetic defects such as facial folds, wrinkles, alveoli or scars and skin gaps, fat transfer has also been used cosmetically in breast augmentation and reconstruction (Bircoll and Novack 1987; Dixon 1988; ). The increase of the hips has also been done using fat transfer approaches (Cárdenas Camarena, Lacouture et al., 1999, Pedroza 2000, Peren, Gómez et al., 2000). The existing fat transfer methods, however, are associated with 5 substantial side effects including infection (Castello, Barros et al., 1999; Valdatta, Thione et al., 2001) and calcifications and scars that can interfere with mammography and other modalities of breast imaging (Huch, Kunzi et al., 1998). Current methods of fat transfer are also frequently associated with inconsistent grafting, where for example either the implanted material is totally or partially resorbed or replaced by scar tissue (Eremia and Newman 2000). In mammoplasty for breast augmentation, for example the use of fat tissue often causes loss of tissue function, which can be attributed in part to implanted fat tissue necrosis during the time it takes for new blood vessels to form and feed the implant 5 (Saunders, Keller et al., 1981; Eppley, Smith et al., 1990; Nishimura, Hashimoto et al., 2000). Similarly, for the long-term correction of soft tissue defects, numerous materials, including autologous fat, have been used to fill scars, wrinkles, and other soft tissue defects (Coleman 2001; Maas and Denton 2001). As described above, however, these adipose tissue transplants also suffer from a lack of neovascularization and necrosis. The transfer of autologous fat has also been applied in non-cosmetic clinical settings where a support structure or soft tissue filling is required. An example is stress urinary incontinence, where the transplanted fat is intended to support the structures of the urethral wall and urinary sphincter (Palma, Riccetto et al., 1997; Lee, Kung et al., 2001). However, the lack of durability of the transplanted fat has prevented widespread acceptance of this technique. A similar approach has been used in fecal incontinence, which is another sphincter disorder (Shafik 1995, Bernard, Favetta et al., 1998). Other examples where fat transfer has been applied in non-cosmetic clinical settings include vocal cord paralysis, vocal atrophy, intubation trauma and post-hemilaryngectomy defects, and vocal implant (Koufman 1991, Mikaelian, Lowry et al., 1991; Hsiung, Woo et al., 2000; Perie, Ming et al., 2002), repair of soft tissue defects caused by irradiation (Jackson, Simman et al., 2001) and war injury (Ghobadi, Zangeneh et al. ., 1995), in lumbar disc surgery (Bemsmann, Kramer et al., 2001; Kanamori, Kawaguchi et al., 2001), and repair of atrophied tissue in the sole of the foot (Chairman 1994, Lauf, Freedman et al., 1998). All these approaches have encountered the problems described above for cosmetic applications.

A number of groups have looked for ways to supplement the graft, in a way that improves long-term survival and retention. One group has reported results using serum free cell culture medium to improve graft survival in an animal model (Ullmann, Hyams et al., 1998) while others have shown that increasing tissue transferred with growth factors can improve the viability of the graft in another model system (Eppley, Snyders et al., 1992; Yuksel, Weinfeld et al., 2000; Yuksel, Weinfeld et al., 2000). A different approach has been proposed by Schoeller et al., Where adipe precursor cells are embedded in fibrin adhesive and then implanted in the hope that the cells will survive and generate new adipose tissue from scratch (Schoeller, Lille et al. , 2001). Others have used a similar approach that involves planting artificial polymers with these cells (Patrick, Chauvin et al., 1999). Problems associated with these approaches are that the approaches can achieve only one component of adipose tissue (the adipe) leaving new production of blood vessels (angiogenesis) to endogenous mechanisms. Furthermore, given the limited capacity for self-renewal of pre-adipes they may be unable to provide long-term adipe production. Accordingly, there remains a need for improved methods for administering adipose tissue to patients, which reduce the problems associated with existing methods. SUMMARY OF THE INVENTION The present invention is based, at least in part, on the discovery that the adipose-derived regenerative cells (e.g., endothelial precursor cells) of the present invention are capable of providing angiogenic support and long-term production of both vascular endothelial cells and adipes. Accordingly, the present invention provides methods for increasing fat transfer, eg, autologous fat transfer. The present invention also provides fast and reliable devices, systems and methods for preparing adult regenerative cells from adipose tissue with increased yield, consistency and purity, with a reduced or non-existent need for post-extraction handling. The present invention further provides compositions, methods and systems for using cells derived from adipose tissue, which can be mixed with intact adipose tissue and placed directly in a recipient together with the additives necessary to promote, engender or support a therapeutic, structural or cosmetic benefit. In one embodiment, regenerative cells prepared according to this disclosure are prepared and subsequently mixed with intact (non-disrupted or unprocessed) adipose tissue fragments to form a composition. In this way, the composition comprises a mixture of adipose tissue and regenerative cells. The composition can be implanted in the receiver to provide an autologous soft tissue filling for correction of contour defects (wrinkles, "recesses of removed pieces", alveoli or pustule marks and larger deficits) or to provide support for damaged structures such as like the urethra. The composition can also be administered to chest regions in connection with breast augmentation procedures and soft tissue defects. Adipose tissue processing occurs in a system that maintains a closed sterile fluid / tissue pathway. This is achieved by the use of a linked assembly, pre-assembled sterile closed containers, and tubing that allows transfer of tissue and fluid elements within a closed path. The system can be linked to a processing device that can automate the addition of reagents, temperature and synchronization of processing, in this way freeing operators by the need to manually manage the process. In a preferred embodiment, any tissue extraction procedure through processing and placement in the receiver will be performed in the same facility, no doubt even within the same room of the patient undergoing the procedure. In certain embodiments, a method for treating a patient includes the steps of: a) providing a tissue removal system; b) remove adipose tissue from a patient using the tissue removal system, the adipose tissue has a concentration of regenerative cells; c) processing at least a portion of the adipose tissue to obtain a concentration of regenerative cells different from the concentration of regenerative cells of the adipose tissue before processing; and d) administering the regenerative cells to a patient, without removing the regenerative cells from the tissue removal system before being administered to the patient in order to treat the patient in this way. In other embodiments, a method for treating a patient includes: a) providing a system for removing adipose tissue; b) remove adipose tissue from a patient using the adipose tissue removal system, the adipose tissue has a concentration of regenerative cells; c) process adipose tissue to increase the concentration of regenerative cells in the adipose tissue; d) mixing the adipose tissue that has the concentrated regenerative cells with another unitary portion of adipose tissue; and e) administering the adipose tissue with the increased concentration of regenerative cells to a patient to thereby treat the patient. In particular modalities, a patient with soft tissue defects is treated. In other modalities, the chest of a patient is treated. In still other modalities, the patient is treated for urinary incontinence. The methods of treatment described herein can be used to treat any cosmetic or non-cosmetic disorder that requires the transfer of both autologous and non-autologous fat. In preferred embodiments, the regenerative cells used to treat a patient are stem cells or progenitor cells. In other embodiments, the regenerative cells are endothelial progenitor cells. In still other embodiments, the regenerative cells are any population of regenerative cells as described herein. Additionally, the population of regenerative cells employed in the treatment methods encompassed by the invention may be a homogeneous or heterogeneous population of cells. According to yet another aspect of the invention, the regenerative cells are placed in the container in combination with other cells, tissues, tissue fragments or other stimulators of growth and / or cell differentiation. For example, regenerative cells can be combined with growth factors and / or cytokines, for example angiogenic or arteriogenic growth factors. Regenerative cells can also be combined with immunosuppressive drugs. These additives may be administered during or after the regenerative cells have been concentrated using the systems and methods of the invention. In yet another aspect of the invention, the regenerative cells are directed to other targets or targets such as implant materials, surgical devices, cell culture devices or purification devices, before being placed in the recipient. In a preferred embodiment, the cells, with any of the aforementioned additives, are placed in the person from which they were obtained in the context of a single operative procedure with the intention of deriving a therapeutic, structural or cosmetic benefit to the patient. Any feature or combination of features described herein are included within the scope of the present invention so long as the features included in said combination are not mutually inconsistent, as will be apparent from the context, this specification and the knowledge of a person with ordinary skill in the art. . Advantages and additional aspects of the present invention are apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an illustration of a system for separating regenerative cells from tissue that includes a filter assembly. The figure 2 is an illustration of a system similar to Figure 1 having a plurality of filter assemblies in a series configuration. Figure 3 is an illustration of a system similar to Figure 1 having a plurality of filter assemblies in parallel configuration. Figure 4 is an illustration of a system for separating regenerative cells from tissue that includes a centrifuge chamber. Figure 5 is a sectional view of a collection chamber including a prefixed filter that is used in a system for separating regenerative cells from tissue.

Figure 6 is a sectional view of a processing chamber of a system for separating regenerative cells from tissue, using a percolation filtration system. Figure 7 is a sectional view of a processing chamber of a system for separating regenerative cells using a centrifugal device to concentrate the regenerative cells. Figure 8 is another sectional view of the processing chamber of Figure 7. Figures 9A, 9B and 9C illustrate an elutriation component in use with the system of the invention. Figure 10 is an illustration of a system for separating tissue regenerative cells that uses vacuum pressure to move fluids through the system. A vacuum system can be constructed by applying a vacuum source or vacuum pump to the output of the system, controlled at a predetermined speed to extract tissue and fluid, using a system of taps or stopcocks, vents and clamps to control the direction and flow synchronization. Figure 11 is an illustration of a system for separating regenerative cells from tissue that uses positive pressure to move fluids through the system. A positive pressure system uses mechanical means such as a peristaltic pump to push or propel the fluid and tissue through the system at a certain speed, using valves and taps, vents and clamps to control the direction and synchronization of the flow. Figure 12A illustrates a filtering process wherein the fluid feed stream flows tangentially to the pores of the filter. Figure 12B illustrates a filtration process in which the fluid feed stream circulates perpendicular to the pores of the filter. Figure 13 is an illustration in exemplary disposable equipment for a system of the invention. Figure 14 is an illustration of an exemplary reusable component for a system of the invention. Figure 15 is an illustration of an exemplary device of the invention assembled using disposable equipment similar to Figure 13 and a reusable component similar to Figure 14. Figures 16A and 16B illustrate the expression of VEGF protein (5A) and PIGF (5B) by cultured adipose-derived stem cells. Figure 17 illustrates detection of endothelial progenitor cells within populations of adipose-derived stem cells. Figures 18A and 18B illustrate in vitro development of vascular structures in both normal (7A) and streptozotocin (7B) mice. Figure 19 illustrates the average increased restoration of blood flow in ischemic mice in the hind limb with adipose-derived stem cells compared to a negative control. Figures 20A and 20B show that increasing the dose of adipose derived stem cells improves graft and angiogenesis survival (20A) and illustrates retention of adipose tissue architecture in histological specimen (20B). DETAILED DESCRIPTION OF THE MODALITIES CURRENTLY PREFERRED The present invention provides methods for increasing autologous fat transfer using adipose-derived regenerative cells ("ADCs"). For example, the present invention demonstrates that the adipose derived regenerative cells of the invention (1) express angiogenic growth factors and cytokines, including PIGF, VEGF, bFGF, IGF-II, Eotaxin, G-CSF, GM-CSF, IL-12p40 / p70, IL-12 p70, IL-13, IL-6, IL-9, Leptin, MCP-1, M-CSF, MIG, PF-4, TIMP -1, TIMP-2, TNF-alpha and Thrombopoietin, (2) comprise endothelial progenitor cells (EPC) that have a well-established role in blood vessel formation, (3) develop into blood vessels in vitro, and (4) support survival of ischemic tissue in vivo. Accordingly, regenerative cells are capable of increasing autologous fat transfer, for example by promoting neovascularization at the site of administration. In order that the present invention can be more easily understood, first certain terms are defined. Additional definitions are established through the detailed description. As used herein, "regenerative cell" refers to any cells that are obtained using the systems and methods of the present invention that cause or contribute to restoration regeneration or complete or partial replacement of structure or function of an organ, tissue or unit. physiological or system, in order to provide a therapeutic, structural or cosmetic benefit.

Examples of regenerative cells include: ASCs, endothelial cells, endothelial precursor cells, endothelial progenitor cells, macrophages, fibroblasts, pericytes, smooth muscle cells, preadipocytes, differentiated or dedifferentiated adipocytes, keratinocytes, progenitor cells and unipotent and multipotent precursors (and their progeny) ), and lymphocytes.

One mechanism by which regenerative cells can provide a structural or cosmetic therapeutic benefit is by incorporating them or their progeny into newly generated, existing or repaired tissues or tissue components. For example, ASCs and / or their progeny can be incorporated into bone, muscle or other newly generated structural or functional tissue and thus cause or contribute to a therapeutic, structural or cosmetic improvement. Similarly, endothelial cells or endothelial progenitor or progenitor cells and their progeny can be incorporated into existing, newly generated, repaired or expanded blood vessels to thereby cause or contribute to a therapeutic, structural or cosmetic benefit. Another mechanism by which the regenerative cells provide a therapeutic, structural or cosmetic benefit by expression and / or secretion of a molecule, for example growth factors, which promote the creation, retention, restoration and / or regeneration of structure or function of a tissue or determined tissue component. For example, regenerative cells can express and / or secrete molecules that result in improved growth of tissues that can then participate directly or indirectly in improved structure or function. Regenerative cells can express and / or secrete growth factors or cytokines, including for example, Endothelial Vascular Growth Factor (VEGF), Placental Growth Factor! (PIGF) and its isoforms, which can perform one or more of the following functions: stimulate development of new blood vessels, ie promote angiogenesis; improve the oxygen supply of pre-existing (collateral) small blood vessels by expanding their blood transport capacity; induce mobilization of regenerative cells from sites distant from the site of injury, in order to improve settlement and migration of these cells to the site of injury; stimulate growth and / or promote the survival of cells within a site of injury, thereby promoting retention of function or structure; provide molecules with anti-apoptotic properties in this way reducing proportion or probability of cell death and permanent loss of function; and interact with endogenous regenerative cells and / or other physiological mechanisms. Regenerative cells can be used in their "native" form as presented in or extracted from the tissue using the systems and methods of the present invention or can be modified by stimulation or priming without growth factors or other biological response modifiers, by gene transfer (transient or stable transfer), by additional sub-fractionation of the resulting population on the basis of physical properties (eg size or density), differential adhesion to a solid phase material, expression of cell surface or intracellular molecules, cell culture or other manipulation, modification or fractionation ex vivo or in vivo as further described herein.

The regenerative cells may also be used in combination with other cells or devices such as synthetic or biological scaffolds, materials or devices that provide factors, drugs, chemicals or other agents that modify or ameliorate the relevant characteristics of the cells as further described herein. As used herein, "regenerative cell composition" refers to the composition of cells typically present in a volume of fluid after a tissue, for example adipose tissue, wash and at least partially disintegrate. For example, a regenerative cell composition of the invention comprises multiple different types of regenerative cells, including ASCs, endothelial cells, endothelial precursor cells, endothelial progenitor cells, macrophages, fibroblasts, pericytes, smooth muscle cells, preadipocytes, differentiated or dedifferentiated adipocytes. , keratinocytes, unipotent and multipotent precursor and progenitor cells (and their progeny), and lymphocytes. The composition of regenerative cells may also contain one or more contaminants, such as collagen, which may also be present in their tissue fragments or residual collagenase or other enzyme or agent employed in or resulting from the tissue disintegration process described herein.

As used herein, "regenerative medicine" refers to any therapeutic, structural or cosmetic benefit that derives from the placement, either direct or indirect, of regenerative cells in a subject. As used herein, the phrase "fat transfer" is a form of regenerative medicine and is intended to include all procedures with which excess fat cells are removed from one area of a body and re-implanted in another area of the body. .

Fat transfer includes both autologous and non-autologous fat transfer. The phrase "autologous fat transfer" is intended to include all procedures with which the removal and re-implantation of fat is performed in the same subject. Exemplary cosmetic fat transfer procedures include fat grafts or implants to the lips, nasolabial (from mouth to nasal folds), wrinkles and other facial folds (depressions around the eyes, between the eyebrows, as well as in the rest of the face), under the eyes, cheeks, chin, temples, breasts, hips, calves, arms, abdomen, hips as well as any other area of the body. Cosmetic fat transfer procedures can be combined with other cosmetic applications such as facial implants, blepharoplasty, brow lift, facial plastic surgery or facelift, neck plastic surgery, botox applications, chemical detachments and laser aesthetic treatment. Non-cosmetic fat transfer procedures include implants to treat sphincter disorders, including fat implants in gastroesophageal, urethral and rectal sphincters.

