Systems and methods for preparing adipose-derived stem cells

Abstract

The present invention relates to systems and methods for preparing adipose-derived stem cells. A device capable of preparing an adipose graft or harvesting a cell fraction is disclosed. The device includes a first centrifuge tube for receiving and processing a biological material, the first centrifuge tube including an upper cylindrical portion and a lower conical portion, a sterile tissue inlet fitting, at least one sterile process fluid inlet fitting, a sterile aspiration fitting, and at least one sterile extraction port connected to the first extraction tube. The first centrifuge tube further includes an interior space, the interior space including a screen positioned therein configured to divide the interior space into two portions, and a filter positioned therein below the screen in the lower conical portion of the first centrifuge tube. The device can further include a second centrifuge tube configured to receive and further process the biological material from the first centrifuge tube.

Description

[0001] Cross-reference to related applications

[0002] This application is a divisional application of Chinese patent application No. 201980006337.4, filed on April 2, 2019 (PCT international application PCT / US2019 / 025325), which entered the Chinese national phase on June 11, 2020, and is entitled "System and Method for Preparing Adipose-Derived Stem Cells." This PCT international patent application claims priority to U.S. patent application No. 15 / 942,793, filed on April 2, 2018, publication number 2018 / 0221550. This application also relates to U.S. application No. 13 / 544,909, filed on July 9, 2012, now U.S. Patent No. 9,931,445, which claims priority to U.S. provisional patent application No. 61 / 505,936, filed on July 8, 2011, entitled "Reagent Kit for Preparing Adipose-Derived Stem Cells." All publicly available information in these applications is hereby incorporated herein by reference. Technical Field

[0003] The present invention discloses a system for preparing and using stem cells derived from adipose tissue, adipose-derived stem cells obtained using such a kit, a method for using such adipose-derived stem cells, and a composition containing such adipose-derived stem cells. Background Technology

[0004] Regenerative medicine can be defined as the clinically targeted use of the body's regenerative mechanisms, utilizing these mechanisms in ways that are not part of the normal healing mechanisms or by artificially amplifying normal mechanisms. A prime example of this process is found in bone marrow transplantation, where hematopoietic stem cells and progenitor cells are harvested from a donor and implanted into a recipient (whose normal hematopoietic regeneration mechanisms have been ablated, or are substantially depleted or impaired), thereby replacing or regenerating the recipient's hematopoietic capacity. This approach has been extended to non-hematopoietic stem cells from various sources for a variety of therapeutic or preventative purposes. In particular, adipose tissue has been shown to be readily available as a source of pluripotent mesenchymal stem cells suitable for therapeutic or preventative use.

[0005] Mesenchymal stem cells (“MSCs”) are pluripotent stem cells that can readily differentiate into lineages including osteoblasts, myocytes, chondrocytes, adipocytes, endothelial cells, and β-islet cells (Pittenger, et al., Science, Vol. 284, pg. 143 (1999); Haynesworth, et al., Bone, Vol. 13, pg. 69 (1992); Prockop, Science, Vol. 276, pg. 71 (1997)). Also referred to in the literature as bone marrow stem cells, skeletal stem cells, and pluripotent mesenchymal stromal cells, MSCs are non-hematopoietic progenitor cells isolated from adult tissues. Their in vitro characteristic lies in their extensive proliferative capacity in an amorphous state, while retaining the potential to differentiate into various lineages along the mesenchymal origin in response to appropriate stimuli, including chondrocyte, osteoblast, and adipocyte lineages. In vitro studies have demonstrated that MSCs have the ability to differentiate into muscle (Wakitani et al., Muscle Nerve, Vol. 18, pg. 1417 (1995)), neuron-like precursors (Woodbury et al., J. Neurosci. Res., Vol. 69, pg. 908 (2002); Sanchez-Ramos et al., Exp. Neurol., Vol. 171, pg. 109 (2001)), cardiomyocytes (Toma et al., Circulation, Vol. 105, pg. 93 (2002); Fakuda, Artif. Organs, Vol. 25, pg. 187 (2001)), and other possible cell types. MSCs are present in multiple tissues of the body and originate from the embryonic mesoderm (e.g., hematopoietic cells and connective tissue). Therefore, pluripotent cells can be isolated from any of these tissue sources and induced to differentiate into any of these cell types.

[0006] In current technology, research on adipose tissue is quite mature, and it is readily available from various sources. For example, the most direct method is to obtain a large number of pluripotent cells from adipose tissue samples needed for cosmetic purposes through simple aspiration or processing. These adipose tissue samples mostly come from the human body, but obtaining adipose tissue from patients or donors also has certain limitations, such as limited donor availability or the need for a minor surgical procedure. Therefore, obtaining adipose tissue as a raw material through in vitro laboratory culture and appropriate processing is entirely feasible. Of course, extraction from the patient is the most direct, safe, and convenient method. In practical applications, the appropriate source of adipose tissue can be selected based on the specific circumstances.

[0007] Adipose tissue has been found to be a particularly rich source of stem cells. This finding may be at least partly attributed to the fact that adipose tissue's main non-stem cell component, namely fat cells, is easily removed. However, in existing systems and methods, processing adipose tissue to isolate pluripotent cells and expand and (optionally) differentiate the resulting stem cell population typically requires expensive specialized equipment and equally expensive specialized processes. Moreover, the cost of these devices, mostly designed for repeated extraction of stem cells from adipose tissue (e.g., through liposuction from living human donors), limits the use of stem cell-rich culture media to large hospitals and research institutions for therapeutic purposes. Therefore, it is characterized by high cost and low practicality for many applications, making these expensive devices and methods unsuitable for widespread adoption, especially for junior physicians or other professional trainees, where the application of adipose-derived stem cells is neither feasible nor economical. Furthermore, the development of stem cell-based cosmetic enhancement technologies is also constrained or limited, primarily by the aforementioned limitations of current equipment and processes in the field, thus hindering the development of current systems and methods for extracting and culturing stem cells derived from adipose tissue.

[0008] As described above, there is an urgent need for a technology and system suitable for the economical and safe extraction or processing of adipose-derived stem cells, whether in pure form or as part of a stem cell-rich culture medium. This is especially important for technologies and systems suitable for use in small-scale professional practices without requiring capital investment in specialized facilities. This application relates to a system for isolating MSCs from adipose tissue. Specifically, it investigates a tissue processing device, a multi-device integrated system for processing tissue in a closed pathway to maintain sterility, and a method for preparing fat grafts and / or resuspension mixtures of cells. As used herein, "adipose tissue" refers to tissue containing multiple cell types, including adipocytes and microvascular cells. Adipose tissue includes stem cells and endothelial progenitor cells. Therefore, adipose tissue refers to fat comprising connective tissue that stores fat. Summary of the Invention

[0009] The inventors have conceived and practically applied systems and various methods for preparing adipose-derived stem cells, solving the challenges and problems in the aforementioned fields. Cell populations from ex vivo adipose tissue from the human body, ex vivo adipose tissue from other organisms, or in vitro cultured adipose tissue can be used to prepare fat grafts and / or fat-resuspended cell mixtures, which can ultimately be used as cell sources for therapeutic and / or cosmetic applications. Furthermore, adipose-derived stem cells in pure form (stem cells only) or enriched form (cell cultures with increased stem cell fractions compared to stem cells generated in adipose tissue in vivo) can be used in regenerative medicine, such as treating diseases that can be improved or cured by using regenerated cells. As described herein, prepared cell populations can be applied to patients without other adipose cells or connective tissue, or these cells can be mixed with adipose tissue for concentrated application. This invention relates to a system for obtaining a purified fraction of adipose-derived mesenchymal stem cells from adipose tissue and administering the purified fraction back to a patient or animal. This invention provides a system for extracting and processing adipose tissue (lipoid) to produce processed adipose tissue (PLA), wherein the PLA contains at least 0.1% stem cells, and more typically more than 0.5%. According to some embodiments of the invention, PLA containing approximately 2-12% stem cells can be obtained, and PLA comprising up to 100% stem cell populations is possible.

[0010] This invention relates to a system for obtaining large quantities of purified or enriched fractions of adipose-derived mesenchymal stem cells in a clinical setting, and also to compositions, methods, and systems using stem cells derived from adipose tissue, which, together with such additives, are directly implanted into a recipient, potentially beneficial for promoting, generating, or supporting therapeutic, structural, or cosmetic purposes. Embodiments of the invention provide a multi-component system for extracting and processing adipose tissue to produce an effective therapeutic amount of adipose-derived stem cells for reintroduction into a patient. Advantageously, this system is easy to prepare, ingeniously assembled, low-cost, convenient to use, has low environmental requirements, and exhibits excellent results. It does not require a complete operating room environment or comprehensive liposuction, and therefore can be performed outside of hospital or laboratory settings. Therefore, the system, methods, and applications of the resulting fat grafts and / or fat-resuspended cell mixtures are also suitable for small to medium-sized medical clinics, outpatient surgical centers, and multidisciplinary physician offices.