Fat transfer procedures can also be used to treat trauma defects (e.g., radiation) or soft tissue defects induced by disease (e.g., abdominal hernia), hemifacial microsomia, vocal cord injury, and spinal cord disorders. Fat transfer procedures can also be used to treat diseases or adipose-related disorders, including but not limited to dyslipidemia, hypoadiponectinemia, hyperlipidemia, lipatrophy and lipohypertrophy. As used herein, "stem cell" refers to a multipotent regenerative cell with the potential to differentiate a variety of other cell types, which perform one or more specific functions and have the capacity for self-renewal. Some of the stem cells described here can be pluripotent. As used herein, "progenitor cell" refers to a multipotent regenerative cell with the potential to differentiate into more than one cell type. "Progenitor cell" as used herein, also refers to a unipotent regenerative cell, with the potential to differentiate into a single type of single cell that performs one or more specific functions and has limited ability or has no ability to self-renew . In particular, as used herein, "endothelial progenitor cell" refers to a multipotent or unipotent cell with the potential to differentiate into vascular endothelial cells.

As used herein, "precursor cell" refers to a unipotent regenerative cell with the potential to differentiate into a cell type. Precursor cells and their progeny may retain extensive proliferative capacity, for example lymphocytes and endothelial cells, which may proliferate under appropriate conditions. As used herein, the term "angiogenesis" refers to the process by which new blood vessels are generated from existing vasculature and tissues (Folkman, 1995). The phrase "repair or remodeling" refers to reformation of existing vasculature. The relief of tissue ischemia critically depends on angiogenesis. The spontaneous growth of new blood vessels provides collateral circulation in and around an ischemic area, improves blood flow and relieves symptoms caused by ischemia. Diseases and disorders mediated by angiogenesis include acute myocardial infarction, ischemic cardiomyopathy, peripheral vascular disease, ischemic attack, acute tubular necrosis, ischemic lesions-including AFT, AFT, sepsis, ischemic intestinal disease, diabetic retinopathy, neuropathy and nephropathy, vasculitis, ischemic encephalopathy, erectile-physiological dysfunction, spinal cord injuries ischemic or traumatic spinal cord, multi-organ system failure, ischemic gum disease and ischemia related to transplantation. As used herein, the term "angiogenic factor" or "angiogenic protein" refers to any known protein, peptide or other agent capable of promoting the growth of new blood vessels from existing vasculature ("angiogenesis"). These angiogenic factors for use in the invention include, but are not limited to, Placenta Growth Factor (Luttun et al., 2002), Macrophage Colony Stimulation Factor (Aharinejad et al., 1995), Stimulation Factor of Colony of Granulocyte Macrophages (Buschmann et al., 2003), Vascular Endothelial Growth Factor (VEGF) -A, VEGF-A, VEGF-B, VEGFC, VEGF-D, VEGF-E (Mints et al., 2002) , neuropilin (Wang et al., 2003), Fibroblast Growth Factor (FGF) -1, FGF-2 (bFGF), FGF-3, FGF-4, FGF-5 (Botta et al., 2000), Angiopoietin 1, Angiopoietin 2 (Sundberg et al., 2002), erythropoietin (Ribatti et al., 2003), BMP-2, BMP-4, BMP-4 (Carano and Filvaroff, 2003), TGF-beta (Xiong et al. , 2002), IGF-1 (Shigematsu et al., 1999), Osteopontin (Asou et al., 2001), Pleiotropin (Beecken et al., 2000), Activin (Lamouille et al., 2002), Endothelin-1 ( Bagnato and Spinella, 2003) and their combinations. Angiogenic factors can act independently or in combination with each other.

When they are in combination, the angiogenic factors also act synergistically, so that the combined effect of the factors is greater than the sum of the effects of the individual factors taken separately. The term "angiogenic factor" or "angiogenic protein" also encompasses functional analogues of these factors. Functional analogues include, for example, functional portions of the factors. Functional analogs also include anti-idiotypic antibodies that bind to the receptors of the factors and, thus, mimic the activity of the factors to promote angiogenesis and / or tissue remodeling. Methods for generating these anti-idiotypic antibodies are well known in the art and are described for example in WO 97/23510, the contents of which are incorporated herein by reference. Angiogenic factors employed in the present invention can be produced or obtained from any convenient source. For example, the factors can be purified from their native sources, or be produced synthetically or by recombinant expression. The factors can be administered to patients as a protein composition. Alternatively, the factors can be administered in the form of an expression plasmid that encodes the factors. The construction of expression plasmids is well known in the art. Suitable vectors for constructing expression plasmids include, for example, adenoviral vectors, retroviral vectors, adeno-associated viral vectors, RNA vectors, liposomes, cationic lipids, lentiviral vectors and transposons. As used herein, "stem cell number" or "stem cell frequency" refers to the number of colonies observed in a clonogenic assay, wherein adipose-derived cells (ADCs) are coated at low cell density (<10,000 cells / well) and develop in growth medium that supports MSC growth (eg, DMEM / F12 medium supplemented with 10% fetal bovine serum, 5% horse serum, and antibiotic / antifungal agents.) Cells are developed for two weeks , after which cultures with hematoxylin are stained and colonies with more than 50 cells are counted as CFU-F.The frequency of stem cells is calculated as the number of CFU-F observed per 100 nucleated coated cells (eg, 15 colonies counted in a plate initiated 1, 000 nucleated ADC cells give a frequency of stem cells of 1.5%.) The number of stem cells is calculated as the frequency of stem cells multiplied by the total number of cells as nucleated ADC obtained. A high percent (approx 100%) of CFU-F developed from ADC cells expresses the cell surface molecule CD105 which is also expressed by stem cells derived from bone marrow (Barry et al., 1999). CD105 is also expressed by adipose-derived stem cells (Zuk et al., 2002).

As used herein, the term "adipose tissue" refers to fat including the connective tissue that stores fat. Adipose tissue contains multiple types of regenerative cells including ASCs and endothelial progenitor and progenitor cells. As used herein the term "adipose tissue unit" refers to a discrete and measurable amount of adipose tissue. A unit of adipose tissue can be measured when determining the weight and / or volume of the unit. Based on the above-identified data, a unit of processed adipose tissue, as removed from the patient, has a cellular component in which at least 0.1% of the cellular component is stem cells; that is, it has a frequency of stem cells determined as described above of at least 0.1%. With reference to the present disclosure, a unit of adipose tissue can refer to the entire amount of adipose tissue removed from a patient, or an amount that is less than the full amount of adipose tissue removed from a patient. In this way, a unit of adipose tissue can be combined with another adipose tissue unit to form an adipose tissue unit having a weight or volume that is the sum of the individual units. As used here, the term "portion" refers to a quantity of material that is less than a whole. A smaller portion refers to an amount that is less than 50%, and a larger portion refers to an amount greater than 50%. In this manner, a unit of adipose tissue that is less than the entire amount of adipose tissue removed from a patient is a portion of the adipose tissue removed. As used herein, the term "processed lipoaspirate" refers to adipose tissue that has been processed to separate the active cellular component (e.g., the component containing stem and progenitor cells) from mature adipocytes and connective tissue. This fraction is referred to herein as "adipose derived cells" or "ADC". Typically, ADC refers to the precipitate of regenerated cells obtained by washing and separating cells from adipose tissue. The precipitate is typically obtained by centrifuging a suspension of cells such that the cells aggregate in the bottom of a centrifuge chamber or cell concentrator. As used herein, the terms "administer", "introduce", "supply", "place" and "transplant" are used interchangeably herein and refer to the placement of the ADC of the invention in a subject by a method or route that results in at least partial location of the ADC at a desired location. The ADC can be administered by an appropriate route that results in delivery to a desired location in the subject where at least a portion of the cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, for example twenty-four hours, a few days, up to several years. As used herein, the term "subject" includes warm-blooded animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human. Reference will now be made in detail to the presently preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings.

Whenever possible, the same or similar reference numbers are used in the drawings and description to refer to the same or similar parts. It should be noted that the drawings are in simplified form and are not on a precise scale. With reference to the present description, for purposes of convenience and clarity only, directional terms, such as upper, background, left, right, above, below, above, above, below, below, back and front, as used herein with respect to to the accompanying drawings. These directional terms are not to be considered as limiting the scope of the invention in any way. Although the present description refers to certain illustrated modalities, it will be understood that these modalities are presented by way of example and not by way of limitation. The intention of the following detailed description, although discussing exemplary embodiments, should be considered to cover all modifications, alternatives and equivalents of the modalities as they may fall within the spirit and scope of the invention as defined by the appended claims. The present invention may be practiced in conjunction with the various tissue or cell separation techniques that are conventionally employed in the art, and only so long as the commonly practiced process steps are included as necessary to provide an understanding of the present invention. As previously stated here, regenerative cells, for example stem and progenitor cells, can be harvested from a wide variety of tissues. The system of the present invention can be used for all these tissues. Adipose tissue, however, is an especially rich source of regenerative cells. Accordingly, the system of the present invention is illustrated herein using adipose tissue as a source of regenerative cells by way of example only and not limitation. The adipose tissue can be obtained by any means known to a person with ordinary skill in the art. For example, the adipose tissue of a patient may be removed by liposuction (syringe or energized) or by lipectomy, for example suction assisted lipoplasty, ultrasound-assisted lipoplasty and excision lipectomy or combinations thereof. The adipose tissue is removed and collected and processed by a system of the invention described herein for the purpose of separating and concentrating regenerative cells. The amount of tissue collected depends on numerous factors, including the body mass index and age of the donor, the time available for collection, the availability of accessible adipose tissue collection sites, concomitant and pre-existing conditions and medications (such as anticoagulant therapy), and the clinical purpose for which the tissue is collected. For example, the percentage of 100 ml stem cells of adipose tissue extracted from a thin individual is greater than that extracted from an obese donor (Table 1). This probably reflects a diluting effect of increased fat content in the obese individual. Therefore, it may be convenient, according to one aspect of the invention, to obtain larger amounts of donor tissue with overweight compared to the amounts that would be withdrawn from thin patients. This observation also indicates that the utility of this invention is not limited to individuals with large amounts of adipose tissue. Table 1: Effect of body mass index on tissue and cell performance Mass Index Status Amount of Tissue Fabric Total Body Performance Cells (? 107) Normal 641 + 142 2.1 + 0.4 Obese 1, 225 + 173 2.4 + 0. 5 Value p 0.03 0.6 After the adipose tissue is processed, the resulting regenerative cells are substantially free of mature adipocytes and connective tissue. Accordingly, the system of the present invention generates a heterogeneous plurality of regenerative cells derived from adipose tissue that can be used for research and / or therapeutic purposes. In a preferred embodiment, the cells are suitable for placement or re-infusion in the recipient's body. In other embodiments, the cells can be used for research, for example the cells can be used to establish stem or progenitor cell lines that can survive for prolonged periods of time and be used for further study. Reference will now be made in detail to the presently preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. When possible, the same or similar reference numbers are used in the drawings and description to refer to the same or similar parts. It should be noted that the drawings are in simplified form and not at precise scale. With reference to the present description, for purposes of convenience and clarity only, directional terms, such as, upper, lower, left, right, above, below, above, above, below, below, background, rear, front, distant and next, they are used with respect to the accompanying drawings. Said directional terms shall not be construed as limiting the scope of the invention in any way. Although the present description refers to certain illustrated modalities, it will be understood that these modalities are presented by way of example and not by way of limitation. The intention of the following detailed description, although discussing exemplary embodiments, will be considered to cover all modifications, alternatives and equivalents of the modalities that fall within the spirit and scope of the invention as defined by the appended claims. The present invention can be used in conjunction with various medical procedures that are conventionally employed in the art. Now with reference to the figures, a system 10 of the present invention is generally constituted by one or more of a tissue collection chambers 20, a processing chamber 30, a debris chamber 40, an exit chamber 50 and a sample chamber 60. The various chambers are coupled together by one or more conduits 12, such that fluids containing biological material can pass from one chamber to another while maintaining a sterile, closed tissue / fluid path. The conduits can comprise rigid or flexible bodies referred to interchangeably here as lumens or pipe. In certain embodiments, the conduits are in the form of flexible tubing, such as polyethylene tubing conventionally used in clinical settings. In other embodiments, the pipes can be constructed of silicone. The flexible pipe used should be able to withstand negative pressure to reduce the likelihood of collapse. The pipe used must also be capable of withstanding positive pressure that is generated, for example, by a positive displacement pump, which can be used in the system.

All the chambers of the system may comprise one or more gates, for example the exit gates 22 or entry 21, which accept standard IV syringe connections and suction piping. The gates can be a sealed gate such as a closed rubber septum with access gate for syringe needle 51. The entry gates can be coupled to one or more cannulas (not shown) via conduits. For example, a tissue entry gate 21 can be attached to an integrated single-use liposuction cannula, and the conduit can be a flexible tubing. Ducts are generally located to provide fluid passages from one system chamber to another. For this purpose, the conduits and gates can be coupled, for example, to a suction device (not shown) that can be operated manually or automatically. The suction device can be, for example, a syringe or an electric pump. The suction device should be able to provide sufficient negative pressure to suck tissue from a patient. In general, any convenient suction device known to a person with ordinary skill in the art, for example a surgeon, may be employed. The conduits 12 may further comprise one or more clamps (not shown) for controlling the flow of material between various components of the system. The clamps are useful to maintain the sterility of the system by effectively sealing different regions of the system. Alternatively, conduits 12 may comprise one or more valves 14 that control the flow of material through the system. The valves 14 are identified as open circles in the Figures. In preferred embodiments, the valves may be electromechanical thrust valves. In another embodiment, the valves can be pneumatic valves. Still in other modalities, the valves can be hydraulic valves or mechanical valves. These valves are preferably activated by a control system that can be coupled with levers. The levers can be manipulated manually so that the levers are activated. In automated modes, the control system can be coupled to the levers as well as a processing device that can activate the valves under pre-determined activation conditions. In certain automated embodiments, the activation of the valves can be partially automated and partially subject to the user's preference, so that the process can be optimized. Still in other modalities, certain valves can be activated manually and others automatically through the processing device. The valves 14 can also be used in conjunction with one or more pumps, for example peristaltic pumps 34 or positive displacement pumps (not shown). The conduits 12 and / or the valves 14 may also comprise sensors 29, for example optical sensors, ultrasonic sensors, pressure sensors or other forms of monitors known in the art that are capable of distinguishing between the various fluid components and fluid levels that circulate through the system. In a preferred embodiment, the sensors 29 can be optical sensors. The system may also include a plurality of filters 36. In certain embodiments, the filters may be within a system chamber 28.

Different cameras within the system can comprise different filters. The filters are effective to separate the regenerative cells, for example stem cells and / or progenitor cells, from undesirable cells and disintegrating agents that can be used according to the system. In one embodiment, a filter assembly 36 includes a hollow fiber filtration device. In another embodiment, a filter assembly 36 includes a percolation filtration device, which may or may not be used with a settling process. In an additional mode, the filter assembly 36 comprises a centrifuge device, which may or may not be used with an elutriation device and process. In yet another embodiment, the system comprises a combination of these filtering devices. The filtering functions of the present invention can be double, with some filters that remove things from the final concentration such as collagen, free lipids, free adipocytes and residual collagenase, and with other filters that are used to concentrate the final product. The system filters may comprise a plurality of pores in the range in diameter and / or length from 20 to 800 μm. In a preferred embodiment, the collection chamber 20 has a prefix filter 28 with a plurality of pores in the range of 80 to 400 μm. In another preferred embodiment, the collection chamber 20 has a pre-set filter 28 with a plurality of pores of 265 μm. In other embodiments, the filters may be removable and / or disposable. The system may also comprise one or more temperature control devices (not shown) that are located to adjust the temperature of the material contained within one or more cameras of the system. The device for temperature control can be a heater, a coolant or both, ie it can be capable of switching between a heater and a refrigerant. The temperature device can adjust the temperature of any of the materials that pass through the system, including the fabric, the disintegrating agents, the resuspension agents, the rinsing agents, the washing agents or additives. For example, heating the adipose tissue facilitates disintegration, while cooling the regenerative cell exit is convenient for maintaining viability. Also, if pre-heated reagents are required for optimal tissue processing, the role of the temperature device would be to maintain the pre-determined temperature instead of increasing or decreasing the temperature. To maintain a sterile and closed tissue / fluid path, all gates and valves may comprise closure that maintains the sealed configuration of the system. The closure may be a membrane that is impervious to fluid, air and other contaminants or may be any other convenient closure known in the art. In addition, all system gates can be designed to accommodate syringes, needles or other devices to remove materials in the chambers without compromising the sterility of the system. As stated herein, tissue can be extracted from a patient by any method recognized in the art. The aspirated tissue is transferred to the collection chamber 20 by a conduit such as 12a where it is rinsed and digested. The aspirated tissue typically enters the collection chamber 20 through a sealed inlet gate, such as an access gate for closed syringe needle with rubber septum (not shown in the collection chamber). The collection chamber 20 may comprise a plurality of flexible or rigid cylinders or cans or combinations thereof. For example, the collection chamber 20 may be constituted by one or more rigid cans of varying size. The collection chamber 20 may also comprise one or more flexible bags. In such systems, the bag of preference is provided with a support such as an internal or external frame, which helps to reduce the likelihood of the bag being crushed before the suction application to the bag. The collection chamber 20 is sized to contain the necessary amount of saline to properly rinse and digest the tissue before the washing and concentration stage of the process performed in the processing chamber 30. Preferably, the volume of the tissue or fluid present in the collection chamber 20 is easily determined by the naked eye. For example, to separate and concentrate regenerative cells from adipose tissue, a convenient collection chamber has the capacity to contain 800 ml of liposuction and 1200 ml of saline. According to this, in one embodiment, the collection chamber 20 has a capacity of at least 2 liters. In another embodiment, to separate and concentrate red blood cells, the collection chamber 20 has a capacity of at least 1.5 liters. In general, the size of the collection chamber 20 will vary depending on the type and amount of tissue collected from the patient. The collection chamber 20 can be sized to contain as little as about 5 ml to about 2 liters of tissue. For smaller tissue volumes, for example 5 ml to 100 ml, the tissue can be collected in a syringe before being transferred to the collection chamber 20. The collection chamber 20 can be constructed using any suitable biocompatible material that can be sterilized. In a preferred embodiment, the collection chamber 20 is constructed of disposable material that satisfies the biocompatible requirements for intravascular contact as described in ISO 10993. For example, acrylic polycarbonate or ABS may be used. The fluid path of the collection chamber 20 is preferably free of pyrogens, ie suitable for use in blood without danger of disease transmission. In one embodiment, the collection chamber 20 is constructed of a material that allows the user to visually determine the approximate volume of tissue present in the chamber. In other embodiments, the volume of tissue and / or fluid in the collection chamber 20 is determined by automated sensors 29. The collection chamber 20 is preferably designed in such a way that the automated system can determine the volume of tissue and / or fluid inside the chamber with a reasonable degree of accuracy. In a preferred embodiment, the system detects the volume within the collection chamber with an accuracy of more or less than fifteen percent. In a particular embodiment that is provided by way of example only, the collection chamber 20 is in the form of a rigid chamber, for example a chamber constructed of medical grade polycarbonate containing an approximately conical prefix filter 28 of medical grade polyester with a mesh size of 265 μm. The rigid tissue collection container may have a size of approximately 20.32 cm (eight inches) in height and approximately a diameter of 12.7 cm (five inches), the wall thickness may be approximately .3175 cm (0.125 inches). The interior of the cylinder can be accessed for example through one or more gates for suction pipe, one or more gates with piping for connection through sterile access technology and / or one or more gates for access through needle drilling through of a rubber septum. The prefix filter 28 inside the collection chamber 20 is preferably structured to retain adipose tissue and pass non-adipose tissue for example the tissues removed from the patient. In more specific forms, the filter 28 can allow the passage of free lipid, blood and saline, while retaining fragments of adipose tissue during or in another mode after the initial collection of adipose tissue. In this aspect, the filter 28 includes a plurality of pores, either the same or different sizes, but in the size range of about 20 μm to 5 mm. In a preferred embodiment, the filter 28 includes a plurality of pores of 400 μm. In a preferred embodiment, the filter 28 is a medical grade polyester mesh with a thickness of approximately 200 μm with a pore size of about 265 μm and about 47% open area. This material retains tissue during rinsing but allows cells to pass through the mesh and after tissue disintegration. In this way, when tissues are aspirated from the patient, non-adipose tissue can be separated from adipose tissue. The same functionality can be achieved with different materials, mesh size and the number and type of gates. For example, mesh pore sizes smaller than 100 μm or as large as several thousands of microns will achieve the same purpose of allowing passage of saline and blood cells while retaining aggregates and fragments of adipose tissue.

Similarly, the same purpose can be achieved by use of a rigid plastic material or by many other modifications that will be known to those skilled in the art. The collection chamber 20 can also be constituted by a means for washing the fabric as well as a means for mixing and / or disintegrating the fabric. The tissue can be washed, mixed or disaggregated by agitation to maximize cell viability and minimize the amount of free lipid released. In one embodiment, the fabric is agitated by rotating the entire collection chamber 20 through an arc with varying degrees (for example, through an arc of about 45 degrees to about 90 degrees) at varying speeds, for example about 30. revolutions per minute. In certain embodiments, the inner surface of the collection chamber 20 to which the vanes 25a or projections are rigidly connected is a prefixed filter 28. In other embodiments, the interior surface of the collection chamber 20 to which the vases are rigidly connected. 25a vanes or projections is a filter cage 27 of a prefixed filter 28.

The rotation of the collection chamber 20 can be achieved by a pulse mechanism connected to or in proximity to the collection chamber 20. The drive mechanism can be a simple gear or belt or other drive mechanism known in the art. The rotation speed can be, for example, 30 revolutions per minute. In general, higher speeds have been found to generate larger volumes of free lipids and may not be optimal. In other embodiments, the fabric is agitated by placing a rotary arrow 25 within the collection chamber 20, wherein the rotary arrow comprises one or more vanes 25a or projections connected lightly to the rotating shaft 25 which pass through the mixture according to the arrow is rotated.

In certain embodiments, the rotary shaft 25 with paddles 25a connected lightly can be supported on the bottom of the collection chamber 20. This can be achieved, for example by placing the pallet type device in a rotating magnetic field (eg, magnetic stirrer). Alternatively, tissue agitation can be achieved using a simple agitator known in the art, i.e. a device that implements upward and downward agitation without rotation. An exemplary collection chamber 20 of the system, illustrated in Figure 5, comprises a vacuum line 11 that can be used to evacuate air from the chamber, which allows the user to remove tissue with a cannula supplied to the user; an input gate 21; an outlet hatch 22 for draining or removing waste; and a rotary arrow 25 with a paddle type device wherein the one or more paddles 25a are rigidly connected to a filter cage 27 of a preset filter 28 for tissue agitation using a magnetic stirrer (not shown).

The system 10 may also comprise one or more sources of wash solution 22. The source of wash solution may comprise a source of saline 23, and a source of tissue disintegrating agent 24, such as collagenase. The wash solution can be any solution known to a person skilled in the art, including saline or any other buffered or un-buffered electrolyte solution. Disintegrating agents that may be employed include neutral proteases, collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, members of the Blendzyme enzyme mixture family, for example Liberase H1, pepsin and / or combinations thereof. The types of fabrics processed will dictate the types or combinations of washing solutions used. Typically, the wash solution such as saline enters the collection chamber 20 after the adipose tissue has been removed from the patient. However, the wash solution can be supplied to the collection chamber 20 before the adipose tissue is removed or can be delivered to the collection chamber 20 concurrently with the adipose tissue. In the collection chamber 20, the wash solution and extracted adipose tissue can be mixed by any means including the methods described above. The containers for the saline agents 23 and / or the disintegrating agents 24 can be any convenient container that can retain the contents is a sterile form, for example a collapsible bag, such as an IV bag used in clinical settings. These containers may have conduits 12, such as conduit 12e, coupled to the collection chamber 20 so that the saline and / or disintegrating agent may be supplied to the interior of the collection chamber 20. The saline and / or agent Disintegration can be supplied to the interior of the collection chamber 20 through any manner recognized in the art, including simple gravity pressure applied to the exterior of the vessels for the saline 23 and / or the disintegrating agents 24 or when placing a pump positive displacement in the conduits, for example conduit 12d in Figure 4. The tissue and / or fluid within the collection chamber should be maintained at a temperature in the range of 30 degrees C to 40 degrees C. In a preferred embodiment , the temperature of the suspension inside the collection chamber is maintained at 37 degrees O In certain modalities, if the surgical procedure or the therapeutic application requires be delayed, the selected fabric can be stored in the collection chamber for later use. The tissue can be stored at or around room temperature or approximately 4 degrees C for up to 96 hours. To assist in the separation and concentration process, the collection chamber 20 allows differentiation of floating and non-floating liquid inside the chamber. In automated system modalities, the collection chamber may be constituted by sensors 29 that can detect when the interface between the floating and non-floating liquids has been reached. For example, the sensor 29 may be an optical sensor that may be capable of detecting a change in light refraction of the effluent circulating in the outlet fluid passage of the collection chamber and sending a signal to the system processing device. to thereby activate or deactivate one or more pumps and / or valves in accordance with the processing device associated with the system. Since the floating layer consists of regenerative cells that require additional washing and concentration, the collection chamber 20 is preferably constituted by an outlet gate 22 at the lowest point of the chamber, so that blood and other components do not Floaters can be drained into the waste container. Accordingly, the collection chamber 20 can be coupled to one or more waste containers 40 by one or more conduits 12 described herein to allow waste from the collection chamber to be drained or removed from the system. Drainage can be passive or active. For example, the non-floating components described above can be drained using gravity, by applying positive or negative pressure, by use of pumps 34 or by use of vents 32. The collection chamber can be located in such a way that the outlet gates 22 are located in, or near the bottom of the collection chamber. The collection chamber of preference is located in this orientation to allow fragments of adipose tissue to float, for example from 15 seconds to several minutes or more. In automated modalities, the system processing device calculates various parameters, for example the volume of saline required to wash the tissue and the time required to wash the tissue, based on information initially supplied by the user (eg, tissue volume). indicted). Based on the control logic of the processing device, certain valves and / or pumps are activated or deactivated in such a way that the waste of the collection chamber 20 is removed from the system. The sensors 29 such as the optical sensors can be positioned such that they are capable of signaling the processing device of the system to proceed with the next step in the tissue processing. In a preferred embodiment, the collection chamber 20 is constituted by a closed fluid path that allows saline and reagents to be added to the tissue in an aseptic manner. Accordingly, the collection chamber 20 further comprises conduits 12, for example flexible or rigid conduits, which are sized appropriately to allow free passage of tissue and liquid. In a preferred embodiment, the conduits 12 are in the form of a pipe. The conduits 12 may vary in size depending on whether the passage of fluid or tissue is desired. The conduits 12 may also vary in size depending on the amount of tissue or fluid that is cycled through the system. For example, for the passage of fluid, the conduits can have a diameter in the range from about 1.524 to 19.05 mm (0.060 to about 0.750 in) and for the passage of tissue, the conduits can have a diameter in the range of 7.925 to 19.05 mm (0.312 to 0.750 in). In general, the size of the ducts is chosen to balance or balance the volume that the ducts can tolerate and the time required to transport the tissue or fluids through the ducts. In automated system modes, the above parameters, ie volume and time for transport, must be identified such that appropriate signals can be transmitted to the system processing device. This allows the device to move precise volumes of fluid and tissue from one chamber to another. The collection chamber 20 also allows removal of washed adipose tissue to a processing chamber 30. Accordingly, the collection chamber 20 must be connected to the necessary pipe 12, valves 14 and pump 34 for movement and storage of the tissue washed adipose In addition, the collection chamber 20 typically includes one or more gates 21 to allow the wash solution to be delivered into the chamber, and one or more gates 22 to allow waste and other materials to be directed out of the chamber. collection 20. For example, the collection chamber may include one or more sealed inlet gates as described herein. The collection chamber 20 may also include one or more layers (not shown), such as the top layer and the bottom layer to further ensure that the system remains sterile while the wash solution is delivered into the collection chamber and / or the waste is transported. The gates 21 can be provided in the layers of the collection chamber or in a side wall of the collection chamber. The washing process with fresh washing solution can be repeated until the residual content of non-floating contaminants in the solution reaches a predetermined level. In other words, the remaining material in the collection chamber 20, which comprises the floating material of the mixture described above, including fragments of adipose tissue, can be washed one or more additional times until the amount of material desired is reduced to a level predetermined. One method to determine the extreme point of the wash is to measure the amount of red blood cells in the tissue solution. This can be achieved by measuring the light absorbed at the wavelength of 540 nm. In a preferred embodiment, a range between about .546 and about .842 mm is considered acceptable. After a desired amount of wash cycles, a tissue disintegrating agent can be supplied to the collection chamber 20 to digest the remaining adipose tissue components. For example, saline such as saline supplied from a saline source 23 as described above, can be added to the adipose tissue together with or immediately followed by addition of collagenase, such as collagenase supplied from a source of collagenase 24 as described above. The washed adipose tissue and the tissue disintegrating agent can then be agitated in forms similar to the agitation methods described above, until the washed adipose tissue is disintegrated. For example, the washed adipose tissue and the tissue disintegrating agent can be agitated by rotating the entire collection chamber through an arc of approximately 90 degrees, having an arrow containing one or more pallets passing through the solution according to the arrow is rotated and / or by turning the entire collection chamber containing pallets or projections on the inner surface of the collection chamber. In one embodiment, the adipose tissue fragments are mixed with an enzyme solution containing collagenase at or about 37 degrees C for about 20-60 minutes. In other embodiments, a higher concentration of collagenase or similar agent may be added to decrease the digestion time. Similar to what has been described above, the collection chamber 20 can then be placed in an upright position in such a way that the outlet gates 22 are located at the bottom of the collection chamber for a sufficient period of time to allow tissue fragments to float and floating cells.

Typically, the time may be in intervals from about 15 seconds to several minutes, but other times may be implemented in modified modes. Depending on the purpose for which cells derived from adipose tissue will be used, the adipose tissue may already be partially disintegrated or completely disintegrated. For example, in modalities in which the adipose-derived cells are combined with a unit of adipose tissue, it may be convenient to partially disintegrate the collected adipose tissue, to remove a portion of the partially disintegrated adipose tissue., and then continue to disintegrate the remaining portion of adipose tissue that remains in the collection chamber. Alternatively, a portion of washed adipose tissue may be removed and set aside in a sample container prior to any digestion. In another embodiment, collected adipose tissue partially breaks down to concentrate cells before being reintroduced back into the patient. In one embodiment, the adipose tissue is mixed with a tissue disintegrating agent for a period of time generally less than about 20 minutes. A portion of the partially disintegrated tissue can then be removed from the collection chamber and the remaining partially disintegrated tissue can be further disrupted by mixing the adipose tissue with a tissue disintegrating agent for another 40 minutes. When adipose-derived cells are to be used as an essentially pure population of regenerative cells, the adipose tissue can be completely disintegrated. During washing and / or disintegration, one or more additives may be added to the various containers as required to improve the results. Some examples of additives include agents that optimize washing and disintegration, additives that improve the viability of the population of active cells during processing, anti-microbial agents (for example antibiotics), additives that lyse adipocytes and / or red blood cells, or additives that enrich cell populations of interest (by differential adhesion to solid phase portions or to otherwise promote substantial reduction or enrichment of cell populations). Other possible additives include those that promote recovery and viability of regenerative cells (e.g., caspase inhibitors) or that reduce the likelihood of adverse reaction to infusion or placement (e.g., inhibitors of re-aggregation of cells or connective tissue).

After a sufficient settling time has elapsed, the non-floating fraction of the mixture resulting from washed adipose tissue fragments and tissue disintegrating agents will contain regenerative cells, for example stem cells and other adipose-derived progenitor cells. As discussed herein, the non-floating fraction containing the regenerative cells will be transferred to the processing chamber 30 where the regenerative cells of interest, such as adipose derived stem cells will be separated from other cells and materials present in the non-floating fraction. mix. Accordingly, the processing chamber 30 is located within the system such that the rinse and digested tissue suspension is moved from the collection chamber 20 to the processing chamber 30 via line 12, valves 14 and pump 34. The processing chamber is sized to accommodate tissue / fluid mixtures in the range of 10 mL to 1.2 L. In a preferred embodiment, the processing chamber is sized to accommodate 800 mLs. In certain embodiments, the entire regenerative cell composition of the collection chamber 20 is directed to the processing chamber 30. However, in other embodiments, a portion of the regenerative cell composition is directed to the processing chamber 30 and another portion is directed to a different region of the system, e.g., sample chamber 60, to recombine with cells processed in processing chamber 30 at a later time. As previously stated herein, the composition of regenerative cells of the present invention is a composition of cells typically present in a volume of liquid after a tissue, for example adipose tissue, is washed and at least partially disintegrates. In other words, the composition of regenerative cells that is transferred from the collection chamber 20 after being mixed with a tissue disrupting agent, comprises multiple different types of cells, including stem cells, progenitor cells, endothelial precursor cells, adipocytes and others. regenerative cells described here. The composition of regenerative cells may also contain one or more contaminants, such as collagen and other connective tissue proteins and their fragments, which were present in the adipose tissue fragments or residual collagenase from the tissue disruption process. The processing chamber 30 can be constructed using any suitable biocompatible material that can be sterilized. In a preferred embodiment, the processing chamber 30 is constructed of disposable material that satisfies or meets the biocompatibility requirements for vascular contact, as described in ISO 10993. For example, polycarbonate, acrylic, ABS, ethylene vinyl acetate or styrene-butadiene copolymers (SBC) can be used. In another embodiment, the fluid path of the disposable processing chamber is pyrogen-free. The processing chamber may be in the form of a plastic bag, such as those conventionally used to process blood in blood banks; or in other modalities, it can be structurally rigid (Figure 6). In one embodiment, the processing chamber 30 may be similar to the processing chamber described in the U.S. patent application. Common Property Serial No. 10 / 316,127, filed on December 7, 2001 and the US Patent Application. Serial No. 10 / 325,728, filed on December 20, 2002, the contents of which are hereby incorporated by reference.