[0011] In one embodiment, a type of This system allows junior physicians and surgical centers to safely extract MSCs or stromal vascular fractions (SVFs) from adipose tissue of any patient for therapeutic or cryopreservation (bank construction) using an economical method. SVFs are heterogeneous cell fractions that can be generated by this system. These fractions are enriched with cells possessing MSC characteristics. If the cell fractions are further cultured, the system can generate MSCs from the further cultured fractions. The system includes apparatus for forming a safe, sterile, and closed process for extracting small amounts of fat samples (e.g., by liposuction) as a closed process for the fat material; materials for on-site processing and purification of MSC or SVF fractions; and optional tools to assist in assessing the quality of the resulting processed fat material. The system can be operated by laboratory technicians with appropriate training following simple procedures.

[0012] In one embodiment, adipose tissue processing occurs within a system that maintains a closed, sterile fluid / tissue pathway. This is achieved using sterile containers and tubing kits that allow for the transfer of tissue and fluid components within the closed pathway. A pre-packaged range of processing reagents (e.g., saline, enzymes, etc.) for introduction into the sterile containers is also provided to facilitate simple control of reagent addition, temperature, and processing time, thereby simplifying the process from manual operator management. In a preferred embodiment, the entire process from tissue extraction to processing and implantation into the recipient is virtually performed within the same apparatus, and can even be conducted in the room where the patient receiving the procedure based on the present invention is located.

[0013] According to one aspect of the invention, a small amount of raw adipose tissue is extracted from a patient and placed into a sterile container of a kit. The raw adipose tissue is processed within one or more sterile containers of the kit to substantially remove mature adipocytes and connective tissue, thereby obtaining a variety of heterogeneous adipose tissue-derived cells suitable for implantation into a recipient. Reagents required for processing the adipose tissue are provided as part of the kit and introduced into one or more sterile containers according to a predetermined schedule to produce a processed adipose material containing a desired concentration of adipose-derived stem cells. The adipose-derived stem cells, or a modified adipose material richer in stem cell fractions than in vivo, can then be combined with other stimulants for cell, tissue, tissue debris, or other cell growth and / or differentiation and implanted into the recipient. In a preferred embodiment, stem cells having any of the aforementioned additives are implanted into the human body, wherein the stem cells are obtained from the human body in a single surgical procedure for the purpose of providing the recipient with therapeutic, structural, or cosmetic effects.

[0014] In one embodiment of the invention, a method of treating a patient includes the following steps: a) providing a disposable tissue extraction system as a component of a kit; b) extracting adipose tissue from the patient using the disposable tissue extraction system, the adipose tissue having a certain concentration of stem cells; c) processing at least a portion of the adipose tissue within one or more sterile containers of the kit to obtain a higher concentration of stem cells than the concentration of stem cells in the adipose tissue before processing; d) applying stem cells to the patient from the sterile containers of the kit.

[0015] In a preferred embodiment of the invention, a system for extracting and processing adipose tissue to produce a therapeutically effective amount of adipose-derived stem cells includes an adipose tissue extraction device and a modified centrifuge tube comprising multiple fat aspiration inlet connectors, multiple processing fluid inlet connectors, and multiple particle extraction tubes. The adipose tissue extraction device is used to extract a certain amount of adipose tissue from the human body, aseptically transfer the adipose material to a first modified centrifuge tube, perform multiple processing steps to clean and separate the adipose material, and centrifuge using the modified centrifuge tube to obtain a stem cell cluster containing increased fractions. The stem cell cluster is resuspended in a liquid and administered to a patient for therapeutic or cosmetic purposes. According to another embodiment, the system further includes a second modified centrifuge tube with a design substantially similar to the first modified centrifuge tube, wherein the second modified centrifuge tube is used for further processing the resuspended cell clusters to obtain a higher concentration of stem cells.

[0016] According to another preferred embodiment of the present invention, a method for preparing adipose-derived stem cells is provided, the method comprising the steps of: (a) extracting adipose tissue from a human body using an adipose tissue extraction device; (b) aseptically transferring the adipose material into a modified centrifuge tube comprising a plurality of adipose material inlet connectors, a plurality of processing fluid inlet connectors, and a plurality of particle extraction tubes; (c) treating the adipose material in the modified centrifuge tube by adding one or more fluids through the plurality of processing fluid connectors, the treatment comprising cleaning and separating one or more of the adipose material; (d) centrifuging the modified centrifuge tube containing the processed adipose material to concentrate a cell cluster containing a fraction of enriched stem cells; (e) resuspending the cell cluster in a fluid to obtain a cell mixture containing more stem cells relative to the stem cell concentration of the original adipose material; and (f) administering the resuspended cell mixture to a human body for therapeutic or cosmetic purposes.

[0017] According to another aspect of this application, a tissue processing apparatus is provided. The apparatus includes a first centrifuge tube configured to receive and process biological material, the first centrifuge tube having an upper cylindrical portion and a lower conical portion, a sterile tissue inlet connector, at least one sterile processing fluid inlet connector, a sterile aspiration connector, and at least one sterile extraction port connected to a first extraction tube. The first centrifuge tube also includes an internal space in which a sieve and a filter are disposed, the sieve being configured to divide the internal space into two halves and / or provide support for the biological material processed therein, the filter being located below the sieve in the lower conical portion of the first centrifuge tube. The apparatus also includes a second centrifuge tube configured to receive and further process the biological material from the first centrifuge tube, the second centrifuge tube having at least one sterile connector. The second centrifuge tube is releasably connected to one of the at least one sterile extraction port of the first centrifuge tube via at least one sterile connector.

[0018] According to another embodiment, a system for processing tissue in a closed environment to maintain sterility is disclosed. The system includes a first centrifuge tube having a generally cylindrical body with a top, an inverted conical apex, and an internal volume. The first centrifuge tube also includes at least two swabable valves; an extraction tube releasably attached to one of the at least two swabable valves, the extraction tube extending a distance towards the apex within the internal volume; and a sieve disposed within the internal volume of the body. The system also includes a second centrifuge tube having a generally cylindrical body with a top, an inverted conical apex, and an internal volume. The second centrifuge tube also includes at least one swabable valve and an extraction tube releasably attached to the at least one swabable valve, the extraction tube extending a distance towards the apex within the internal volume. The second centrifuge tube is releasably connected to one of the at least two swabable valves of the first centrifuge tube via the at least one swabable valve.

[0019] In yet another embodiment, a multi-device integrated system for processing tissue in a closed pathway to maintain sterility is disclosed. The system includes a centrifuge tube having a generally cylindrical body with a top, an inverted conical apex, and an internal volume. The system also includes at least two swabable valves; an extraction tube releasably attached to one of the at least two swabable valves, extending a distance towards the apex within the internal volume; a sieve disposed within the internal volume of the body; and a filter located below the sieve at the inverted conical apex of the centrifuge tube. The system further includes a container having at least one swabable valve releasably connected to one of the at least two swabable valves of the centrifuge tube for aseptic transfer of materials contained within the closed system.

[0020] In yet another embodiment, a method for preparing a fat graft is provided. The method includes the following steps:

[0021] (a) Aseptically transferring fat into a centrifuge tube, the centrifuge tube including an upper cylindrical portion and a lower conical portion, a sterile tissue inlet connector, at least one sterile processing fluid inlet connector, a sterile aspiration connector and at least one sterile extraction port connected to a first extraction tube, the centrifuge tube also including a sieve and a filter located therein, the filter being located below the sieve in the lower conical portion of the first centrifuge tube;

[0022] (b) Wash the fatty substances in the centrifuge tube by adding one or more fluids through at least one fluid inlet connector;

[0023] (c) Centrifuge the centrifuge tube containing fatty substances at low speed to separate contaminants and dense fatty fractions, wherein the dense fatty fraction is retained at the top of the screen and the liquid is collected in the lower conical fraction;

[0024] (d) Centrifuge the centrifuge tube containing washed dense fat at a higher speed to force the dense fat through the holes of the screen by centrifugal force and accumulate broken fat particles in the compartment between the screen and the filter, wherein the filter surface serves as the bottom for accumulating fat particles, and the broken fat and debris are filtered downward through the filter into the lower conical section.

[0025] (e) Collecting the accumulated fat grafts using a syringe, by connecting the syringe to at least one inlet and allowing the corresponding tubing to flow into the section between the filter and the screen; and

[0026] (f) The prepared fat grafts can be used on patients, such as for therapeutic or cosmetic purposes.

[0027] In yet another embodiment, a method for preparing fat-derived cell fractions is disclosed. The method includes the following steps:

[0028] (a) The fat is aseptically transferred into the first centrifuge tube of the device;

[0029] (b) Adding one or more fluids through at least one fluid inlet connector to treat fatty substances in a first centrifuge tube, the treatment including one or more of cleaning and separating fatty substances operations;

[0030] (c) Centrifuge the first centrifuge tube containing processed fats to concentrate cell clusters containing stem cell fractions;

[0031] (d) Aseptically transfer the concentrated cell cluster containing stem cell fractions into the second centrifuge tube of the device;

[0032] (e) Wash the cell fractions in the second centrifuge tube by adding one or more fluids through at least one fluid inlet connector;

[0033] (f) Centrifuge the second centrifuge tube containing cell fractions to concentrate cell clusters containing stem cell-rich fractions;

[0034] (g) Resuspending the cell clusters in a fluid to obtain a cell mixture containing more stem cells relative to the original fat content; and

[0035] (h) The prepared cell mixture can be applied to patients, such as for therapeutic or cosmetic purposes.

[0036] The above and other objects, features and advantages of the present invention will become apparent from the following detailed description. Attached Figure Description

[0037] The accompanying drawings illustrate several embodiments of the invention and, together with the description, can be used to explain the principles of the invention based on these embodiments. Those skilled in the art should recognize that the specific embodiments shown in the drawings are merely exemplary and are not intended to limit the scope of the invention.

[0038] Figure 1 This is a diagram of a modified centrifuge tube for extracting stem cells from adipose tissue according to a preferred embodiment of the present invention.

[0039] Figure 2 This is a diagram of a modified centrifuge tube for extracting stem cells from adipose tissue according to a preferred embodiment of the present invention, the centrifuge tube including a novel nebulizer.

[0040] Figure 3 This is a system diagram of a preferred embodiment of the present invention for preparing mesenchymal stem cells or SVF from adipose tissue for clinical use.

[0041] Figure 4 This is a diagram of an advanced process for preparing mesenchymal stem cells or SVF from adipose tissue for clinical use, according to an embodiment of the present invention.

[0042] Figure 5 This is a diagram illustrating a detailed process for preparing mesenchymal stem cells or SVF from adipose tissue for clinical use, according to a preferred embodiment of the present invention.

[0043] Figure 6 This is a diagram of a centrifuge tube according to another embodiment of the present invention.

[0044] Figure 7 yes Figure 6 An exploded view of the centrifuge tube shown.

[0045] Figure 8This is a diagram of a centrifuge tube according to another embodiment of the present invention.

[0046] Figure 9 yes Figure 8 An exploded view of the centrifuge tube shown. Detailed Implementation

[0047] The inventors have conceived and practically applied systems and various methods for preparing adipose-derived stem cells, addressing the challenges and problems in the aforementioned fields. As illustrated in the accompanying drawings, various techniques will now be described in detail with reference to some exemplary embodiments therein. Numerous specific details are set forth in the following description to provide a comprehensive understanding of one or more aspects and / or features described or referenced herein. However, it will be apparent to those skilled in the art that one or more aspects and / or features described or referenced herein can be practiced without some or all of these specific details. In other instances, well-known processing steps and / or structures have not been described in detail so as not to obscure one or more aspects and / or features described or referenced herein.

[0048] One or more different inventions may be described in this application. Furthermore, many alternative embodiments may be described for the one or more inventions described herein; it should be noted that these are presented for illustrative purposes only. The described embodiments are not intended to be limiting in any sense. It will be apparent from this disclosure that one or more inventions can be broadly applied to many embodiments. Generally, the embodiments are described in sufficient detail to enable those skilled in the art to implement one or more inventions; it should be noted that other embodiments can be utilized, and structural, logical, software, electrical, and other changes can be made without departing from the specific scope of the invention. Therefore, those skilled in the art will recognize that various modifications and changes can be made to one or more inventions. Specific features of one or more inventions may be illustrated with reference to one or more specific embodiments or drawings that form part of this disclosure, and specific embodiments of one or more inventions are shown by way of example in the drawings. However, it should be noted that such features are not limited to their use in the one or more specific embodiments or drawings described with reference to them. This disclosure is neither a verbal description of all embodiments of one or more inventions nor a mandatory list of one or more inventive features in all existing embodiments.

[0049] The titles of the various parts provided in this patent application and the name of this patent application are merely for convenience and are not to be construed in any way as limiting the disclosure of this invention.

[0050] The description of an embodiment having several interconnected components does not imply the necessity of all such components. Instead, various optional components may be described to illustrate multiple possible embodiments of one or more inventions in order to more fully illustrate one or more aspects of the invention. Similarly, although processing steps, method steps, algorithms, etc., are described sequentially, unless specifically and explicitly stated otherwise, the aforementioned processes, methods, and algorithms may generally be operated in different orders. In other words, any sequence or order of steps described in this patent application may not itself imply a requirement to operate in that order. The steps of the process may be operated in any actual order. Furthermore, although described or implied to occur non-simultaneously, some steps may be operated simultaneously (e.g., because one step is described after another). Moreover, the description of a process by way of its illustrations in the drawings does not imply that the process excludes modification and alteration, nor does it imply that the process or any of its steps are necessary for one or more inventions, nor does it imply that the process is preferred. Additionally, each step is generally described once per embodiment, but this does not mean that these steps must occur once, or that they occur only once each time a process, method, or algorithm is performed or operated. In some implementations or under certain circumstances, certain steps may be omitted, or certain steps may be performed multiple times in a given implementation or under certain circumstances.

[0051] When describing a single device or article, it is obvious that multiple devices or articles can be used in place of the single device or article. Similarly, when describing multiple devices or articles, it is obvious that a single device or article can be used in place of multiple devices or articles.

[0052] The function or feature of the device can be further described by one or more other devices that are not explicitly described as having such function or feature. Therefore, other embodiments of one or more inventions do not need to include the device itself.

[0053] For clarity, the techniques and mechanisms described or referenced in this invention will sometimes be described in the singular. However, it should be noted that, unless otherwise indicated, a particular embodiment includes multiple iterations of the technique or multiple instances of the mechanism. The process illustrations or blocks in the drawings should be understood as representing modules, fragments, or portions of code, including one or more executable instructions for a specific logical function or step in the implementation process. As will be understood by those skilled in the art, alternative embodiments are included within the scope of embodiments of the invention; for example, functions may be performed not in the order shown or discussed, depending on the functionality involved, including substantially simultaneous or reverse order.

[0054] As will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art, any feature or combination of features described herein is included within the scope of the invention, provided that the features included in any such combination do not contradict each other. Certain aspects, advantages, and novel features of the invention have been described herein to summarize it. It should be understood, of course, that not all of these aspects, advantages, or features will necessarily be apparent in any particular embodiment of the invention. Other advantages and aspects of the invention will become apparent from the following detailed description and claims.

[0055] This document cites numerous published documents and patents. Each of these cited documents and patents is incorporated herein by reference. The full text of all cited documents, patents, and patent applications is incorporated herein by reference to the same extent that each individual document, patent, or patent application is specifically and individually indicated to be incorporated herein by reference in its full text.

[0056] definition

[0057] As described in this invention, a "unit of adipose tissue" refers to a discrete or measurably quantity of adipose tissue. A unit of adipose tissue can be measured by determining the weight and / or volume of the unit. Based on the data determined above, a unit of processed adipose tissue extracted from a patient has a cellular component, wherein at least 0.1% of the cellular component is stem cells. Referring to relevant published literature, a unit of adipose tissue can refer to the total amount of adipose tissue extracted from a patient, or an amount less than the total amount of adipose tissue extracted from a patient. Therefore, a unit of adipose tissue can be combined with another unit of adipose tissue to form a unit of adipose tissue whose weight or volume is the sum of the individual units.

[0058] As described in this invention, "part" refers to an amount smaller than the total amount of material. A small portion refers to an amount less than 50%, and a large portion refers to an amount greater than 50%. Therefore, a unit of adipose tissue smaller than the total amount of adipose tissue removed from the patient is a part of the removed adipose tissue.

[0059] As described in this invention, "stem cell" refers to a pluripotent cell with the potential to differentiate into various other cell types, and the differentiated cells perform one or more specific functions and have the ability to self-renew. Some of the stem cells disclosed in this invention may be pluripotent.