In certain embodiments, the regenerative cell composition of the collection chamber 20 is introduced into the processing chamber 30 wherein the solution can be filtered to separate and / or concentrate a particular regenerative cell composition. Cell filtration is a method for separating particular components and cells from other different components or cell types. For example, the composition of regenerative cells of the invention comprises multiple different types of cells, including stem cells, progenitor cells and adipocytes, as well as one or more contaminants, such as collagen, which is present in the adipose tissue fragments or residual collagenase of the process of tissue disintegration. The filters 36 present in the processing chamber 30 can allow separation and concentration of a particular sub-population of regenerative cells, for example stem cells or endothelial progenitor cells, etc. Some variables that are associated with filtration of cells from a liquid include, but are not limited to, pore sizes of filter media, geometry (shape) of the pore, surface area of the filter, direction of flow of the filtering solution, pressure trans-membrane, dilution of the particular cell population, size and shape of particles as well as cell size and cell viability. In accordance with the present disclosure, the particular cells that are described to be separated or filtered are typically adipose-derived stem cells. However, in certain embodiments, the particular cells may include adipose-derived progenitor cells, such as endothelial precursor cells, alone or in combination with the stem cells. The composition of regenerative cells can be directed through a filter assembly, such as a filter assembly 36. In certain embodiments, the filter assembly 36 comprises a plurality of filters that are structured to perform different functions and separate the cell composition. regenerative in different parts or components. For example, one of the filters can be configured to separate collagen from the regenerative cell composition, one of the filters can be configured to separate adipocytes and / or lipid components from the regenerative cell composition and one of the filters can be configured to separate residual enzymes , such as the tissue disaggregation agent, of the composition of regenerative cells. In certain embodiments, one of the filters is capable of performing two functions, such as separating collagen and the tissue disintegrating agent from the composition. The plurality of filters are typically arranged in series; however, at least a portion of the filters can be arranged in parallel equally. A series arrangement of the filters of the filter assembly 36 is illustrated in Figure 2. A parallel assembly of the filters of the filter assembly 36 is illustrated in Figure 3. In one embodiment, the filter assembly 36 comprises a first filter , a second filter and a third filter. The first filter is configured to remove collagen particles present in the composition of regenerative cells. These collagen particles typically have a diameter of approximately .00254 mm (0.1 miera) and can be up to .508 mm (20 microns) in length. The collagen particles can be of varying sizes depending on the digestion. They can also be fibrils, which means they have twists and turns or twists. Any of the filters described herein can be made of polyethersulfone, polyester, PTFE, polypropylene, PVDF or possibly cellulose. There are two possibilities to filter the collagen. One is to try to remove the larger particles first, allowing the cells to pass, which requires for example a filter probably in the range of 10 microns. The second method is to use a filter of smaller size such as 4.5 microns, with the intention that the collagen is well digested, to trap the cells and allow the collagen to pass. This will require a means to float the cells back to the filter. There may also be the possibility of implementing a filter that attracts and retains the collagen fibers. The second filter is configured to remove free immature adipocytes that are not floating in the composition of regenerative cells. In one embodiment, the second filter can be constructed of polyester and have a pore size between about 30 and about 50 microns with a preferred pore size that is about 40 microns. Although referred to as a second filter, the placement of this device may be in a first position instead of second, to facilitate an initial removal of larger cells and particles. The third filter is configured to remove the unused or residual collagenase or other tissue breakdown agent present in the composition. In a preferred implementation, the collagenase can degenerate with time. In one embodiment, the third filter comprises a plurality of pores having a diameter, or length less than 1 μm. In certain embodiments, the pores may have diameters that are smaller than 1 μm. In other embodiments, the pores have diameters between 10 kD and 5 microns. In certain embodiments, the third filter can be configured to concentrate the population of regenerative cells in a small volume of saline or other wash solution, as discussed herein. As currently preferred, only the final filter is the hollow fiber unit. It is not necessary that any of the filters be of the hollow fiber type. The hollow fiber unit is used for the final filter in a preferred implementation because it is more efficient to remove the collagenase with the lesser deleterious effect to the regenerative cells. In a modality where the device is a collection of inventory items, the three filters are in separate shelters. It is feasible to have the first and second filters combined in a housing if the hollow fiber unit is used for the third filter. If the final filter is not a hollow fiber configuration, then all three filters can be contained in a housing. The filters of the filter assembly 36 can be located in the processing chamber 30 or can be provided as separate components of the processing chamber 30. In addition, the filters of the filter assembly 36 can be provided in multiple processing chambers or in an online form . In certain embodiments, the conduits or pipe may act as a camera or processing chambers. The processing chamber may be reduced in size such that it becomes the interior volume of the ducts connecting the filters. This type of system will work correctly if the volume of tissue solution is appropriately sized. In this way, the ducts can act as the processing chamber by containing the fluid with cells as it passes through the filters. Care should be taken to minimize the volume of the ducts, so that the cells / tissue are not lost unnecessarily in the process of priming and operation of the system. With reference to the embodiment described above, the composition of regenerative cells, containing the washed cells and the residual collagen, adipocytes and / or undigested tissue disrupting agent, can be directed through the first filter to remove at least a portion of and preferably substantially all of the collagen particles of the composition, such that less and preferably no collagen particles are present in the filtered solution. The composition of filtered regenerative cells containing the adipocytes and / or undigested tissue disrupting agent can not be directed through the second filter to remove at least a portion of and preferably substantially all of the free adipocytes from the regenerative cell composition. filtered. Subsequently, the double filtered regenerative cell composition, which contains the undigested tissue disintegrating agent, can be directed through the third filter, such as a hollow fiber filtering device, as discussed herein, to remove or reduce the agent of disintegration of undigested tissue from the composition of regenerative cells. The triple filtered regenerative cell composition (i.e., the composition remaining after passing through the first, second and third filters) can then be directed to multiple outputs, which can include a portion of the processing chamber 30 comprising multiple exits These outlets can serve to maintain the necessary pressure, as well as provide connections via conduits to other containers which may include the collection chamber 20, the outlet chamber 50 and / or the waste container 40. In one embodiment, a filter of the assembly of filter 36 comprises a hollow fiber filtration member. Or, in other words, the filter comprises a collection of hollow tubes formed with the filter medium. Examples of filter media that can be employed with the described system 10 include polysulfone, polyethersulfone or a mixed ester material and the like. These hollow fibers or hollow tubes of filter media may be contained in a cylindrical cartridge of the filter assembly 36. Individual tubes or fibers of filter media typically have an inner diameter that is in the range of from about 0.1 mm to about 1. mm with a preferred value that is approximately 0.5 mm.

The diameter and length of a convenient cylindrical cartridge will determine the number of individual tubes of filter media that can be placed inside the cartridge. An example of a convenient hollow fiber filter cartridge is the FiberFlosMR Tangential Flow Filter, catalog # M-C-050-K (Minntech, Minneapolis, Minnesota). Pore sizes of the filter medium may be in the range between about 10 kiloDaltons and about 5 microns with a preferred pore size that is about 0.5 miera. In the hollow fiber filter, each hollow tube had a body with a first end, a second end and a lumen located in the body and extending between the first end and the second end. The body of each hollow tube includes a plurality of pores. The pores are generally oriented in the body, such that a composition of regenerative cells is filtered by circulating through the lumen of the body, and the products to be filtered pass tangentially through the pores, as illustrated in Figure 12A . In other words, the smallest particles in the liquid pass tangentially through the pores with respect to the flow of fluid through the lumen of the body. The composition with the regenerative cells passes through the lumen of each hollow tube when the composition is filtered. Preferably, the flow of the composition is tangential to the pores of the body of each hollow tube. By using a tangential flow of fluid, the filtration efficiency of stem cells can be improved over other filtration techniques. For example, according to some filtration techniques, the pores of the filter media are positioned such that the filter is oriented perpendicular to the fluid flow, such that the filter medium blocks the route of the seeping fluid. , as illustrated in Figure 12B. In this type of filtration, particles that are filtered out of the composition of regenerative cells, for example stem cells, tend to accumulate on one side of the filter and block the flow of fluid through the pores. This blockage can reduce the efficiency of the filter. In addition, the cells are constantly compressed by the fluid flow pressure as well as the weight of the cells accumulates on the upstream side of the filter. This can lead to increased lysis of stem cells. In this way, in these filtration techniques where the fluid flow is parallel to the orientation of the pores in the filter, both large and small particles can undesirably be directed against the filter medium as the fluid is passed through. of the pores. Consequently, larger products in the liquid such as cells can block the pores, thereby decreasing the filtering effect and increasing an occurrence of injury or rupture of the cells. In contrast, in the hollow fiber configuration of the present system 10, the filtering fluid flows into the lumen of the hollow tube. The portion of the fluid that has the ability to pass through the pores of the body of the filter does so with the help of positive pressure of the fluid inside the body as well as a negative pressure that is applied to the outside of the body. In this embodiment, the cells typically do not undergo the fluid flow pressure or the weight of other cells, and therefore the shear forces in the stem cells are reduced. In this way, the efficiency and effectiveness of filtering can be improved by the reduction in filling rates and the reduction in lysis of regenerative cells. Due to the size of the saline and the unwanted protein molecules, during filtration, these molecules and other small components pass through the pores of the hollow tube bodies to the outside of the hollow tubes and are directed to the waste container 40. In one embodiment, filtering is improved by generating a vacuum on the outside of the hollow tube filter medium. Due to the size of the regenerative cells, for example stem cells or progenitor cells, these cells typically can not pass through the pores of the body and therefore remain inside the hollow tube filter (e.g., in the lumens of the tubes) and are directed from new to the processing chamber 30 by a conduit between the filter and the processing chamber, or to the outlet chamber 50. In a specific embodiment, the hollow fiber filter has approximately a pore size of 0.05 microns and contains approximately 550 cm2 of surface area of the filter medium. A single medium tube typically has a diameter of approximately 0.5 mm. By processing 130 ml of the regenerative cell composition, approximately 120 ml of additional saline can be added to the composition. The processing time or filter can be approximately 8 minutes. The differential of the pressures on both sides of the hollow fiber tube body (for example, the pressure inside the lumen of the body, and outside the body) is considered the trans-membrane pressure. The trans-membrane pressure may be in the range from about 1 mm Hg to about 500 mm Hg with a preferred pressure that is about 200 mm Hg. The viability and recovery of average nucleated cells using hollow fiber filtration can be about 80% viable cells. The amount of collagenase that is typically removed in said system is equal to a three log reduction. For example, if the initial concentration of collagenase in the composition of regenerative cells that are transferred from the collection chamber to the processing chamber is 0.078 U / ml, the co-genase concentration of the final regenerative cell composition would be 0.00078 U / ml . The collagenase is removed in the hollow fiber filter and the hollow fiber filter corresponds to the third filter discussed above. Processing chambers that illustrate one or more cell filtration methods described above are illustrated in the Figures, particularly Figures 1-3. With reference to Figures 1-3, between the processing chamber 30 and the filter chamber of the filter assembly 36, a pump, such as the pump 34, may be provided. In addition, ventilation and pressure sensors, such as ventilation 32 and the pressure sensor 39, can be provided in line with the processing chamber 30 and the filter assembly 36. Accessories for the exit chamber 50 can also be provided. These optional components (for example, the pump 34, the vent 32, the pressure sensor 39 and the accessories for the outlet chamber 50) can be provided between the processing chamber 30 and the filter assembly 36, such that the liquid contained in the processing chamber 30 can flow to one or more of these optional components before flowing through the filter assembly 36. For example, liquid can flow through the pump 34 before it is passed to the filter assembly 36. Or, liquid can pass through the pressure sensor 39 before passing through the filter assembly to obtain a pre-filter liquid pressure in the system. In certain situations, one or more of these components can also be provided as an element of the processing chamber 30, such as the vent 32 as illustrated in FIGURE 6. In the illustrated embodiment, the pressure sensor 39 is in line for determining the pressure of the composition of regenerative cells that is generated by the pump 34 as it enters the filter chamber of the filter assembly 36. This construction can facilitate the monitoring of the trans-membrane pressure through the filter membrane. Additional salt or other buffer and wash solution can be added to the regenerative cell composition to aid in the removal of unwanted proteins as the composition is filtered through the filter assembly 36. This repeated washing can be performed multiple times to improve the purity of the composition of regenerative cells. In certain modalities, the saline can be added at any stage considered necessary to improve filtration. In a specific embodiment, which is provided by way of example and not limitation, the unwanted proteins and the saline or other wash solution are removed in the following manner. The composition with the regenerative cells, as well as collagen and connective tissue particles or fragments, adipocytes and collagenase, are cycled through a series of filters until a minimum volume is reached. The minimum volume is a function of the total system retention volume and some predetermined constant. The retention volume is the volume of liquid that is contained in the pipe and ducts if all the processing chambers are empty. In one embodiment, the minimum volume is 15 ml. When the minimum volume is reached, a predetermined volume of wash solution is introduced into the system to mix with the composition of regenerative cells. This mixture of washing solution and the composition of regenerative cells is then cycled through the filters until the minimum volume is reached again. This cycle can be repeated multiple times to improve the purity of the regenerative cells, or in other words, increase the proportion of regenerative cells in the composition to the other materials in the composition. See Figures 10 and 11.

After it has been determined that the regenerative cell composition has been cleared of unwanted proteins and sufficiently concentrated (in exemplary embodiments, minimal concentrations can be used within a range of about 1x10 5 to about 1 x 10 7 cells / ml and, in a preferred embodiment, the minimum concentration may be about 1 x 10 7 cells / ml), an exit chamber 50, such as an outlet bag may be connected to the exit port of the processing chamber 30 and / or the filter 36, depending on the specific mode. Ventilation, such as ventilation 32, can then be opened to facilitate the exit of the concentrated regenerative cells. In an implementation, this determination of when a minimum concentration has been reached is made empirically after having run experiments and programmed into the electronic controls of the device. The determination can be a feeding to the process of what you want to produce, that is to say that so many stem / progenitor cells are desired, or the interval of cellular concentration.

Based on scientific data, a predefined amount of adipose tissue needs to be obtained and placed in the system to achieve the desired output. With the vent 32 open, a pump, such as the pump 34, can operate to transfer the concentrated regenerative cells into the outlet bag. In one embodiment, the outlet bag 50 is similar to an empty blood bag having a tube with an attachment at one end. In a sterile form, the accessory in the outlet bag can be connected to the outlet gate and the concentrated regenerative cells can be transferred to the outlet bag. As illustrated in Figures 1-3, a vacuum pump 26 may be provided in the system 10 to change the pressure in the system, among other things. For example, the vacuum pump 26 may be coupled to the collection chamber 20 via a conduit, such as the conduit 12b, to cause a decrease in pressure within the collection chamber 20. The vacuum pump 26 may also be coupled to the processing chamber 30 by a conduit, such as conduit 12g. Regarding the operation of the vacuum pump 26 in connection with the pump 34, two separate vacuum sources or pumps can be implemented, or a single one can be implemented by using valves that direct the vacuum application or vacuum extraction to the different conduits that require it at specific points in the process. In addition, the vacuum pump 26 can be coupled to the waste container 40 via a conduit, such as the conduit 12f. With reference to Figures 10 and 11, the pressure generated by the vacuum pump 26 can be used to direct the flow of fluids, including the regenerative cells, through the conduits 12. This pressure can be supplied in multiple directions, for example by automatic or manual control of the position of one or more valves 14 in the system 10. The system 10 can be made to function adequately with the use of positive pressure or through the use of negative pressure, or combinations thereof. For example, the regenerative cells can be extracted through the first and second filters described above in a container with soft sides that is connected to the third filter. The vessel with soft sides can be in line (serial) connected in front of the third filter. The final outlet chamber can be a container with a soft side, which is on the other side (for example, the downstream side) of the third filter. In this embodiment, pressure is used to move the regenerative cells from a container with a soft side to a second container with a smooth side through the filter.

In another embodiment of system 10, filtration of adipose-derived stem cells and / or progenitor cells can be accomplished using a combination of percolation filtration and sedimentation. For exampleSaid system uses saline which is passed through a composition of tissue regenerative cells (for example, the composition containing the stem cells and / or the progenitor cells derived from adipose tissue) and then through a filter. Some of the variables that are associated with percolation filtration of cells of a regenerative cell composition include, but are not limited to, pore size of the filter medium, geometry or pore shape, filter surface area, flow direction of the regenerative cell composition that leaked, infusion saline flow expense, trans-membrane pressure, dilution of cell population, cell size and viability. In one embodiment of the system 10, the processing chamber 30 utilizes a filter assembly 36 that implements percolation filtration and sedimentation to separate and concentrate the regenerative cells. By way of example, and not by way of limitation, the processing chamber 30 is defined as a generally cylindrical body having a side wall 30a, an upper surface 30b and a bottom surface 30c, as shown in Figure 6. A sterile vent 32 is provided in the upper surface 30b. In the embodiment of Figure 6, the processing chamber 30 is illustrated to include a filter assembly 36, which includes two filters, such as a large-pore filter 36a, and a small-pore filter 36b. The pore sizes of the filters 36a and 36b are typically in a range between about 0.05 microns and about 10 microns. The large pore filter 36a can comprise pores with a diameter of about 5 μm and the small pore filter 36b can comprise pores with a diameter of about 1-3 μm. In one embodiment, the filters have a surface area of about 785 mm2. The filters 36a and 36b divide an interior of the processing chamber 30 to include a first chamber 37a, a second chamber 37b, and a third chamber 37c. As shown in Figure 6, the first chamber 37a is located between the second chamber 37b and the third chamber 37c. In addition, the first chamber 37a is illustrated in the region of the processing chamber 30 having an inlet gate 31a and an outlet gate 3lb. The illustrated processing chamber 30 includes a plurality of gates that provide communication paths from an exterior of the processing chamber 30 to the interior of the processing chamber 30, such as the gates 31a, 31b and 31c. The gates 3la, 31b and 31c, are illustrated placed on the side wall 30a of a body of the processing chamber 30. However, the gates 31a, 31b and 31c can be located in other regions, equally. The gate 3la is illustrated as a sample inlet gate, which is constructed to be coupled to a conduit, such that a composition containing regenerative cells can be passed into the processing chamber 30. The gate 31b is illustrated as a outlet gate, constructed to be coupled to a conduit, such that separate and concentrated cells can be removed from the interior of the processing chamber 30. The gate 31c is illustrated as an inlet gate constructed to be coupled to a conduit for supply of a fresh wash solution, such as saline, into the processing chamber 30. In use, the regenerative cells may be introduced into the central chamber 37a via the inlet gate 3la. Saline or other buffer is introduced into the bottom chamber 37b through the inlet gate 31c.

Saline can be directed through the composition of regenerative cells in chamber 37a at a rate of about 10 ml / min. The flow rate of the saline is such that the force of gravity counterattacks. The flow of saline gives the cells in the chamber the ability to separate based on the density of the cells. Typically, since the saline is forced upwardly through the composition, the larger cells in the composition will settle to the bottom of the central chamber 37a, and the smaller cells and proteins will be entrained through the second filter 36b in the upper chamber 37c. This filtering is achieved by adjusting the flow rate of the saline so that the larger cells roll up on site, allowing the smaller particles to be released and washed away with the saline. Sterile ventilation 32 is included in chamber 30 to ensure that the correct pressure gradient is maintained in all three chambers within the processing unit. The upper chamber 37c may comprise an absorbent medium 33. The purpose of the absorbent medium is to trap the unwanted proteins in the solution to ensure that they do not cross the filter medium back into the processing solution, for example if the flow rate of the sample is decreased. saline. An absorbent medium can be a type of filter material that is absorbent or attracts materials or components to be filtered. An outflow gate can be added over the upper filter to help extract the waste. Another mode of this may be to apply a slight vacuum from the top to assist in extracting the waste. Absorbent media can be implemented when, as in the illustrated embodiment, the flow costs are relatively small. Excesses of saline and proteins are then dragged into the waste container. When larger cells, (for example, stem cells derived from adipose tissue and / or progenitor cells) have separated sufficiently from smaller cells and proteins, the composition containing the separated cells can be concentrated, as discussed. here. The composition can also be concentrated after it has been removed from the chamber 37a through the outlet gate 31b, or while it is in the chamber 37a. In one embodiment, the concentration of cells in the composition is increased in the following manner. After the cells have separated sufficiently, the filters such as the filters 36a and 36b can move towards each other. This movement has the effect of reducing the volume between the two filters (for example, the volume of the chamber 37a). A vibration member may also be provided in connection with the processing chamber 30 to facilitate concentrating the cells in the composition. In one embodiment, the vibration member may be coupled to the filter 36b (e.g., the small pore filter). The vibration can reduce an incidence of cells that are trapped in the filters. The reduction in volume of the composition allows the excess saline to be removed as waste and the cells to concentrate in a smaller volume. In another embodiment, the concentration of the regenerative cells is achieved in the following manner. After the cells have separated sufficiently, the composition of regenerative cells can be transferred to another chamber (not shown) that uses gravity to filter out excess saline. In a preferred embodiment, sedimentation may occur at the same time as percolation. This sedimentation can be achieved by introducing the composition to the top of a filter having a pore size in the range of about 10 kD to about 2 microns. In one embodiment, a suitable filter has a pore size of about 1 miera. The force of gravity will allow the saline and smaller particles to pass through the filter while preventing the cells in the composition from flowing through the filter. After the desired concentration of cells has been obtained and after the smaller filtered particles have been removed under the filter, the composition of regenerative cells can be agitated to remove the cells from the filter and subsequently the concentrated regenerative cells can be transferred to the output bag. Smaller particles can be extracted as waste through an outlet. In a particular embodiment of the invention, the processing chamber comprises a centrifugal chamber or a cell concentrator (Figures 4, 7 and 8) which facilitates the separation of the regenerative cells from the regenerative cell composition. For example, the cell concentrator may be a centrifugal device or part of a centrifugal device that can separate regenerative cells from the composition of regenerative cells based for example on size or density (Figures 7 and 8). The cell concentrator can also be a centrifuged membrane filter. It is recognized in the art that centrifugation is a means to separate and concentrate solutions having multiple components with varying densities. This is done by imparting a centripetal force on the solution, which for example is higher than gravity. The imparted force causes the tissue solution to separate based on the density of the cells and / or particles. After the cells have been concentrated enough, excess saline and proteins can be removed. Some of the variables that are associated with the centrifugal separation of cells from the solution include, but are not limited to, rotor speed, distance from the solution of the center of rotation and time.

The centrifugal device can be a component of the processing chamber 30, or it can be separated from the processing chamber. The centrifugal device may also be partially inside the processing chamber and partially separated from the processing chamber (see Figures 14 and 15). Typically, the centrifugal device causes a container containing the cell solution, eg, an exit chamber 50, to rotate about an axis thereby increasing the force in the cells in the solution to be greater than gravity. The denser or heavier materials in the solution typically settle at one end of the outlet chamber 50 to form a spin. The centrifugation can then be re-suspended to obtain a solution with a desired concentration of cells and / or desired volumes of cells and medium. In other embodiments, the processing chamber 30 itself may be in the form of a centrifuge chamber or cell concentrator. In general, said processing chamber is constructed to separate and concentrate cells using both gravitational and centrifugal forces. Specifically, during centrifugation, the centrifugal force directs the denser components of the regenerative cell composition, for example the regenerative cells, towards the outermost ends of the centrifugal chamber. Since the centrifugal chamber is braked and eventually stopped, the gravitational force helps the regenerative cells to remain at the outermost ends of the centrifugal chamber and form a cellular precipitate. Accordingly, the undesired components of the regenerative cell composition, i.e. the waste, can be removed without disturbing the cell pellet.

In a further embodiment of the centrifugation process, centrifugal elutriation can also be applied. In this embodiment, the cells can be separated based on the rate of sedimentation of individual cells such that the directional force (for example outward) applied by centrifugation causes the cells and solutes to settle at different rates. In elutriation, the sedimentation rate of the target or target cell population is opposed by a flow expense (for example inward) applied when pumping solution in the opposite direction to the centrifugal force. The counterflow is adjusted so that the cells and particles within the solution separate. Elutriation has been applied in many cases of cell separation (Inoue, Carsten et al., 1981; Hayner, Braun et al., 1984; Noga 1999) and the principles and practices employed to optimize flow and centrifugal parameters can be applied here in light of the present disclosure by a person skilled in the art. Figure 9 illustrates principles associated with an elutriation implementation in accordance with the present invention. The elutriation mode can be similar to a centrifugal implementation as long as a force is applied to the solution using a centrifuge rotor. Some of the variables that are associated with the currently incorporated elutriation separation include, but are not limited to, the size and shape of the spin chamber, the diameter of the rotor, the speed of the rotor, the diameter of the opposite flow pipe. , the flow expense of the opposite flow, as well as the size and density of the particles and cells that are to be removed from the solution. As in centrifugation, regenerative cells can be separated based on densities of individual cells. In one embodiment of the regenerative cell composition, for example the solution containing regenerative cells and collagenase, is introduced into a rotating rotor chamber as illustrated in Figure 9.1. After the solution is added to the chamber, additional saline is added to the chamber at a predetermined flow rate. The flow rate of the saline can be predetermined as a function of the rotor speed, the diameter of cells and the constant of the camera that has been established empirically. The flow rate will be controlled, for example, with a device similar to an IV pump. One purpose of the additional salt is to provide a condition inside the rotor chamber, where the larger particles will move to one side of the chamber and the smaller particles will move to the other, as illustrated in Figure 9.2. The flow is adjusted so that, in this application, the smallest particles will leave the chamber and move to a waste container, as illustrated in Figure 9.3. This movement results in the solution in the rotor chamber having a substantially homogeneous population of cells, such as stem cells. After it has been determined that the stem cells have been separated from the rest of the items in the solution (with the unwanted proteins and free lipids that have been removed from the chamber), the opposite flow is stopped. The cells within the chamber will then form a concentrated precipitate on the outer wall of the chamber. The opposite flow is reversed and the cell pellet is transferred to the outlet bag. An exemplary processing chamber 30 in the form of a centrifuge chamber or cell concentrator, illustrated in Figures 7 and 8, comprises a rotating seal 30.1 comprising an outer housing 30.2, one or more seals 30.3, one or more bearings 30.4 and a connection point 30.6, to connect the processing chamber to the centrifugal device of the system; one or more fluid paths 30.5, in the form of conduits extend out from the rotary seal and terminate in a centrifugal chamber at each end which is in the form of an outlet chamber 50 housed in a frame 53, wherein the frame it comprises one or more gates 52 and one or more handles for manually relocating the exit chamber 50. In certain embodiments, the processing chamber 30 in the form of a centrifugal chamber is constituted by one or more fluid routes 30.5 directing inside and outside the various components of the processing chamber, for example the exit chambers 50. In one embodiment, a fluid path 30.6 radiates out from the central axis of the processing chamber 30 and terminates near the outer ends of the chamber 30. processing chamber 30, ie within the centrifugal chambers housing the outlet chambers 50. Said fluid path can be used for example to transport the cell composition regenerating from a collection chamber 20 to the processing chamber 30. The fluid path can also be used to re-suspend the cell pellet that is formed after centrifugation. Accordingly, the placement and size of the fluid path must be optimized. The processing chamber may also comprise a fluid path 30.5 terminating in the central bottom portion of the processing chamber. Said fluid path can be used to remove supernatant or waste from the outlet chambers 50. Alternately, the processing chamber comprises a fluid path that terminates in the central bottom portion of the processing chamber that is used to remove supernatant or waste generated by the processing chamber itself 30. In certain embodiments, the processing chamber 30 has two fluid routes. Both fluid paths pass through the upper part of the processing chamber (ie, the center of the arrow on the rotary seal). One fluid path continues directly to the bottom of the processing chamber and the other is divided in two if it extends to the outer ends of the processing chamber, ie to the centrifugal or centrifuge chambers that house the exit chambers. In a reconfigured embodiment, the processing chamber 30 has the same general shape but one of the fluid paths moves or changes. In this mode, a fluid path continues to be straight from the arrow on the rotating seal to the bottom of the processing chamber. The second fluid path however divides out of the processing chamber and then connects to the outer ends of the processing chamber. In this embodiment, large output volumes can be generated since the fluid path can be used to add additives and re-suspension solutions to the centrifuge chamber and / or the output chambers directly. Processing chambers comprising a centrifuge device described above are illustrated in Figures 4.7-9 and 14-15. With reference to Figures 4 and 7-9, between the collection chamber 20 and the processing chamber 30, a pump 34 and one or more valves 14 may be provided. In a preferred embodiment, the valves 14 are electromechanical valves. In addition, sensors such as the pressure sensor 29 can be provided in line with the processing chamber 30 and the collection chamber 36. By using a processing chamber 30 shown in Figures 7 and 8, the composition of regenerative cells can be pumped from the collection chamber 20 on a route through the rotating seal network 30.1, comprising an outer housing 30.2, one or more seals 30.3 (for example lip seals), and one or more bearings 30.4.

In a preferred embodiment, the rotating seal network 30.1 including a rotating shaft further comprises two or more bearings 30.4, three or more lip seals 30.3, and an outer housing 30.2. In this embodiment, the bearings 30.4 further comprise an outer and inner arrow (not shown) referred to herein as channels. The channels can be separated by precision ground spheres. The channels and spheres comprising the bearings are preferably made of material suitable for contact with body fluid, or are coated with material suitable for contact with body fluid. In a preferred embodiment, the channels and spheres are manufactured using for example, silicon nitride or zirconium oxide. In addition, in this embodiment, the three lip seals are formed of a circular "U" shaped channel (not shown) as well as a circular spring (not shown). The circular "U" -shaped channel is preferably manufactured using flexible material such that a leak-proof joint is formed with the rotating shaft of the rotating seal network 30.1. Additionally, the lip seals are preferably oriented in such a way that the pressure of the composition of regenerative cells circulating through the processing chamber causes the seal assembly to tighten its junction with the rotary shaft by increased tension. The seals can be held in place by one or more circular fasteners (not shown) which are capable of expansion and / or collapse as required in order to couple a groove in the outer housing 30.2 of the rotating seal network 30.1. In general, the rotary seal network 30.1 is preferably designed in such a way that the multiple fluid paths, for example the fluid path 30.5, can be maintained in a sterile condition and can be accessed while the centrifugal chamber of the processing chamber tour. Accordingly, an advantage of this embodiment is that all areas of the processing chamber illustrated in Figures 7 and 8 can be accessed at any given time during the phase of separation and concentration of the system. Finally, the heat generated by or near the rotating seal network 30.1 must be controlled to avoid lysis of the cells in the solution traveling through the passage. This can be achieved, for example, by selecting a hard material to construct the rotating shaft, joining the area of the rotating shaft that comes into contact with the seals and minimizing the contact between the rotating shaft and the seal. In one embodiment, the rotary seal network 30.1 comprises a single rubber seal 30.3 and an air seal (not shown). This seal and packaging provide a tortuous path for any biological material that may compromise the sterility of the system. In another embodiment, the rotary seal network 30.1 comprises multiple spring-loaded seals 30.3 that isolate the individual fluid paths. Seals 30.3 are made of a material that can be sterilized as well as seal the rotating shaft without lubricant. In another embodiment, the rotating seal network 30.1 is comprised of a pair of ceramic discs (not shown) that create the different fluid paths and can support the rotation of the system and not cause cell lysis. In other embodiments, the fluid path is flexible and is allowed to wind and unbundle relative to the processing chamber. This is achieved by making the flexible fluid path rotate one revolution for every two revolutions of the processing chamber 30. This eliminates the need for a full rotary seal. In one embodiment, the processing chamber 30 is designed such that the fluid path enters through the axis of rotation of the rotating seal network 30.1 and then divides into a minimum of two fluid paths 30.5 each of which leads to opposite ends of the processing chamber 30 towards the exit chambers 50. According to this, in a preferred embodiment, the processing chamber 30 comprises two or more exit chambers 50 as illustrated in Figures 7 and 8. These exit chambers 50 are located such that they are in one orientation during 30.7 processing and another orientation to recover concentrated regenerative cells 30.8. The two positions of the outlet chamber 50 can be manually manipulated through a handle or handle 53 projecting out of the processing chamber 30. Once the regenerative cell composition is transferred to the processing chamber of FIG. 4, the composition is subjected to a load of, for example, approximately 400 times the force of gravity for a period of about 5 minutes. The outlet chamber 50 is constructed in such a way that the outer ends of the chamber form a small deposit for the particles and dense cells. The exit chamber 50 retains the dense particles in what is referred to as a "cell pellet," while allowing the lighter supernatant to be removed through the second fluid path (not shown). The exit chamber is further constructed such that the supernatant can be removed without disturbing the cell pellet. This can be achieved by the fluid path that is controlled with valves 14 and a pump 34 that helps remove the supernatant. The second fluid path is on the axis of rotation of the rotating seal network 30.1. This fluid path travels from the low point in the center of the processing chamber 30 through the rotary seal to the waste container 40. The cell pellet comprises the concentrated regenerative cells of the invention. In some embodiments, after the supernatant is removed and directed to the waste chamber 40, additional solutions and / or other additives may be added to the processing chamber 30 from the collection chamber 20 in the manner described above, for this way re-suspend the cellular precipitate. The re-suspension of the cellular precipitate in this way allows for greater washing of the regenerative cells to remove undesired proteins and chemical compounds as well as to increase the oxygen flow to the cells. The resulting suspension may be subjected to another charge of approximately 400 times the force of gravity for another period of about 5 minutes. After a second "cell pellet" is formed and the resulting supernatant is removed to the waste chamber 40, a final wash may be performed in the manner described above with saline or some other appropriate buffer solution. The final precipitate present in the outlet chamber 50 can then be recovered using an appropriate syringe after the outlet chamber 50 is located in the proper orientation for cell removal. In other embodiments, the final precipitate can be automatically moved to a container in the outlet chamber 50 that can be removed and stored or used as required. This container can be in any suitable shape or size. For example, the container can be a syringe. In all modalities, the final precipitate is removed aseptically. For example, an outlet container 50 can be automatically sealed and isolated from the other components of the processing chamber for subsequent recovery and use in proprietary therapeutic applications as described herein. In the illustrated embodiment of Figure 4, the pressure sensor 29 is in line to determine the pressure of the regenerative cell composition that is generated by the pump 34 as it enters the processing chamber 30. Additional salt or other buffer and solution Washings can be added to the composition of regenerative cells to aid in the removal of unwanted proteins as the solution is processed in the processing chamber 30. This repeated washing can be performed multiple times to improve the purity of the regenerative cell solution. In certain modalities, saline can be added at any stage deemed necessary to improve processing. In other embodiments, the processing chamber 30 or the exit chamber 50 may include one or more gates, for example gates 51 or 52. One or more of these gates may be designed to direct the regenerative cells, or a portion thereof, to other objects such as implant materials (e.g., scaffolds or bone fragments), surgical devices, cell culture devices, or purification devices. In these embodiments, the processing chamber 30 or the exit chamber 50 may additionally comprise a device for mixing the regenerative cells and additives. Mixing can be accomplished by any means known to those skilled in the art, including but not limited to agitation, rolling, inversion or by movement or pulsed compression rolls. The gates can also be used to add one or more additives, for example, growth factors, re-suspension fluids, cell culture reagents, cell expansion reagents, cell preservation reagents or cell modification reagents including agents that transfer genes to cells.