[0060] As described in this invention, "processed adipose tissue" (PLA) refers to adipose tissue that has been processed to separate viable cellular components (e.g., components containing stem cells) from mature adipocytes and connective tissue. Typically, PLA refers to cell clusters obtained by washing and separating cells from the adipose tissue. Cell clusters are usually obtained by centrifuging a cell suspension to cause the cells to aggregate at the bottom of a centrifuge container.

[0061] As described in this invention, the term "effective dose for prevention or treatment" refers to the number of cells of the invention contained in the pharmaceutical composition capable of producing the desired therapeutic effect. Those skilled in the art will recognize that the number of cells (e.g., dosage) will vary depending on a variety of factors, including but not limited to the site of application, the severity of the disease, and the method of application. Experience with hematopoietic stem cell (bone marrow or umbilical cord blood-derived stem cells for regenerating recipient blood cells) transplantation has shown that transplantation is cell dose-dependent and has a threshold effect.

[0062] Therefore, it is possible to apply the general principle of "the more the better" within limits set by other variables, and where feasible, the harvest will collect as much tissue as possible. Depending on the patient's condition and response, the cell dosage described in this invention can be repeated at intervals of days, weeks, or months, as determined by the treating physician or other healthcare professional.

[0063] The term "pharmaceutically acceptable mediator" refers to a drug approved by a federal or state regulatory agency or as specified in the United States Pharmacopeia or other relevant pharmacopoeia.

[0064] The ingredients listed in the European Pharmacopoeia or other recognized pharmacopoeias for use in animals (including humans). The term "mediator" refers to a diluent, adjuvant, excipient, or carrier used in conjunction with the cells of the present invention; therefore, the mediator must be compatible with the cells. If desired, the pharmaceutical ingredients of the present invention may also contain, where necessary, additives and / or adjuvants or pharmaceutically acceptable substances, such as small amounts of pH buffers, tonic agents, solubilizers, preservatives, etc., wherein the additives are used to enhance, control, or otherwise dominate the intended therapeutic effect of the cells containing the pharmaceutical ingredient. Additionally, metal chelators may be added to stabilize the cell suspension. The stability of cells in a liquid medium of the pharmaceutical ingredients of the present invention can be improved by adding other substances, such as amino acids (e.g., aspartic acid, glutamic acid, etc.). Generally, pharmaceutically acceptable substances that can be used in the pharmaceutical ingredients of the present invention are known to those skilled in the art and are commonly used in the preparation of cell components. Examples of suitable pharmaceutical mediators are described in EW Martin's "Remington's Pharmaceutical Sciences". Illustrative, non-limiting examples of mediators for applying cells contained in the pharmaceutical ingredients of the present invention include, for example, sterile saline solution (0.9% NaCl), PBS, etc.

[0065] Numerous devices have been developed for harvesting and processing cells from adipose tissue; however, these devices may have one or more problems that hinder their widespread application. These problems include difficulty in use and cost. Therefore, there is a need for alternative methods that can rapidly and reliably prepare viable cell populations with increased yield, consistency, and / or purity, thereby reducing or eliminating the need for post-extraction manipulation. Ideally, the cell populations could be obtained through a method suitable for direct implantation into the recipient.

[0066] Reference will now be made in detail to the presently preferred embodiments of the invention. In the drawings and description, the same or similar reference numerals are used wherever possible to refer to the same or similar parts. It should be noted that the drawings are simplified forms and not precise scales. Referring to the disclosure of the invention, directional terms such as top, bottom, left, right, up, down, from above, above, from below, below, back, and front are used relative to the drawings only for convenience and clarity. Such directional terms should not be construed in any way as limiting the scope of the invention.

[0067] While the disclosure of this invention refers to certain illustrated embodiments, it should be understood that these embodiments are presented by way of example and not limitation. Although exemplary embodiments are discussed, the following detailed description is intended to cover all modifications, alternatives, and equivalents that may fall within the spirit and scope of the invention as defined by the appended claims. The invention can be practiced in conjunction with various cell or tissue isolation techniques conventionally used in the art, and the processing steps of common practice included in the invention are merely necessary to provide an understanding of the invention.

[0068] Systems for extracting and processing adipose tissue

[0069] Various embodiments of the present invention relate to a system 300 for obtaining adipose-derived mesenchymal stem cells or SVF fractions in a clinical setting, and also to stem cell compositions, methods, and systems derived from adipose tissue, which, together with additives, are directly implanted into a recipient to facilitate, generate, or support therapeutic, structural, or cosmetic effects. In various embodiments, the present invention provides a system 300 for extracting and processing adipose tissue to produce therapeutically effective amounts of adipose-derived stem cells for reintroduction into a patient, wherein the system is easy to operate, low-cost, and suitable for small to medium-sized medical clinics with multidisciplinary specialties.

[0070] In a preferred embodiment of the present invention, such as Figure 1As shown, a modified centrifuge tube 100, used in conjunction with various accessories known in the art, is included in a system or kit that allows junior physicians and surgical centers to operate cost-effectively to safely extract mesenchymal stem cells (MSCs) or SVFs from any patient or donor or in vitro cultured adipose tissue for direct therapeutic use or for cryopreservation (bank establishment). The kit includes the centrifuge tube 100 and various other components for performing safe, aseptic, and closed procedures to aspirate small amounts of adipose tissue samples, as well as components for on-site processing and purification of MSC fractions or SVFs, and components for quality assessment. Laboratory technicians or similarly qualified personnel can operate the components, including the centrifuge tube 100, by following the detailed instructions below or by following simple methodological steps with some training.

[0071] Centrifuge tube 100 maintains a closed, sterile fluid / tissue pathway, receiving adipose tissue samples and providing more or less mesenchymal stem cell or SVF-enriched samples as output, as needed. This is achieved using a sterile container and tubing kit that allows for the transfer of tissue and fluid components within the closed pathway. A range of processing reagents (e.g., saline, enzymes, etc.) can also be provided pre-packaged to be introduced into centrifuge tube 100, enabling simple control over reagent addition and processing temperature and time, thereby simplifying process management for the operator. In a preferred embodiment, the entire process from tissue extraction to processing and implantation into the recipient is performed in the same device (in fact, even in the same room, preferably in the patient's room receiving the MSC treatment procedure obtained through this invention).

[0072] Tube 100 includes a conventionally designed body and an internal volume 160, which is generally available in various sizes from approximately 10 mL to up to 500 mL and is suitable for use with a standard centrifuge. Figure 1 The tubes shown are of conventional geometry (although, according to the invention, any suitable centrifuge tube geometry known in the art and compatible with centrifuges available in the art may be used).

[0073] In one embodiment, the kit or system comprises three main components: (1) a set of permanent equipment known in the art: aspiration pump, vibrator, centrifuge, and miniature incubator; (2) multiple disposable equipment items: aspiration needles / cannulas, collection bags, syringes, filters, flasks, and centrifuge tubes 100; and (3) various processing solutions / reagents. These components are better suited to work together to facilitate the extraction and processing of adipose tissue to obtain MSCs while maintaining sterility and simplifying the process. Tube 100 is provided with a cap 150, which is penetrated by multiple openings 115, 120, 121, and 122, and a separate connector 110, which is typically mounted on top of tube 100 near cap 150 (in practice, it should be understood that a person skilled in the art can arrange various connectors (although all are fixed to the upper part of tube 100) in various ways, some of which, all or none of which penetrate cap 150; in practice, cap 150 may be optionally omitted to facilitate the integrated structure of tube 100, which can be discarded after use). The connector 120 is penetrated by a tube 129 fitted with a sterile connector 125 (such as a Luer lock) to allow the introduction of various reagents or other fluids through the tube 129 without disrupting the sterile environment within the tube 100.

[0074] Typically, fluid is added to tube 100 via tube 129 using a sterile syringe. Tubes 130 and 131 penetrate caps 150 at connectors 121 and 122, respectively, and are provided with sterile connectors 126 and 127 to allow cells or other material to remain in a cell clump at the bottom (the point forming the inverted cone at the bottom of tube 100) after centrifugation (it is well known in the art that cell clumps of material with a relatively high specific gravity are generated during centrifugation). Connectors 126 and 127 can be of any type known in the art, such as Luer locks, and are adapted to establish a sterile connection between tubes 130, 131 and a syringe or other device capable of applying suction to remove cells from the cell clump at the bottom of tube 100. Connector 115 is penetrated by tube 116, at the end of which a sterile connector 117 is fitted, and can be used to apply suction from the top of tube 100 to create a vacuum within tube 100 (e.g., to facilitate the rapid introduction of fatty substances into tube 100 through connector 110). Connector 110 connects to connector 111 via a short tube. This connector (like all other connectors entering tube 100) is adapted to maintain a sterile state within tube 100 at all times. Connector 110 is typically used to inject fatty material into tube 100 as a first step in a process of extracting MSCs from the fatty material and potentially purifying or concentrating them into cell clusters, which can be withdrawn via either tube 130 or 131. In a preferred embodiment, two tubes 131 are provided to ensure that cell clusters can be extracted even if one tube becomes blocked.

[0075] Figure 2This figure shows a modified centrifuge tube 200 for extracting stem cells from adipose tissue according to an embodiment of the invention, the centrifuge tube including a novel nebulizer. Those skilled in the art will find that tube 200 is very similar to tube 100, but more illustrative details are provided regarding connectors 125-127. Tube 116 is also securely connected to a vacuum pump 220 (details of the aseptic connection between tube 116 and pump 220 are not shown, but are well known in the art). The vacuum pump, already aseptically connected to tube 116, can be configured as a component of a portable kit for separating MSCs from adipose material, or more generally, it can be connected to tube 116 as needed during the extraction of MSCs from adipose material. Generally, the object of the invention is to facilitate the rapid breakdown of adipose material into small droplets upon entering tube 100, so that the breakdown of adipose tissue and the washing of cells (without damaging the cells) can be carried out efficiently without the aid of special equipment. Therefore, in some embodiments, a turbine or screen 210 may be provided, comprising a plurality of holes 211 through which the fatty material must pass when it enters the tube 100 through the connector 110; the presence of the holes 211 facilitates the breaking down of fatty tissue clumps, which constitute at least a portion of the entering fatty material. It should be noted that the fatty material can be aspirated into the tube 100 using a vacuum provided by the pump 220 or another device for suction through the tube 116, or by direct injection of the fatty material through the connector 110 (such as with a plunger-type syringe). In either case, as the fatty material is forced through the connector 110 and then through the holes 211, it breaks down into small droplets or small fragments of cells and tissue that are easier to wash and process according to the methods described elsewhere in the invention (e.g., see reference). Figure 4 ).

[0076] Figure 3 This is a diagram of a system 300 for preparing mesenchymal stem cells or SVF from adipose tissue for clinical use, according to an embodiment of the present invention.

[0077] According to this embodiment, as described above, a kit comprising various components is provided for extracting adipose tissue from a patient 301 (but according to this embodiment, a non-patient donor can also use system 300 to extract adipose tissue). As described above, adipose tissue can be obtained in a variety of ways known in the art. According to an exemplary embodiment, such as Figure 3As shown, system 300 includes a syringe 302 and a sterile sealed bag 303 for obtaining adipose tissue from a patient 301. The syringe 302 can be any conventional type known in the art, suitable for aspirating varying amounts of adipose tissue, typically from 1 ml to several hundred ml, by manual aspiration. Depending on the different kits of system 300, one or more devices for dissecting the adipose tissue may be provided, such as an ultrasound chamber 305. As shown, the adipose tissue can be placed in the ultrasound chamber 305 while still in the sterile bag 303, or it can be passed through the ultrasound chamber 305 via a sterile tube. System 300 typically includes at least one medium-sized (approximately 100 ml) modified centrifuge tube 310 (as described above). Figure 1 and 2 (As explained). Fatty tissue can be aspirated into tube 310 by applying a vacuum 350 to the internal regions 340-341 of tube 310, or by injection directly from a syringe or via a sterile tube from a syringe. The fatty material typically enters through a turbine or sieve 210, which is perforated with multiple holes 211, as described above, which helps to break down the incoming fatty tissue sample into smaller droplets more suitable for efficient processing within tube 310. After the liposuction sample is placed into tube 310, it can be passed through one or more sterile fittings (e.g., Figure 1 and 2 The accessory 125 shown injects various cleaning agents 320 or reagents 321 (e.g., reagents that aid in tissue dissociation) from a sterile container provided as part of the system 300 kit.

[0078] As described above, tube 310 is suitable for easy use within a centrifuge (which may be supplied as part of a system 300 kit, or may be used in conjunction with a standard benchtop or other centrifuge; centrifuges are well known in the art and are generally readily available in a wide range of medical facilities), thus allowing for easy centrifugation of fatty substances in tube 310, for example, separating stem cells from other cells or tissues. Tube 310 is typically provided with one or more connectors 330, 331 for removing isolated stem cells or other cells or materials contained in the cell cluster at the apex of the conical portion 340 of tube 310. Examples of such connectors 330, 331 are shown in... Figure 1 and 2The diagram shows connectors 126 and 127, which aspirate spherical material from tubes 130 and 131, respectively. In some embodiments, the fatty material processed in tube 310 (by any of washing, dissociation, and centrifugation) is applied directly to patient 301; in other embodiments, system 300 may include a generally smaller second tube 360, which, after being removed from tube 310, can be used for further processing of the fatty material. For example, partially enriched fatty material (i.e., processed fatty material having a higher stem cell fraction than that present when the fatty material was initially extracted from patient or donor 301) may be injected (or aspirated by vacuum 350) into tube 360, for example, in a volume of 10 to 20 ml (but other volumes are possible, including volumes the same as or larger than that of tube 310; the relative dimensions shown and described are merely exemplary and should not be considered limiting, especially where different specific tube sizes may be required for various treatment regimens). Therefore, Figure 3 The system 300 shown is exemplary and demonstrates that it can be included in a kit (suitable for a single treatment involving aspiration of fat from patient 301) along with various reagents and detergents for on-demand use in a sterile container, processed in a portable, continuously sterile environment (which includes elements of the system 300 delivered as a kit), and applied to the same or another patient 301 without stem cell culture or differentiation. It should be understood that in some embodiments, it may be desirable to use techniques known in the art to differentiate the stem cell fractions obtained with system 300, for example, to obtain and culture precursor osteocytes or other precursor cell types differentiated from mesenchymal stem cells or SVF obtained according to the invention.

[0079] Methods for extracting and processing adipose tissue

[0080] In implementing the methods disclosed in this invention, cells (such as mesenchymal stem cells or SVF) intended for use in patient 301 can be obtained from adipose tissue. Adipose tissue can be obtained by various methods known to those skilled in the art. For example, adipose tissue can be removed from the patient by liposuction, ultrasonic liposuction, and fat excision. Furthermore, combinations of these procedures, such as a combination of fat excision and liposuction, may also be included. Since the tissue or certain fractions of the tissue will be used for reimplantation into the patient, adipose tissue should be collected in a manner that preserves the viability of the cellular components and minimizes the possibility of contamination by potentially infectious organisms (such as bacteria and / or viruses). Therefore, tissue extraction is more preferably performed aseptically to minimize contamination.

[0081] Figure 4 This is a diagram of an advanced process 400 for preparing mesenchymal stem cells or SVF from adipose tissue for clinical use, according to an embodiment of the present invention.

[0082] According to this embodiment, in step 401, a disposable tissue extraction system is provided as a component of a sterile kit designed to obtain processed, stem cell-rich lip aspirate (e.g., syringe 302). Then, in step 402, a technician, physician, or other professional extracts adipose tissue (or cultured adipose tissue) with an initial concentration of stem cells (specifically MSCs) from a patient (or donor). For negative pressure liposuction, the adipose tissue can be collected by inserting a cannula into or near adipose tissue deposits present in the patient's body 301, and then aspirated into an aspiration device. In one embodiment, a small cannula can be attached to syringe 302, and the adipose tissue can be manually aspirated. Syringe 302 or other similar devices can be used to harvest relatively small to moderate amounts of adipose tissue (e.g., from 0.1 to several hundred milliliters).

[0083] Negative pressure liposuction can be used to remove adipose tissue from a patient 301 because it provides a minimally invasive method for tissue collection with a relatively low risk of damaging stem cells. Furthermore, the widespread use of liposuction in this field has spurred the development of sophisticated and safe techniques for removing large amounts of adipose tissue from donors or patients. In a preferred embodiment, the system includes a disposable aspiration system that extracts the adipose tissue into a sterile, sealed bag for further processing. This system is more suited to relatively small devices, such as a miniature syringe 302, with the corresponding advantage that fat aspiration can be performed under local (or even no) anesthesia, rather than general anesthesia.

[0084] Adipose tissue removed from a patient can be collected in a sterile container (such as a sterile sealed container 303) for further processing. As described in this invention, in one embodiment, a device is designed specifically for collecting tissue to create a processed adipose tissue cell population, comprising stem cells and / or endothelial precursor cells. In a preferred embodiment, the device is a disposable aspiration system that extracts adipose tissue from a patient into a sterile sealed bag 303. In an alternative embodiment, the device can be any commonly used device typically used by the physician performing the extraction procedure to collect tissue.