Other examples of additives include agents that optimize washing and disintegration, additives that improve the viability of the population of active cells during processing, anti-microbial agents (for example, antibiotics), additives that lyse adipocytes and / or red blood cells, or additives that enrich the cell population of interest (by differential adhesion to solid phase portions or otherwise promote the reduction or substantial enrichment of cell populations). For example, to obtain a population of homogeneous regenerative cells, any convenient method for separating the particular type of regenerative cells, such as the use of cell-specific antibodies that recognize and bind antigens present for example, in stem cells or progenitor cells, can be employed. , for example endothelial precursor cells. These include both positive selection (select target cells), negative selection (selective removal of unwanted cells), or combinations thereof. Intracellular markers such as enzymes can also be used in screening using molecules that fluoresce when activated by specific enzymes. In addition, a solid phase material with adhesive properties selected to allow differential adhesion and / or elusion of a particular population of regenerative cells within the final cell pellet can be inserted into the exit chamber of the system. An alternative embodiment of this differential adhesion approach would be to include the use of antibodies and / or combinations of antibodies that recognize differentially expressed surface molecules in target regenerative cells and unwanted cells. Selection based on expression of specific cell surface markers (or combinations thereof) is another commonly applied technique where antibodies are connected (directly or indirectly) to a solid phase support structure (Geiselhart et al., 1996; Formanek et al. al., 1998, Graepler et al., 1998, Kobari et al., 2001, Mohr et al., 2001). In another embodiment, the cell pellet can be re-suspended, placed in layers on (or below) a fluid material formed in a continuous or discontinuous density gradient and placed in a centrifuge for separation of cell populations based on density cell phone. In a similar modality, continuous flow approaches such as apheresis (Smith, 1997), and elutriation (with or without countercurrent) (Lasch et al., 2000) (Ito and Shinomiya, 2001) can also be employed. In all of the above embodiments, at least a portion of the separated adipose tissue-derived cells can be cryopreserved, as described in co-pending US patent application. Serial No. 10 / 242,094, with title PRESERVATION OF NON EMBRYONIC CELLS FROM NON HEMATOPOIETIC TISSUES (CONSERVATION OF NON-EMBRYOIC CELLS FROM NON-HEMATOPOYETIC TISSUES), presented on September 12, 2002, which claims the benefit of the provisional patent application of the US. Serial No. 60 / 322,070 filed September 14, 2001, commonly assigned and all of the contents of which are hereby expressly incorporated by reference. In a preferred embodiment, the entire system is automated. In another modality, the system has both automated and manual components. The system may comprise one or more disposable components mounted on a component or module of reusable physical equipment. The automated systems of the invention provide display displays (see Figure 16) that signal the proper operation of the system. The automated systems also provide a screen that provides procedural status and / or step-by-step instructions regarding the proper configuration of the disposable components of the system. The screen can also indicate problems or failures in the system if they occur and provide, if appropriate, guidance for "troubleshooting". In a modality, the screen that allows the user to interconnect with the system is a touch sensitive screen. Partially and fully automated systems may include a processing device (eg, microprocessor or personal computer) and associated software programs that provide the control logic to the system to operate and automate one or more stages of the process based on the selection of the user. The processing device can be operatively linked to one or more components or steps of the system. By way of example, steps susceptible to said automation include, but are not limited to, controlling the ingress and egress of fluids and tissues onto the particular pipe routes by controlling pumps and valves of the system or processing device.; control the appropriate sequence and / or direction of activation; detect blockages with pressure sensor; mixing mechanisms, measuring the amount of tissue and / or fluid to be moved on a particular route using volumetric mechanisms; maintain temperatures of the various components using thermal control devices; wash and concentrate the cells and integrate the process with synchronization mechanisms and software or programs. The automated system may also include pressure sensors to detect blockages and similar quality and safety control mechanisms. In a modality, program or software can control the parameters of the process to allow the production of a cell population prepared for parameters defined by the specific operator. For example, the processing device can control the centrifuge speeds based on the type of tissue processed and / or population or cell sub-population that is harvested. In this way, the automation device or devices improve the performance of the procedures and provide automatic harvesting of the adipose tissue and processing of the adipose tissue for administration to a patient. The processing device may also include parallel or standard serial gates or other means of communication with other computers or networks. Accordingly, the processing device can be a stand-alone unit or associated with another device. In certain embodiments, one or more aspects of the system may be programmable by the user through programs or software residing in the processing device. The processing device may have one or more pre-programmed in Read Only Memory (ROM). For example, the processing device may have software or software tailored to process blood, another program for processing adipose tissue to have small volumes of regenerative cells and another program for processing adipose tissue to obtain large volumes of regenerative cells. The processing device may have a program or pre-programmed software that provides the user with appropriate parameters to optimize the process based on the user's feeding of relevant information such as the number of regenerative cells required, the type of tissue that is required. process, the type of post-processing manipulation required, the type of therapeutic application, etc. The software can allow automated collection of "operation data" including, for example, the numbers of batches of disposable components, measurements of temperature and volume, tissue volume and parameters of cell numbers, dose of enzyme applied, incubation time, identity of the operator, date and time, patient's identity, etc. In a preferred embodiment of the device, a barcode reading system will be integrated to allow data entry of these variables (eg batch number of disposable equipment and expiration date, batch number and expiration date of the collagenase, patient / sample identifiers, etc.) in the processing device as part of the processing documentation. This will reduce the opportunity for data feeding errors. Said bar code reading system can be easily incorporated into the processing device using a USB gateway or other interface known in the art. In this way, the device will provide integrated control of data entry and process documentation. A printed report of these parameters will be part of the parameters defined by the user of a programmed operation of the system. Naturally, this will require integration of a printer component (tooling and controller) or printer driver into the program plus a printer output connector for a printer (for example a USB port) into the hardware or tooling of the device. In certain modalities, the system is a simple integrated system that does not require any user intervention to perform the various stages of the separation and concentration process or separate devices. In other embodiments, the system can be operated in a fully automatic mode without user power. The system can also be operated in a semi-automatic mode during which the system goes through certain stages without user intervention but requires user intervention before certain processes can occur. In other embodiments, the system is a simple integrated system that displays instructions to guide the user in performing predetermined operations at predetermined times. For example, the processing device can signal to the users the steps necessary for an adequate insertion of tubing, chambers and other components of! system. Accordingly, the user can ensure that the proper sequence of operations is carried out. Said system may additionally require confirmation of each operational stage by the user to avoid activation or accidental termination of stages in the process. In an additional mode, the system can initiate automated testing to confirm correct insertion of pipes, cameras, absence of blockages, etc. In yet another embodiment, the system of the present invention is capable of being programmed to perform multiple separation and concentration processes through automated control of tissue flow through the system. This feature may be important, for example during surgery in a patient where tissue that would otherwise be lost is collected in the system and tissue regenerative cells are separated and concentrated and returned to the patient. As stated previously, system components may be disposable (here referred to as "disposable equipment") such that portions of the system may be discarded after a single use. This implementation can help in ensuring that any surface that comes in contact with the patient's tissue will be discarded or disposed of properly after being used. Exemplary disposable equipment is illustrated in Figure 13. In a preferred embodiment, the disposable components of the system are pre-sterilized and packaged, to be usable "from inventory" which are easy to use and easy to load and which eliminate the need for many pipe connections and complex addressing of pipe connections. These disposable components are relatively inexpensive to manufacture, and therefore do not create a substantial expense due to their disposal. In one embodiment, the disposable system (referred to interchangeably herein as "disposable equipment"), comprises, consists essentially of or consists of, the collection chamber 20, the processing chamber 30, the waste chamber 40, the outlet 50, filter assemblies 36, sample bag 60 and associated conduits 12 or pipe. In preferred embodiments of the disposable equipment of the system, the collection chamber 20 and the processing chamber 30 are connected by conduits 12 which are housed in a rigid frame. The rotary seal network (Figures 7 and 8) of a processing chamber 30 can also be housed in the same rigid frame. In another preferred embodiment, the various chambers and containers of the disposable equipment are constituted by necessary interfaces that are capable of communicating with the processing device of the system in such a way that pumps, valves, sensors and other devices that automate the system are activated or deactivate appropriately as required without user intervention. The interfaces also reduce the time and dexterity required to configure the system and also reduce errors by indicating how to properly configure the system and alert the user in the case of an erroneous configuration. Disposable equipment may further comprise one or more needles or syringes suitable for obtaining adipose or other tissue from the patient and returning regenerative cells to the patient. The type and variety number of needles and syringes included, will depend on the type and amount of processed tissue. The disposable equipment may further comprise one or more rigid or flexible containers for containing washing fluids and other processing reagents employed in the system. For example, disposable equipment may comprise containers for containing saline, enzymes and any other treatment or replacement fluids required for the process. In addition, convenient washing solutions, re-suspension fluids, additives, agents or transplant materials can be provided with the disposable equipment to be used in conjunction with the systems and methods of the invention. The reusable component of the system comprises, consists essentially of, or consists of the stirring mechanism for the collection chamber, the pump and assorted sensors that activate valves and pump controls, the centrifugal motor, the rotating frame of the centrifugal motor, the User interface screen and USB gates and other associated devices. An exemplary reusable component is illustrated in Figure 14. Reusable tooling can be employed with a variety of disposable equipment. For example, reusable tooling can be used with disposable equipment to separate and concentrate regenerative cells from a wide variety of tissues as described herein. In one embodiment, disposable equipment for use in the system comprises a collection chamber 20 that can accommodate approximately 800 mL of tissue; a processing chamber 30 capable of processing the regenerative cell composition generated by approximately 800 mL of washed and digested tissue in the collection chamber 20; an exit chamber 50 that can accommodate at least 0.5 mL of regenerative cells; and a waste container 40 that can accommodate approximately 10 L of waste. In this modality, the physical equipment device is not greater than 60.96 x 45.72 x 91.44 cm (24 X 18 X 36 inches), length, width and height, respectively. Alternate dimensions of the various components of the disposable equipment as well as the physical equipment device may be constructed as required and are intended to be encompassed by the present invention without limitation. The disposable components of the system are easy to place on the device. An illustration of a disposable equipment used assembled in conjunction with a corresponding reusable component is illustrated in Figure 15. The preferred system is designed such that it can detect an improperly loaded disposable component. For example, the components of each disposable equipment may have color-guided markings to properly align and insert tubing, chambers, etc., into appropriate locations in the system. In additional embodiments, the system described herein is a portable unit. For example, the portable unit may be able to move from one site to another where adipose tissue collection has occurred, to another site for adipose tissue collection. In certain implementations, the portable unit is suitable for collecting and processing adipose tissue next to a patient's bed. In this way, a portable unit can be part of a system that can be passed from one patient to another. Accordingly, the portable unit can be wheeled, which is locked in place and thus can be easily placed and used in a convenient location in a stable and secure position throughout the process. In other embodiments, the portable unit is designed for configuration and operation on a flat surface such as a counter. The portable unit can also be circumscribed in a housing unit. The portable unit may also comprise hooks, hangers, labels, scales and other devices to aid in the procedure. All the reusable system components described herein such as the centrifuge, processing device, display screen, can be mounted on the portable unit of the system. Alternative manual modes for obtaining regenerative cells are also within the scope of this invention. For example, in one embodiment, adipose tissue can be processed using any combination of the components of the system, equipment and / or supplies described herein. The regenerative cells obtained by the above methods can be mixed with fragments of adipose tissue and administered to a patient without further processing, or they can be administered to a patient after mixing with other tissues, cells, implants or devices. In certain embodiments, the regenerative cells are mixed with one or more units of adipose tissue that have not been similarly processed. In this way, when practicing the methods of the invention, a composition comprising adipose tissue with an improved concentration of regenerative cells, can be administered to the patient. The volumes of the various adipose tissue units may be different. For example, a volume can be at least 25% greater than the volume of another adipose tissue unit. In addition, a volume can be at least 50%, such as at least 100% and even 150% or more, greater than the volume of another adipose tissue unit. In addition, the desired composition can be obtained by mixing a first unit of adipose tissue with the population of concentrated regenerative cells, which can be a cell pellet containing the regenerative cells., with one or more other adipose tissue units. In certain embodiments, these other units will not have an increased concentration of regenerative cells, or in other words, will have a lower concentration of regenerative cells than that contained in the first adipose tissue unit. In other embodiments, one of the units is cryopreserved material containing, for example, an increased concentration of regenerative cells. At the end of processing, the concentrated cells can be loaded into a delivery device, such as a syringe, to be placed in the recipient either subcutaneously, intramuscularly or another technique that allows delivery of the cell / tissue mixture to the target site within the patient, for example the periurethral region, the subcutaneous space below a wrinkle, or within the chest. In other words, cells can be placed in the patient by any means known to persons of ordinary skill in the art. Preferred modalities include placement by needle or catheter, or by direct surgical implant.

In the surgical implant modality, the mixture of cells and tissue can be applied in association with additives such as a preformed matrix. The population of active cells can be applied alone or in combination with other cells, tissue, tissue fragments, growth factors such as VEGF and other known angiogenic or arteriogenic growth factors, inert or biologically active compounds, re-absorbable plastic scaffolds, or other additives intended to improve the supply, efficacy, tolerability or function of the population. The cell population can also be modified by insertion of DNA or by placing cell culture in such a manner as to change, improve or supplement the function of the cells for derivation of a therapeutic or structural purpose. For example, techniques for gene transfer for stem cells are known to those of ordinary skill in the art, as described in (Morizono et al., 2003; Mosca et al., 2000), and may include viral transfection techniques. and more specifically, adeno-associated virus gene transfer techniques as described in (Walther and Stein, 2000) and (Athanasopoulos et al., 2000). Techniques that are not viral based can also be performed as described in (Muramatsu et al., 1998). In another aspect, the cells can be combined with growth factors that encode genes, for example a factor or several angiogenic growth factors that can allow the cells to act as their own source of the growth factor. The addition of the gene (or combination of genes) can be by any technology known in the art, including but not limited to adenoviral transduction, "particle accelerators", liposome-mediated transduction and lentivirus-mediated transduction or retrovirus, plasmid, adeno virus -associated. The cells can be implanted together with a carrier material containing a gene delivery vehicle capable of releasing and / or presenting genes to the cells over time so that transduction can be continued or initiated in situ. When the cells and / or tissue containing the cells are administered to a patient other than the patient from whom the cells and / or tissue were obtained, one or more immunosuppressive agents can be administered to the patient receiving the cells and / or tissue for reduce and preferably avoid rejection of the transplant. As used herein, the term "immunosuppressant agent or drug" is intended to include pharmaceutical agents that inhibit or interfere with normal immune function. Examples of suitable immunosuppressive agents with the methods described herein include agents that inhibit T cell / B cell co-stimulation pathways, such as agents that interfere with the coupling of T cells and B cells by the CTLA4 and B7 pathways, as is described in the US patent publication No. 20020182211. A preferred immunosuppressive agent is cyclosporin A. Other examples include myophenylate mofetil, rapamycin, and anti-thymocyte globulin.