[0085] Because only a small amount of adipose tissue is typically required, the application of this invention is not limited to individuals with large amounts of adipose tissue. The amount of tissue to be collected will depend on many variables, including but not limited to the donor's body mass index, the availability of suitable sites for harvesting adipose tissue, accompanying and pre-existing medications and conditions (such as anticoagulation therapy), and the clinical purpose of collecting the adipose tissue. Furthermore, the concentration of stem cells present in adipose tissue varies considerably from individual to individual; therefore, when a specific amount of stem cells is required, a larger or smaller volume of adipose tissue may be needed to obtain the desired amount. For example, the concentration of stem cells in tissue extracted from a lean individual will typically be greater than that extracted from an obese donor (this reflects the typical dilution effect of increased fat content in the adipose tissue of obese individuals).

[0086] A preferred method for obtaining human or donor adipose tissue is through excision or liposuction, as is well known in the art. Regardless of the method of obtaining the adipose tissue, the pluripotent cells of the present invention are present in the initially excised or extracted adipose tissue. The adipose tissue can then be processed to facilitate the isolation / concentration of stem cells. For example, pluripotent cells can be obtained by washing the collected adipose tissue with a physiologically compatible solution such as phosphate-buffered saline (PBS). Typically, the washing step includes rinsing the adipose tissue sample with PBS or lactated Ringer's solution, stirring or centrifuging the sample, and separating the tissue from the fluid in the sample. In addition to washing, the adipose tissue can be dissociated. Dissociation can occur by enzymatic degradation (e.g., treatment with collagenase, trypsin). Other dissociation methods, such as mechanical stirring, acoustic or ultrasonic energy, or thermal energy methods, can be used as alternatives to or in combination with such enzymatic treatment. The cells can then be centrifuged, and the resulting cell clusters (containing pluripotent cells) can be resuspended in a suitable solution (e.g., PBS) for further processing.

[0087] In step 403, a portion of the adipose tissue obtained in step 402 is processed (e.g., as described above) to obtain a higher concentration of stem cells, and in step 404, the processed stem cells are applied from a sterile container to patient 301, which may be located in the kit of system 300 or may be available in a medical facility. Pluripotent cells in a resuspended cell mass can be separated from other cells in the resuspended cell mass by methods including, but not limited to, cell sorting, size grading, particle size, density, molecular, morphological, and immunohistochemical methods (e.g., panning using magnetic beads, fluorescence-activated cell sorting / FACS, magnetically activated cell sorting / MACS, or affinity chromatography). In some immunologically based cell separation methods, pluripotent cells can be obtained through positive screening using antibodies or other specifically binding proteins that bind to epitopes on the cell surface. In such methods, as known to those skilled in the art, cells can be recovered from antibody adherents by washing once or multiple times with a suitable buffer. Alternatively, pluripotent cells can be separated by negative screening. According to various embodiments of the invention, stem cell collection kits may include sterile containers and / or reagents to facilitate the isolation or concentration of stem cells from fat aspiration samples. The presence of pluripotent cells is more suitable for assessment using, for example, specific cell surface labeling and / or counting techniques known in the art prior to their reintroduction.

[0088] Pluripotent cells obtained using the system 300 of the present invention are typically introduced (or re-introduced) into a patient without expansion or differentiation. However, in some embodiments, pluripotent cells can be expanded and / or differentiated in vitro. That is, after isolation, pluripotent cell activity can be maintained and they can be proliferated in a culture medium. Using techniques known in the art, pluripotent cells can be induced to differentiate (or phenotypically alter) into the desired cell type. Modifying or adjusting the concentration of the culture medium and / or culture medium supplements according to the needs of the cells used is within the skill of those skilled in the art. In another embodiment, the pluripotent cells of the present invention can be expanded in a culture medium of a defined composition, wherein serum is replaced by a combination of serum albumin, serum transferrin, selenium, and recombinant proteins, including but not limited to insulin, platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF), as known in the art. The maintenance conditions for the pluripotent cell population of the present invention may also include one or more cytokines that allow the cells to remain in an undifferentiated form.

[0089] If differentiation into pluripotent cells is desired, one or more other substances can be added to the culture to induce the desired specific phenotypic changes. Such substances may include activators of one or more intracellular signaling pathways (e.g., mitogen-activated protein kinases and SMAD), activators of transcription factors (e.g., sox9, L-sox5, and L-sox6), activators that produce and interact with extracellular matrix proteins (e.g., collagen II, aggregates, and chondroitin oligomeric matrix proteins), growth factors, cytokines, chemokines, hormones, and environmental factors (e.g., oxygen tension).

[0090] Figure 5This is a detailed process diagram 500 for preparing mesenchymal stem cells or SVF from adipose tissue for clinical use, according to a preferred embodiment of the present invention. According to this embodiment, in step 501, adipose tissue is extracted from a donor or patient 301. In step 502, the fat is injected or aspirated into a modified centrifuge tube 310 via a vacuum 350. Alternatively, in step 503, the extracted fat can be washed using a detergent known in the art, such as PBS. In step 504, the fat may be selectively dissociated to break down adipose tissue and cell clumps for subsequent processing. In step 505, the cells or fat may be centrifuged to separate cell and tissue fractions with different specific gravities. For example, in properly prepared fat, MSCs can form cell clumps at the apex of the cone-shaped volume 340 of tube 310 and thus separate to a considerable extent from lighter cell or tissue fragments. In many cases, the cell clusters resulting from step 505 may have a slightly enriched concentration of stem cells (relative to the concentration of stem cells present in the adipose tissue extracted from the donor or patient in step 501), and further enrichment may be expected. In step 506, the stem cell-rich cell clusters may be resuspended in a suitable solution for further processing (including possibly repeating steps 502 through 505). In step 507, stem cells may be selectively further separated from other cell fractions using techniques known in the art. Pluripotent cells in the resuspended cell clusters can be separated from other cells in the resuspended cell clusters by methods including, but not limited to, cell sorting, size grading, particle size, density, molecular, morphological, and immunohistochemical methods (e.g., panning using magnetic beads, fluorescence-activated cell sorting / FACS, magnetically activated cell sorting / MACS, or affinity chromatography). In some immunologically based cell separation methods, pluripotent cells can be obtained through positive screening using antibodies or other specifically binding proteins that bind to epitopes on the cell surface. In such methods, as known to those skilled in the art, cells can be recovered from the antibody adherent by washing once or multiple times with a suitable buffer. Alternatively, pluripotent cells can be isolated by negative screening. In step 508, the concentration of stem cells in the processed adipose sample can be determined using any of the various methods known in the art. The presence of pluripotent cells is more suitable for assessment using, for example, specific cell surface labeling and / or counting techniques known in the art before their reintroduction. In step 509, it can be determined, at least in part, based on the concentration determined in step 508 whether further processing of the adipose material is needed for further concentration (i.e., to increase the concentration of stem cells), which, if desired, can be performed by repeating one or more of steps 502 to 508. In step 510, stem cells can be selectively cultured using techniques well-known in the art to obtain a larger quantity of stem cells.

[0091] Similarly, in step 511 (which may be performed before or after step 510, or actually simultaneously with step 510), pluripotent stem cells may be differentiated into one or more types of precursor cells using techniques known in the art. Finally, in step 512, the stem cells or precursor cells extracted and processed as described above with reference to steps 501 to 511 may be administered to one or more patients 301, or a bank may be established for later use in treatment, cosmetic or other applications.

[0092] Figure 6 and 7 Another embodiment of centrifuge tube 600 is shown, which can be used as a tool for preparing injectable fat grafts as described above or as a tool for cell fractionation collection. Centrifuge tube 600 includes a body 602 having an internal volume 604. Body 602 includes a generally cylindrical portion 601 and a generally conical portion 603. Centrifuge tube 600 also includes a cap 606, which is secured to the tube by a cap ring 608 via a fastening device 609. Although in Figure 6 The diagram shows a threaded fastener, but it should be understood that any suitable fastener can be used to secure the cap ring to the tube 600.

[0093] Cover 606 includes multiple openings 607 (see...) Figure 7 Each opening may include a connector, which may be a needleless shut-off valve 610 connected thereto. The needleless shut-off valve 610 is similar to the connectors 125-127 described above and allows for the aseptic extraction of samples from the body 602. For example, the needleless shut-off valve 610 may include at least one sterile tissue inlet connector, at least one sterile processing fluid inlet connector, at least one sterile aspiration connector, and at least one sterile extraction port. The valve 610 also allows for the extraction and introduction of reagents into or from the body 602. In other embodiments, there may be more or fewer needleless shut-off valves 610.