In one embodiment, the immunosuppressant drug is administered with at least one other therapeutic agent. The immunosuppressant drug is administered in a formulation that is compatible with the route of administration and is administered to a subject at a dose sufficient to achieve the desired therapeutic effect. In another embodiment, the immunosuppressant drug is administered transiently for a sufficient time to induce tolerance to the regenerative cells of the invention. In certain embodiments of the invention, the cells are administered to a patient with one or more cell differentiation agents, such as cytokines and growth factors. Examples of various cell differentiation agents are described in (Gimble et al., 1995; Lennon et al., 1995; Majumdar et al., 1998; Caplan and Goldberg, 1999; Ohgushi and Caplan, 1999; Pittenger et al., 1999; Caplan and Bruder, 2001; Fukuda, 2001; Worster et al., 2001; Zuk et al., 2001). In another aspect, the cell population can be placed in the recipient and enclosed by a re-absorbable plastic liner or other materials such as those manufactured by MacroPore Biosurgery, Inc. (U.S. 6,269,716; 5,919,234; 6,673,362; 6,635,064; 6,653,146; 6,391,059; 6,343,531; 6,280,473). In this scenario, the lining would prevent prolapse of muscle and other soft tissues in the area of a defect processed with cells derived from adipose tissue to promote controlled repair of the defect. This approach can be used in reconstructive surgery where the liner can be pre-molded to the desired final shape allowing the tissue to be molded in vivo to the desired shape. In this regard, the beneficial effect can be improved by supplementing with additional components such as pro-adipogenic or angiogenic protein growth factors or biological or artificial scaffolds. As described herein, numerous defects and disorders can be treated with the regenerative cells that are obtained using the systems and methods of the invention. For example, in mammoplasty for breast augmentation, correction of soft tissue defect and / or urinary incontinence treatments, improved adipose tissue with regenerative cells can improve neovascularization and decrease necrosis in the implant, thus resulting in improved grafting and reduction in the risk of formation of liponecrotic pseudo-cysts. Improved adipose tissue with regenerative cells can be used to correct soft tissue defects and the like as described above. The addition of this population of concentrated regenerative cells to the normal adipose tissue graft can improve graft longevity without providing a supporting microenvironment. In addition, the compositions described herein can also be used to provide structural support of the lower esophageal sphincter as well as the external anal sphincter to treat gastroesophageal reflux disease (GERD) and fecal incontinence (Bernardi, Favetta et al., 1998), respectively. Typically, a person who is considered a candidate for conventional augmentation mammoplasty is a candidate for breast augmentation by increased autologous fat transfer with cells derived from adipose tissue. In addition to those candidates who are considered eligible for conventional augmentation mammoplasty, these methods may apply to the population of people seeking a small / moderate liking, shape change or contour alteration of one or both breasts, which may not be technically possible or aesthetically acceptable with the existing implant technology. Candidates for soft tissue augmentation are similarly candidates for autologous fat transfer procedures using autologous adipose tissue enhanced with cells. Examples of soft tissue augmentation procedures include but are not limited to: contour deformities of the face including but not limited to facial folds (e.g., glabellar, nasolabial) perioral lines, nasolabial folds, skin pieces; buttocks; calves; genitals; retro-orbital and plant fat cushions. A person who is considered a candidate for urethral bulging injection is also a candidate for autologous fat transplantation with the improved adipose tissue with cells described herein. These procedures may include transurethral as well as periurethral injection in women, as well as transurethral or anti-male injection in men. A pre-operative evaluation typically includes routine history and physical examination in addition to full informed consent describing all the relevant risks and benefits of the procedure. After identification of a patient candidate, the patient typically undergoes adipose tissue collection. The patient's habits can be examined for a proper cycle for collection and adipose tissue.

The procedure can be performed in bed or in an operations suite with appropriate hemodynamic monitoring of the patient's clinical status. Some preferred collection sites will be characterized by: one or several potential spaces limited by normal anatomical structures, without visceral or vascular structures greater in risk for damage and easy access. While virgin collection sites are typically preferred, a prior collection site does not prevent additional adipose tissue collection. These preferred sites include but are not limited to the following: lateral thigh and middle regions of bilateral lower limbs, cloth (pannus) of anterior abdominal wall, and bilateral flank regions. These procedures can often be performed concomitantly with liposculpture. The adipose tissue collection site can also be determined by the patient's aesthetic expectations as well as the safety profile as determined by the physician. The area to be collected is injected subcutaneously, for example with a standard tumescent fluid solution, which may or may not contain a combination of lidocaine, saline and / or epinephrine in different standardized dosing regimens. Using a scalpel with scalpel 11 (or other standard scalpel), a small perforated lesion is made in order to cross the dermis near the harvest area. The knife is rotated, such as turning 360 degrees to complete the wound. A 14-gauge blunt-tipped cannula (or appropriate size) can then be inserted into the subcutaneous adipose tissue plane. The cannula can be connected to an energized suction device or to a syringe for manual aspiration. The cannula is then passed through the plane to break the connective tissue architecture. The volume of aspirate obtained could be in the range from about 0 cc to about 1500 cc. A fraction or portion of adipose tissue collected in this way will be processed for isolation and concentration of regenerative cells using the methods described herein. The rest of the adipose tissue can be processed to re-implant in the patient according to the currently accepted standard of care or attention. Alternatively, the patient can be removed adipose tissue through a lipectomy procedure. After removal of adipose tissue, hemostasis will be achieved with standard surgical techniques and the wound is closed primarily. Adipose tissue collection may take 1-2 hours before transplant procedures. However, the timing of the collection may vary and may depend on the standards of quality of care or attention. Finally, the physician responsible for managing patient care will determine the timing of collection. In another embodiment, cells derived from adipose tissue can be used, they will have to be cryopreserved in a cell bank installation derived from adipose tissue. The volume of adipose tissue collected will typically vary from about 1 cc to about 1500 cc. Preferred methods of tissue collection will be followed to meet the standards accepted in quality of care. The volume of fat removed will vary from patient to patient and may depend on a number of factors including but not limited to: amount of adipose tissue required for augmentation mammoplasty, esthetic expectations, age, body habits, coagulation profile, hemodynamic stability, -morbidity and doctor's preference. After completion of tissue processing, the patient may be prepared to undergo adipose tissue transplantation in connection with augmentation mammoplasty, soft or soft tissue augmentation and / or urinary incontinence treatment. Some aspects that surround the transplant include synchronization, cell dose, route, method, location and supervision. In certain embodiments of the invention, the adipose tissue enhanced with regenerative cells is administered to the patient at the time of transplant procedures. These methods do not exclude the need for multiple injections of material over time. Finally, the synchronization used will follow quality standards in the service. In additional modalities, an alternate synchronization regime may exist if the cells to be applied undergo modification, purification, stimulation or other manipulation as discussed above. The cellular dose to be delivered to an individual patient will typically be determined from cell performance after adipose tissue processing. All collected cells may not be required for particular procedures, and the remaining portions of the cells may be cryopreserved as described herein. In one embodiment, the minimum number of cells to be delivered to the patient is expected to be 5.5 x 105 per 50 cc of transplanted fat. However, it can be expected that this value will be changed by orders of magnitude to achieve the desired effect. The injection of increased adipose tissue (overcorrection) is not an uncommon practice, since a percentage of the injected volume is expected to return over time. In addition, because the methods described herein do not exclude the need for a series of doses, more cells than previously indicated can be administered to the patient. In breast augmentation procedures, the delivery route may include open delivery through a standard 14 gauge blunt cannula inserted into the chest tissue through an armpit, periareolar, inframammary in soft tissue augmentation procedures. In soft tissue augmentation procedures the delivery route may include open delivery through a standard 14 gauge blunt cannula inserted into the soft tissue by an appropriately placed incision. In urinary incontinence procedures, the delivery route may include direct injection into the neck of the bladder and nearby urethra through cystoscopic visualization. Alternatively or additionally, the transplant can be delivered by an antiseptic route. Alternatively or additionally, tissue enhanced with cells can be delivered through an intravenous route that will be accessed by currently accepted methods. In the intravenous method, controlling the directional flow of transplanted material can be achieved through endocrine and paracrine traffic resulting from the inflammatory process initiated by surgical intervention. The routes discussed here do not exclude the use of multiple routes to achieve the desired clinical effect or umbilical incision. Alternatively or in addition, tissue enhanced with cells can be delivered through a transaxillary endoscopic subpectoral approach. In one embodiment, regenerative cells obtained from adipose processing are mixed with fat to be transplanted in the above-described proportion. This mixing can occur through automated means (e.g., centrifugation or device controlled agitation) or through manual methods (e.g., syringes with luer interlock, whirlpool). Adipose tissue enhanced with regenerative cells is preferably administered in a tear-like form to maximize the ratio of surface area to volume. In another embodiment, regenerative cells can be resuspended in an artificial or natural medium or tissue scaffold, which is then inserted into the implant region, such as the chest region and / or the intrinsic sphincter region to achieve the desired effect. In a preferred embodiment, the combined cells and tissues are injected following the process generally referred to as the "Coleman Technique" (Coleman 1991).; Coleman 1995; Coleman 2001). The collection of adipose tissue can be carried out in any appropriate clinical setting, such as the following sites: clinic, doctor's office, emergency department, hospital corridor, intensive care unit, operating rooms, catheterization suite and radiological suite. The augmentation mammoplasty is typically performed in an external patient configuration, but can be performed in the internal environment. A urethral bulge injection is typically performed in an external patient configuration but can be performed in the internal patient environment. In breast augmentation procedures, the increased autologous fat transfer effect with regenerative cells may be manifested by one or more of the following clinical measures: increased breast size, altered breast shape, altered breast contour, sustained graft, decreased proportion of formation of liponecrotic cysts, improved patient satisfaction and decreased use of implantable foreign material. In other soft tissue augmentation procedures, the increased autologous fat transfer effect with regenerative cells may be manifested by one or more of the following clinical measures: improved soft tissue shape, improved tissue function, improved soft tissue contour, sustained graft, improved quality of life of the patient and decreased use of implantable foreign material. In urinary incontinence procedures, the increased autologous fat transfer effect with regenerative cells for sphincter support may be manifested by one or more of the following clinical measures: decreased incontinence frequency, sustained graft, improved quality of life of the patient and decreased use of implantable foreign material. The effect of cell therapy typically takes place over the course of days to weeks. However, a beneficial effect can be observed as early as several hours and may persist for years. Patient supervision before, during and after the delivery of transplanted adipose tissue may include, but is not limited to, the following: coagulation studies, oxygen saturation, hemodynamic monitoring and wound status. Patients may be notified that they should receive pre-operative diagnostic procedures, such as mammograms, as there may be considerations that calcifications may form that distort the ability to detect malignant breast calcification. However, notification may be optional due to the use of magnetic resonance imaging training to overcome this limitation. Additional supervision will be specific to the desired clinical effect. The supervision of the patient before, during and after the delivery of the transplanted adipose tissue may include the following: urinalysis, pelvic examination, cystoscopy and urodynamic evaluation, coagulation studies, oxygen saturation, hemodynamic monitoring and wound status. Additional supervision will be specific to the desired clinical effect. The following examples are provided to demonstrate particular situations and environments in which this technology may be applied and are not intended to restrict the scope of the invention and the claims included in this disclosure. EXAMPLES EXAMPLE 1: Expression of Angiogenic Growth Factor, VEGF by ADC The Vascular Endothelial Growth Factor (VEGF) is one of the key regulators of angiogenesis (Nagy et al., 2003; Folkman, 1995). The Placenta Growth Factor, another member of the VEGF family, plays a similar role in both angiogenesis as well as arteriogenesis. Specifically, transplantation of wild type cells (P1GF + / +) in a mouse with inoperable PIGF genes restores the ability to induce rapid recovery of ischemia of the hind limb (Scholz and collaborators, 2003). Given the importance of angiogenesis and arteriogenesis in the revascularization process, the expression PIGF and VEGF by regenerative cells of the present invention was examined using an ELISA assay (R & amp; amp; amp; amp;; D Systems, Minneapolis, MN) using regenerative cells derived from adipose from three donors. One donor had a history of hyperglycemia and type 2 diabetes (a condition highly associated with microvascular and macrovascular disease). Regenerative cells from each donor were coated at 1,000 cells / cm2 in DMEM / F-12 medium supplemented with 10% FCS and 5% HS and developed to confluence. Samples of supernatants were taken and assayed for expression of PIGF and VEGF protein. As illustrated in Figures 16A and 16B, the results demonstrate robust expression of both VEGF (Figure 16A) and PIGF (Figure 16B) other adipose-derived regenerative cells of the invention. In a separate study, the relative amount of angiogenic related cytokines secreted by regenerative cells grown from normal adult mice, their measure. Regenerative cells were grown in alpha-MEM with 10% FBS at five days beyond cell confluence, at which time the cell culture medium was collected and evaluated by orderly antibody analysis (RayBio Mouse Cytokine Antibody Array II ( RayBiotech, Inc.). The following angiogenic related growth factors were detected: Vascular Endothelial Growth Factor (VEGF), bFGF, IGF-11, Eotaxin, G-CSF, GM-CSF, IL-12p40 / p70, IL-12 p70, IL-13, IL-6, IL-9, Leptin, MCP-1, M-CSF, MIG, PF-4, TIMP-1, TIMP-2, TNF-a, and Thrombopoietin. These data demonstrate that the regenerative cells of the present invention express a broad set of angiogenic and arteriogenic growth factors. Furthermore, the finding that a diabetic patient expresses VEGF and P1GF at equivalent levels with those of normal patients, suggests that diabetic patients may be candidates for angiogenic therapy by regenerative cells derived from autologous adipose. EXAMPLE 2: ADC Contains Cell Populations That Participate in Angiogenesis Endothelial cells and their precursors, endothelial progenitor cells (EPCs), are known to be involved in angiogenesis. To determine whether EPCs are present in adipose-derived regenerative cells, adipose-derived regenerative cells were evaluated for EPC cell surface markers, for example CD-34. ADCs were isolated by enzymatic digestion of human subcutaneous adipose tissue. ADCs (100 μl) were incubated in phosphate buffered saline (PBS) containing 0.2% fetal bovine serum (FBS), and incubated for 20 to 30 minutes at 4 degrees C with fluorescent labeling antibodies directed toward human CD endothelial markers -31 (differentiated endothelial cell marker) and CD-34 (EPC marker), as well as human ABCG2 (ATP binding cassette transporter), which is selectively expressed in multipotent cells. After washing, cells were analyzed in a FACSAria Sorter classifier (Beckton Dickenson-Immunocytometry). Acquisition of data and analysis were then performed by the FACSDiva program (BD-lmmunocytometry, CA). The results (not shown) showed that adipose-derived regenerative cells from a healthy adult expressed the CD-34 marker EPC and ABCG2, but not the endothelial cell marker CD-31. Cells expressing the EPC CD-34 marker were detected at a similar frequency in regenerative cells derived from a donor with a history of diabetes. To determine the frequency of EPCs in human adipose-derived regenerative cells after their culture in endothelial cell differentiation medium, ADCs were coated on fibronectin-coated plates and cultured in endothelial cell media for three days to remove mature endothelial cells. Non-adherent cells were removed and re-coated. After 14 days, colonies were identified by staining with Ulex europaeus Aglutinin-1 conjugated with FITC (Vector Labs, Burlingame, CA) and acetylated LDL Dil-labeled (Molecular Probes, Eugene, OR). As illustrated in Figure 17, the results indicate an EPC frequency of approximately 500EPC / 106 ADC cell. The presence of EPCs within regenerative cells derived from adipose tissue indicates that these cells can directly participate in the development of new blood vessels and enhanced angiogenesis and reperfusion. EXAMPLE 3: In Vitro Development of Vascular Structures in ADC A recognized assay in the art for angiogenesis is that in which endothelial cells grown in a fibroblast feeder layer develop a complex network of CD31 -positive tubes that resemble a nascent capillary network (Donovan et al., 2001). Since adipose-derived regenerative cells contain endothelial cells, EPCs and other stromal cell precursors, we tested the ability of these regenerative cells to form capillary-like structures in the absence of a feeder layer. Regenerative cells that are obtained from inguinal fat pads of normal mice developed capillary networks two weeks after culture (Figure 18A). Remarkably, regenerative cells of hyperglycemic mice with type 1 diabetes induced by streptozotocin (STZ) eight weeks after administration of equivalent capillary networks formed with STZ as those formed by cells from normal mice (Figure 18B). In a subsequent study, adipose-derived regenerative cells were cultured in complete culture medium (alpha-MEM supplemented with FCS at 10%) and without additional growth factors. These regenerative cells also formed capillary networks. In addition, the vascular structures formed were stained positive for the endothelial cell markers CD31, CD34, VE-cadherin and Willebrand factor / Factor VIII, but not the pan-lymphocyte marker, CD45. To compare the ability of regenerative cells of young versus older mice to form capillary networks, regenerative cells from normal and older young mice (ages 1, 12, or 18 months) were cultured for 2 weeks in complete culture medium (alpha-MEM) supplemented with 10% FCS) and without additional growth factors. Equivalent capillary-like networks were observed in regenerative cell culture of all donors (not shown). The above data demonstrate that adipose-derived regenerative cells from normal and diabetic patients, as well as young and old can form vascular structures consistent with the formation of nascent capillary networks. Accordingly, the regenerative cells of the invention can be used to treat angiogenic insufficiencies. EXAMPLE 4: In Vivo Development of Vascular Structures in ADC Angiogenic potential in vitro, while promising, is of little value if the cells do not exert an angiogenic activity in vivo. Surgical induction of ischemia in the hind limb is an in vivo model capable of identifying the angiogenic potential of a given therapy. This model was developed in immunodeficient mice (NOD-SCID) where the ability of human cells to promote reperfusion can be observed. Pre-operative blood flow values were determined for both hind limbs as described below. The vasculature of anesthetized mice was ligated with 4-0 silk at the following sites: 1) iliac artery near its bifurcation, 2) just distant to the origin of the deep femoral artery, 3) just proximal to branch of the superficial femoral artery. After ligating, the vasculature was removed in block. Before closure of the wound, roughly observable collaterals that branch off the ligated femoral arteries were also ligated. Twenty-four hours later, 129S mice were injected with 5 X 10 6 regenerative cells derived from syngeneic mouse adipose and non-SCID mice were injected with human adipose-derived regenerative cells through the tail vein. The flow was imaged immediately after surgery and at intervals after treatment using a Doppler Flow Imager (Moor Instruments Inc., Wilmington, DE). Measurements, taken three times a week for 24 days, were normalized to the pre-operative value for that extremity and presented with respect to the control extremity (not operated). The model of ischemia of the hind limb or posterior is extremely sensitive to the mouse strain used. NOD SCID mice are immunodeficient animals, lacking the ability to activate an acute inflammatory response. For these mice, this surgical approach generates severe ischemia such that two-thirds of untreated animals lose posterior limb structures below the femoral excision site. No animal treated with cells lost any structure on the toe or toe. However, for immunocompetent 129S mice, no untreated animal lost any structure on the phalanges and exhibited an endogenous ability for partial regeneration of reperfusion. This could be due to intrinsic angiogenesis associated with an acute inflammatory response. This may explain why reperfusion was less extreme when comparing treated animals versus those of control of different strains. However, the results showed that mice treated with adipose-derived regenerative cells showed significantly improved perfusion compared to untreated mice from both strains. On day 12, blood flow was restored to 50 + 11% in NOD-SCID mice treated with human regenerative cells, compared to 10 + 10% in untreated mice (p <; 0.05). Similarly, 129S immunocompetent mice exhibited 80 + 12% flow restoration on day 14, compared to 56 + 4% in untreated mice. In addition, the net dissection of mice revealed the appearance of collateral vessels in the hind limbs of mice treated with regenerative cells, but not in those of untreated mice or in the healthy extremities of any mouse. EXAMPLE 5: Increased ADC Dosage Is Associated with Improved Graft Survival and Angiogenesis Autologous adipose tissue transplantation is a relatively common procedure in plastic and reconstructive surgery. { Fulton, 1998; Shiffinan, 2001. #} . However, this procedure is limited by the fact that adipose tissue fragments are transferred without vascular supply and as a result, graft survival depends on neovascularization (Coleman, 1995; Eppley et al., 1990). In this way, in a limited way, the transplanted tissues represent an ischemic tissue. A study in Fisher rats was performed where fragments of adipose tissue were transplanted in the subcutaneous space on the muscles of the outer thigh. The right leg was transplanted with 0.2 g of adipose tissue fragments alone, the left leg was 0.2 g of adipose tissue fragments supplemented by the addition of adipose-derived stem cells at three different doses (1.7x105- 1.3x106 cells / graft; animals per dose); in this way, the contralateral leg acted as control. Then the animals were kept for a month after which they were euthanized and the grafts were recovered, weighed, fixed in formalin and embedded in paraffin for histological analysis. As illustrated in Figure 9A, the results show minimal retention of grafted tissue in the control paw and a dose-dependent increase in graft weight retention in the treated paw. In addition, immunohistochemical analysis of the grafts showed considerable neoangiogenesis and perfusion in the grafts treated with adipose-derived stem cells (Figure 20B, arrows). This was also associated with retention of adipose tissue morphology (Figure 20B). Accordingly, Examples 1-5 demonstrate that the adipose derived regenerative cells of the invention secrete angiogenic and arteriogenic growth factors; form nascent capillary networks in vitro; improves the survival of fat grafts; and improve ischemic reperfusion. In this manner, the regenerative cells of the invention are capable of promoting angiogenesis and arteriogenesis and can be functional to treat multiple diseases with underlying circulatory insufficiencies. EXAMPLE 6: Increase in Autologous Fat Transfer by Adipose-derived Regenerative Cells in Mice The potential of adipose-derived regenerative cells (ADCs) to increase autologous fat transfer was tested in mice. ADCs were obtained from mice that transport the transgene elacZ (Rosa 26 mice (B6; 129S-Gt (ROSA) 26Sor), usually known as Rosa 26 mice). ADCs were mixed with adipose tissue obtained from inguinal fat pads of C57 B16 / S129 F1 histocompatible mice according to the methods described herein and implanted in the subcutaneous space on the back of the skull of additional F1 mice. After one month, the implants were recovered and stained overnight in X-gal solution. Cells that express the lacZ transgene (ADCs) will stain blue upon exposure to X-gal. The implant showed blue staining through the tissue. The implant was thus embedded in paraffin, sectioned and stained with an antibody to the receptor for Vascular Endothelial Growth Factor 1 of Mouse (VEGFR1). Figure 19 shows the staining results where the implant is shown to contain numerous circular structures consisting of cells containing blue granules (arrows) and which were also positive VEGFR1 (dark coloration on cell cytoplasm). These data confirm the ability of processed liposuction to induce the formation of new blood vessels within the implant. EXAMPLE 7: Increase in Autologous Fat Transfer by Adipose-derived Regenerative Cells in Rats Adipose tissue fragments extracted from Wistar rats spawned by inbreeding were mixed with adipose derived regenerative cells according to the methods described herein. This composition is then implanted subcutaneously in the thigh and under the scalp of recipient rats. As controls, an equal amount of animals received adipose tissue alone (without ADCs) under the scalp, while animals that receive an implant in the thigh had the contralateral thigh implanted with adipose tissue alone. Grafts were harvested one month after the implant. The results (Figure 20) show an increased graft weight tendency of the thigh implants with increased dose of adipose derived regenerative cells. Histological examination of the implants showed improved vascularity of the grafts supplemented with ADC. A similar correlation is observed with scalp implants although with less total retention due to the low vascularity of the dorsal cranium in these rats. EXAMPLE 8: Autologous Fat Transfer in Mammoplasty for Breast Augmentation: A person wants to alter the shape of their breasts. Pre-operative evaluation of the patient includes routine history and physical examination in addition to complete informed consent that describes all the relevant risks and benefits of the procedure. To begin the procedure, the patient undergoes adipose tissue collection. The habit or disposition of the patient is examined by a suitable site for adipose tissue collection. The procedure is performed next to the patient's bed. Adipose tissue is chosen to be collected from the lateral and middle thigh regions of the patient. The area to be collected is injected subcutaneously with a standard tumescent fluid solution, which may or may not contain a combination of lidocaine, saline and / or epinephrine at different standardized dosing regimens. Using a scalpel with blade 11 (or other standard blade), a small perforating wound is made to pass through the dermis. The blade is rotated 360 degrees to complete the wound. A 14-gauge blunt tip cannula (or appropriate size) is then inserted into the subcutaneous adipose tissue plane. The cannula can be connected to an energized suction device or to a syringe for manual aspiration. The cannula then moves through the plane to break the connective tissue architecture. The volume of aspirate obtained is between 700 ce and 1000 ce. A fraction of adipose tissue collected in this manner is processed for isolation and concentration of regenerative cells derived from adipose tissue using the methods described above. The rest of the adipose tissue is processed to re-implant in the breasts. Alternatively, the patient can be removed adipose tissue through a lipectomy procedure. After removal of adipose tissue, hemostasis of the patient is achieved with standard surgical techniques and the wound is closed primarily. The collection of adipose tissue occurs approximately 1-2 hours before the augmentation mammoplasty in a clinic. However, the timing of collection is expected to vary and will depend on the quality standards of care. Finally, the practitioner responsible for managing the patient's care will determine the timing of collection. The regenerative cells obtained from the processing of adipose tissue are mixed with a unit of adipose tissue (approximately 100-300 ce) to be transplanted in the above described proportion. After the tissue processing is completed, the patient is prepared to undergo augmentation mammoplasty. The cellular dose delivered to the patient is determined from cellular performance after adipose tissue processing.

Approximately 5.5 x 105 cells per 50 cc of autologous fat are transplanted to the breast. The composition is delivered through a standard 14 gauge blunt tip cannula inserted into the chest tissue through a periareolar incision. Improved adipose tissue with regenerative cells is administered in tear-like form to increase the ratio of surface area to volume. The patient is monitored and approximately 7 days after the procedure, the transplant appears to have been successfully grafted with the effects of cell therapy that become noticeable to the attending physician. EXAMPLE 9. Autologous Fat Transfer and Soft Tissue Defects A patient exhibits a desire for soft tissue augmentation, in particular treatment of dermal patches. A physician evaluates the patient when conducting a history and physical assessment and determines that the patient is a candidate for autologous fat transfer. To begin the procedure, the patient undergoes adipose tissue collection. The habit or disposition of the patient is examined by a suitable site for adipose tissue collection. The procedure can be performed next to the patient's bed. The adipose tissue is collected from the pannus of the patient's anterior abdominal wall. The area to be collected is injected subcutaneously with a standard tumescent fluid solution, which may or may not contain a combination of lidocaine, saline and / or epinephrine at different standardized dosing regimens. Using a scalpel with blade 11 (or other standard blade), a small perforation lesion is practiced in order to traverse the dermis. The blade is rotated 360 degrees to complete the wound. A 14-gauge blunt-tipped cannula (or appropriate size) is then inserted into the subcutaneous adipose tissue plane.

The cannula can be connected to an energized suction device or to a syringe for manual aspiration. The cannula then moves through the plane to break the connective tissue architecture. The volume of aspirate obtained is in the range from about 400 cc to about 800 cc. A fraction of adipose tissue collected in this manner is processed for isolation and concentration of cells using the methods described above. The rest is processed to re-implant in the soft tissue. After removing adipose tissue, hemostasis of the patient is achieved with standard surgical techniques and the wound closed primarily. Collection can take 1-2 hours before soft tissue augmentation.

The regenerative cells obtained from adipose tissue processing are mixed with a unit of adipose tissue to be transplanted in a form and proportion as described above. After the tissue processing is completed, the patient is prepared to undergo soft tissue augmentation. The cellular dose delivered to the patient is determined from cellular performance after adipose tissue processing. Approximately 5.5 x105 per 50 cc of autologous fat are transplanted into the soft tissue. The composition is delivered through a standard 14 gauge blunt tip cannula inserted into the soft tissue through appropriately placed incision. Adipose tissue enhanced with regenerative cells is preferably administered in tear-like form to increase the ratio of surface area to volume. The patient is monitored before, during and after the supply of transplanted adipose tissue. After 2 days, the patient noticed an almost complete elimination of the skin patches. EXAMPLE 10: Autologous Fat Transfer for Urinary Incontinence Stress A person who experiences urinary incontinence requests treatment from a doctor. The pre-operative evaluation of the patient includes routine history and physical examination in addition to a complete informed consent that describes all the risks and benefits relevant to their procedure. To start the procedure, the patient undergoes adipose tissue collection. The patient's habit is examined for a suitable site for adipose tissue collection. The procedure can be performed in an operation suite with appropriate hemodynamic supervision for clinical collection of the patient. Adipose tissue is collected from the lateral and middle thigh regions of the patient of the bilateral lower extremities, and pannus of the anterior abdominal wall. The area to be harvested is injected subcutaneously with a standard tumescent fluid solution that may or may not contain a combination of lidocaine, saline and / or epinephrine at different standardized dosing regimens. Using a scalpel with blade 11 (or other standard blade), a small perforation injury is performed in order to traverse the dermis. The blade is rotated 360 degrees to complete the wound. A 14-gauge blunt-tipped cannula (or appropriate size) is then inserted into the subcutaneous adipose tissue plane. The cannula can be connected to an energized suction device or to a syringe for manual aspiration. The cannula then moves through the plane to break the connective tissue architecture. The volume of aspirate is measured at approximately 1200 ce. A fraction of adipose tissue collected in this manner is processed for isolation and concentration of regenerative cells using the methods described above. The rest of the adipose tissue is processed for re-implantation in proximity to the bladder neck and the patient's nearby urethra. Alternatively, the patient can remove the tissue through a lipectomy procedure. After removing the adipose tissue, hemostasis is achieved with standard surgical techniques and the wound is closed primarily. Adipose tissue collection occurs approximately 1-2 hours before the procedure. The regenerative cells obtained from the processing of adipose tissue are mixed with a unit of adipose tissue to be transplanted in the proportion described above. After completing the tissue processing, the patient is prepared to undergo transplantation. The dose of cells delivered to the patient is determined inter alia of cellular performance after processing of adipose tissue. Approximately 5.5 x 105 cells per 50 cc of autologous fat are transplanted in proximity to the patient's bladder neck and nearby urethra through cystoscopic visualization. The patient is monitored after the procedure. Approximately three days after the transplant, the patient experiences a decreased frequency of incontinence. Approximately one month after the procedure, the patient indicates that their quality of life has improved. The doctor evaluates the grafted tissue and determines that the long-term graft was successful. A number of publications and patents have been previously cited. Each of the publications and patents cited herein are incorporated by reference in their entirety. EQUIVALENTS Those skilled in the art will recognize or be able to use no more than routine experimentation to evaluate many equivalents to the specific embodiments of the invention described herein. These equivalents are intended to be encompassed by the following claims.

Claims (28)

1. CLAIMS 1. A method for treating a patient, characterized in that it comprises: removing adipose tissue from a patient; processing a first portion of the adipose tissue to obtain a population substantially isolated from regenerative cells; mixing a second portion of the adipose tissue with the population substantially isolated from regenerative cells to form an adipose tissue composition; and administering the composition of adipose tissue to the patient from which the adipose tissue was removed, in order to treat the patient in this way. Method according to claim 1, characterized in that the adipose tissue composition is administered to the patient's chest. Method according to claim 1, characterized in that the adipose tissue composition is administered to a soft tissue region of the patient to treat a soft tissue defect. Method according to claim 1, characterized in that the adipose tissue composition is administered to a urethral region of the patient to treat urinary incontinence. Method according to claim 1, characterized in that the regenerative cells comprise stem cells. 6. Method according to claim 1, characterized in that the regenerative cells comprise progenitor cells. Method according to claim 1, characterized in that the regenerative cells comprise a combination of stem cells and progenitor cells. 8. Method according to claim 1, characterized in that the method comprises administering multiple doses of adipose tissue composition. 9. Method according to claim 1, characterized in that the adipose tissue composition further comprises one or more angiogenic factors. 10. Method according to claim 1, characterized in that the adipose tissue composition further comprises one or more arteriogenic factors. 11. Method according to claim 1, characterized in that the adipose tissue composition further comprises one or more immunosuppressive drugs. Method according to claim 1, characterized in that the regenerative cells are grown in cell culture before being mixed with the second adipose tissue portion. Method according to claim 12, characterized in that the cell culture conditions promote differentiation towards an endothelial phenotype. 14. Method according to claim 12, characterized in that the cell culture is performed on a scaffold material to generate a bi or three-dimensional construction that can be placed on or inside the patient. 15. Method according to claim 14, characterized in that the scaffold material is reabsorbed in vivo. Method according to claim 1, characterized in that the regenerative cells are modified by gene transfer such that the expression of one or more genes in the modified regenerative cells is altered. Method according to claim 16, characterized in that the modification results in alteration of the level of angiogenesis in the subject. Method according to claim 16, characterized in that the modification results in alteration of the level of arteriogenesis in the subject. 19. Method according to claim 16, characterized in that the modification results in alteration of the level of apoptosis in the subject. 20. Method according to claim 1, characterized in that the administration of the adipose tissue composition promotes neovascularization. 21. The method according to claim 20, characterized in that the neovascularization remains stable after the adipose tissue composition administered is no longer present. 22. Method according to claim 1, characterized in that the administration of adipose tissue composition reduces necrosis. 23. Method according to claim 1, characterized in that the subject is a human. 24. A process for preparing a composition comprising regenerative cells for administration to a patient, characterized in that it comprises: removing adipose tissue from a patient; and separating the regenerative cells present in the adipose tissue from the cellular components and lipids of the tissue or adipose, to form a composition comprising regenerative cells for immediate administration to the patient from which the adipose tissue was removed. 25. Process according to claim 24, characterized in that the patient requires or desires a fat transplant procedure. 26. A device for preparing a composition comprising 5 regenerative cells for administration to a patient, characterized in that it comprises: a tissue collection chamber for containing, rinsing and digesting adipose tissue removed from a patient; and a processing chamber for separating and concentrating the regenerative cells of the cellular components and lipids of the adipose tissue, to thereby form a composition comprising regenerative cells for immediate administration to the patient from which the adipose tissue was removed. 27. The device according to claim 26, characterized in that the device is automated. 28. The device according to claim 27, characterized in that the patient requires or desires a fat transplant procedure.