[0094] The centrifuge tube 600 also includes a plurality of tubes 612, similar to tubes 130, 131 as described above, and provides channels for removing samples from the body 602.

[0095] The centrifuge tube 600 also includes a mixing ring 614 located within its internal volume, which promotes the homogenization of fats prior to centrifugation and also traps filaments of connective tissue during enzymatic digestion. The mixing ring prevents these residues from clogging the screen 616 and filter 618 (described below).

[0096] The screen 616 is located below the mixing ring 614. In one embodiment, the screen 616 is configured to divide the internal volume 604 of the body 602 into two parts (e.g., substantially two halves) and to provide support for viscous tissue introduced into the container 600. In an alternative embodiment, the screen 616 is configured to provide support for the viscous material introduced into the container 600 and to divide the internal volume 604 of the body 602 proportionally rather than into two equal parts (e.g., 60% of the internal volume 604 above the screen and 40% below the screen; 70% of the internal volume 604 above the screen and 30% below the screen; 80% of the internal volume 604 above the screen and 20% below the screen; 40% of the internal volume 604 above the screen and 60% below the screen; 30% of the internal volume 604 above the screen and 70% below the screen; 20% of the internal volume 604 above the screen and 80% below the screen). In some embodiments, the sieve 616 has a plurality of holes 617 with a size of about 1 mm, thereby allowing liquid to be rinsed away while dense fractions (e.g., fat grafts) are retained on top. The sieve 616 is also used to break down tissue into small particles smaller than 1 mm, such as when centrifugal force is applied during centrifugation.

[0097] Therefore, in one embodiment, the size of the plurality of holes 617 is set to have a diameter of approximately 1 mm. In a particular embodiment, the size of the holes 617 is set to have a diameter of no more than 1.2 mm (e.g., in the range of 1 mm to 1.2 mm). Additionally, the size of the holes is set to have a diameter of less than 1 mm (e.g., 0.9 mm to 1 mm; 0.8 mm to 1 mm; 0.7 mm to 1 mm; 0.6 mm to 1 mm; 0.5 mm to 1 mm; 0.5 mm to 0.6 mm; 0.6 mm to 0.7 mm; 0.7 mm to 0.8 mm; 0.8 mm to 0.9 mm). In another embodiment, the size of the holes 617 is set to have a diameter of less than 500 micrometers. Specifically, the size of the holes 617 is set to be between 350 micrometers and 450 micrometers.

[0098] Additionally, the distribution and / or density of the holes can be configured according to the application. In one embodiment, the holes are uniformly distributed on the surface of the screen 616. Furthermore, in one embodiment, more than 50% of the surface area of ​​the screen 616 is holes 617. Therefore, in this embodiment, more than half of the surface area of ​​the screen 616 is holes. In a first specific embodiment, the screen 616 has holes 617 that are greater than or equal to 60% of its surface area (e.g., 60% to 70% of the surface area is holes 617). In a second specific embodiment, the screen 616 has holes 617 that are greater than or equal to 70% of its surface area (e.g., 70% to 80% of the surface area of ​​the screen 616 is the surface area of ​​holes 617). In a third specific embodiment, the screen 616 has holes 617 that are greater than or equal to 80% of its surface area (e.g., 80% to 90% of the surface area of ​​the screen 616 is the surface area of ​​holes 617). In a fourth specific embodiment, the screen 616 has holes 617 that are greater than or equal to 90% of its surface area (e.g., 90% to 95% of the surface area of ​​the screen 616 is the surface area of ​​the holes 617).

[0099] As described above, the sieve 616 can be configured to: provide support for the liquid / non-liquid fraction of a tissue; and, when a force (such as centrifugal force) is applied, prepare a fat graft with certain properties. For example, a sieve with pores 617 of a certain aperture and surface area can, upon centrifugation, prepare an injectable fat graft that is sufficiently consolidated for local application. In particular, the prepared fat graft has a certain viscosity, such as a certain centipoise (cP) or a certain pascal second (Pa·s) selected for the current application. Furthermore, the fat graft can be characterized by viscoelasticity, which includes viscosity (e.g., fluid properties and a measure of flow resistance) and elasticity (properties of solid materials). Therefore, one or both of the aperture of pores 617 and the percentage of the total area of ​​pores 617 on the surface area of ​​the sieve 616 can affect the properties of the material prepared by centrifugation. As an example, reducing the size of the pores 617 (e.g., making the pores 617 have a diameter between 350 and 450 micrometers) and increasing the total surface area of ​​the pores on the screen 616 (e.g., making the surface area of ​​the pores 617 80% to 90%) can produce a more liquefied fat graft by centrifugation. This fat graft is viscous enough that its stem cells are still preserved, so that the liquefied fat graft can be used as a bio-ink for printing.

[0100] Therefore, due to the various properties of the sieve 616, the subsequent tissue produced by centrifugation can be used in different situations. As an example, the aperture of the pores 617 of the sieve 616 is approximately 1 mm (e.g., 1.2 mm), and centrifugation can cause the tissue to break down into pieces smaller than 1.2 mm. This fragmented tissue, with an aperture of approximately 1 mm like the pores 617 of the sieve 616, can preserve stem cells, thus making the fragmented tissue clinically applicable. For example, with tissue of this size, the fragmented tissue can be used in various situations, such as for injection through needles of a specific size and / or for topical application to promote wound healing.

[0101] In one application, a 3D bioprinter can be used to 3D bioprint fat grafts generated via centrifugation. 3D bioprinting utilizes specialized materials to create three-dimensional tissues and organs from these centrifuged fat grafts. These specialized materials may be referred to as bioinks. In a particular embodiment, the bioink may be hydrogel-based, capable of retaining cells after printing. In one embodiment, bioprinting involves the precise three-dimensional deposition of cells using a method compatible with automated, computer-aided 3D prototyping equipment (e.g., a bioprinter). Various 3D bioprinting procedures are envisioned in this invention. One example bioprinting procedure includes microextrusion, which uses applied force to extrude bioink through a printhead.

[0102] Specifically, the pore size of 617, ranging from 350 to 450 micrometers, allows for the preparation of micronized fats that are compatible with 3D bioprinting, such as with the nozzle diameter of a 3D printer (which can be approximately 450 micrometers or less), to 3D print 3D tissue structures together with hydrogels. This fat graft contains abundant endothelial progenitor cell fractions, which can support and maintain angiogenesis in ischemic tissues, organs, and wounds. Therefore, centrifuge tubes 600 with pores 617 of a specific pore size (e.g., 350 to 450 micrometers) can produce materials suitable for microextrusion.

[0103] In this regard, any one, any combination, or all of the size, distribution, or density of the pores 617 (e.g., the total surface area of ​​the pores 617 on the surface of the screen 616) can be adjusted depending on the application. In particular, any one, any combination, or all of the size, distribution, or density of the pores can be adjusted to prepare different types of fat grafts with different physical properties (e.g., including different viscosities and / or flowability).

[0104] The centrifuge tube 600 also includes a filter 618 located in the lower conical portion of the internal volume 604, below the sieve 616. The filter 618 serves as a collection platform for tissue material that permeates the sieve 616. The filter 618 may also include tubes 619 extending therefrom, configured to connect to tubes 612 within the body 602. Figure 6 and 7 As shown, there are two tubes 619. Alternatively, centrifuge tube 600 may consist of only a single tube 619.

[0105] In some embodiments, filter 618 includes pores of approximately 100 micrometers. Therefore, during centrifugation, tissue is squeezed through the sieve and collected at the top of the filter surface, while the liquid fraction moves downwards to the bottom of tube 600. Thus, this "micronized" tissue (typically fat) can be collected through the compartment in the middle section of 602, between sieve 616 and filter 618. The correct tubing is then connected to the top cap inlet. In this way, the device is used as a tool for preparing injectable fat grafts.

[0106] If digestive enzymes are applied to adipose tissue, stromal cell fractions will be separated. Cells smaller than 100 micrometers in size can be collected at the bottom of the conical portion 603 after centrifugation and passing through filter 618. Therefore, filter 618 allows for the separation and purification of specific cell fractions from digested tissue.

[0107] The fat grafts and cell fractions collected through centrifuge tube 600 can then be further washed and concentrated in a second, smaller centrifuge tube 700, such as... Figure 8-9 As shown. In some embodiments, centrifuge tube 700 may be similar to centrifuge tube 360 ​​described above. Centrifuge tube 700 may include, for example, a body 702 having an internal volume of about 50 mL. Tube 700 may also include a cap 704 having a plurality of openings 706, which may be a needleless shut-off valve or connector as described above. Centrifuge tube 700 also includes one or more tubes 708, similar to tubes 130, 131, 612 described above, and providing a channel for removing samples from body 702.

[0108] In one embodiment, the second tube 700 can be continuously connected to the tube 600 by applying negative pressure via a pump or syringe. Alternatively, material collected in the first tube 600 can be transferred to the second tube 700 by collecting it directly from the first tube 600 into a syringe and then releasing it into the second tube 700, where the material is washed and concentrated by centrifugation. The final product is collected through a corresponding inlet mounted on top of the second centrifuge tube 700, similar to the inlet in tube 600. In some embodiments, the second centrifuge tube 700 is not used for fat graft preparation.

[0109] In some embodiments, a method for preparing fat grafts is disclosed. The method includes aseptically transferring a fatty substance into a first centrifuge tube 600. Next, one or more fluids are added through at least one fluid inlet connector or needleless shut-off valve 610 to wash the fatty substance in the first centrifuge tube 600. The first centrifuge tube 600 containing the fatty substance is then centrifuged at a low speed to separate contaminants and dense fat fractions. The dense fat remains at the top of a sieve 616, while the liquid collects in the lower conical portion 603. The first centrifuge tube 600 containing the washed dense fat is then centrifuged at a higher speed, causing the dense fat to pass through the pores of the sieve 616 by centrifugal force, and accumulating broken fat particles (fat grafts) in the compartment between the sieve 616 and the filter 618. Thus, in one embodiment where the sieve 616 has approximately 1 mm of pores, the first centrifuge tube 600 is first centrifuged at a low speed, and then centrifuged at a higher speed (e.g., 400 g). The filter surface serves as a bottom to accumulate fat particles. Broken fat (oil) and debris are filtered downwards through filter 618 to the lower conical portion 603. The accumulated fat graft can then be collected by attaching a syringe to at least one of the inlet or needleless shut-off valve 610 and directing the corresponding tubing to the intermediate compartment between filter 618 and screen 616. The resulting fat graft can then be applied to a patient for therapeutic or cosmetic purposes.

[0110] Furthermore, centrifuge tubes 600 with sieves 616 of different pore sizes can be centrifuged. As an example, when the pore size of the sieve 616 is approximately 1 mm, the first centrifuge tube 600 can be rotated first at a lower speed and then at a higher speed (e.g., 400 g). Alternatively, when the pore size of the sieve 616 is approximately 1 mm or smaller (e.g., 350 to 450 micrometers), the first centrifuge tube 600 can be rotated first at a lower speed and then at a higher speed of less than 400 g (e.g., 200 g). The higher (or second) speed can be selected based on the desired rheological properties (e.g., flowability). In particular, a higher speed (e.g., 400 g) can provide a final graft with higher density and higher dry weight. Conversely, a lower speed (e.g., 200 g) can provide a final graft with lower density and lower dry weight. Thus, depending on the application of the bio-ink (e.g., the carrier) and the crosslinking parameters, the system can select the properties of the sieve 616 (e.g., pore size adjustment processing parameters) to obtain the desired rheological properties in order to form fat grafts with printable properties suitable for a variety of applications (e.g., centrifugation can be performed on a machine that includes multiple discrete higher (or second) speed settings, where a higher speed is selected for the rheological properties required for the corresponding fat graft).

[0111] therefore, Figure 6 and 7The system has a dual function, allowing both fat graft preparation and cell-level harvesting. Similar to the system disclosed above, the process is performed in a completely closed and aseptic manner, while remaining transparent and flexible to the user. In some implementations, Figure 6 and 7 The system can be with Figure 8-9 Use the tube 700 shown.

[0112] Therefore, in one implementation, such as Figure 6 and 7 The centrifuge tube 600 shown is configured to prepare fat grafts and / or cell-grade extracts. Specifically, the centrifuge tube is configured for processing fat graft formulations and for processing cell-grade harvests, thereby achieving greater flexibility as described in this invention. In this respect, the centrifuge tube provides the ability to prepare cell-rich fat grafts. Specifically, cell-rich fat grafts can be prepared by returning / mixing the extracted cell fractions into the fat graft (e.g., fat grafts previously prepared via the centrifuge tube). In practice, this can be accomplished by separating the raw fat material into at least two fractions, the first fraction for preparing the fat graft and the second fraction for enzymatic digestion to prepare the cell-grade extract.

[0113] In one embodiment, the same centrifuge tube can be used to prepare fat grafts and as at least part of a cell fractionation extraction process to prepare cell-rich fat grafts. Therefore, in one specific embodiment, the same centrifuge tube can be used sequentially, such as by sequentially applying a fat grafting protocol and a cell extraction protocol to perform at least one of the steps of preparing fat grafts and preparing cell fractionation extracts (e.g., preparing a fat graft in a centrifuge tube using a fat grafting protocol, removing the prepared fat graft from the centrifuge tube, then applying a cell extraction protocol using the same centrifuge tube to prepare a cell fractionation extract, and then returning / mixing the extracted cell fractions back into the fat graft). As described above, in one embodiment, two centrifuge tubes can be used to prepare cell fractionation extracts, including: first processing the biomaterial in a first centrifuge tube; after processing in the first centrifuge tube, removing the processed biomaterial from the first centrifuge tube and then inserting it into a second centrifuge tube for further processing. In this respect, at least one of the centrifuge tubes used in the patient cell fractionation extraction process can also be used to prepare fat grafts for the same patient.

[0114] In an alternative implementation, different centrifuge tubes can be used to prepare fat grafts (e.g., using...). Figure 6 and 7 The fat grafts prepared in the first centrifuge tube of the type shown in the figure) and the cell-level extracts prepared (e.g., in Figure 6 and7 (As explained above). Afterwards, the cell-level extract can be mixed into the fat graft. This allows for the use of centrifuge tubes (regardless of the type used). Figure 6 and 7 The commonly used centrifuge tubes disclosed in the literature (e.g., those common to the steps of performing fat grafting and cell fractionation extraction protocols) or those used in other ways... Figure 6 and 7 Multiple centrifuge tubes of the same type disclosed herein are used to mix cells into fat grafts, thereby providing additional implementation for the regenerative properties of fat grafts.

[0115] As described above, one application includes 3D printing, which may involve the sequential printing of fat and cells. For example, 3D printing may include multilayer fabrication to achieve a target geometry of the implant and have a desired concentration of cells within a monolithic porous structure composed of bio-ink and fat. In practice, a 3D printer may include multiple nozzles, such as a first nozzle and a second nozzle. For example, a combination of a fat graft with desired rheological properties and another material (e.g., a polymeric material that serves as a carrier and provides the ability to 3D print structures) can be delivered via the first nozzle. The 3D printer may operate the first nozzle together with the second nozzle, wherein the second nozzle delivers extracted cell fractions encapsulated in a carrier (such as a printable polymeric carrier). For example, the 3D printer may operate the second nozzle in parallel with the first nozzle. Alternatively, the 3D printer may operate the second nozzle in series with the first nozzle. In another embodiment, the 3D printer may operate the second nozzle in at least partially in series and at least partially in parallel with the first nozzle. Example carriers include, but are not limited to, polymers, including collagen, gelatin, alginate and hyaluronic acid (HA), as well as synthetic polymers, such as PVA and polyethylene glycol (PEG), and are cross-linked by UV or chemical means.

[0116] As an example, the task of a 3D printer is to print a 3D shape composed of fat grafts (using bio-inks to achieve structural stiffness) and one or more structures within the 3D shape (such as one or more blood vessels inside the printed fat). The 3D printer can print cells and fat in specific patterns, thereby creating locations where cells are more concentrated within the 3D printed structure (e.g., the location of blood vessels in the 3D printed structure in the blood vessel example).

[0117] same, Figure 6 and 7 The centrifuge tube 600 shown is particularly well-suited for use with 3D printing because it can produce fat grafts and cell-level extracts (using a single centrifuge tube 600 or multiple centrifuge tubes 600). When combined with bio-inks and carriers respectively, it can be used in an efficient and cost-effective manner with the nozzles of 3D printers.

[0118] Reintroducing fat-derived stem cells

[0119] The kit of system 300 according to the invention can be used to produce pharmaceutical compositions containing, as described in the invention, a preventive or therapeutically effective amount of mesenchymal stem cells or SVF, preferably in a fully purified form, while containing an appropriate amount of a suitable carrier to provide stem cells in a form suitable for safe and effective administration to patients.

[0120] Compared to other treatments using adipose tissue or stem cells derived from adipose tissue, patients receiving treatment according to various embodiments of the invention may receive different concentrations of stem cells. Therefore, adipose tissue taken from a patient can be processed to alter the concentration of stem cells administered to the patient. In a preferred embodiment of the invention, the patient receives a higher concentration of stem cells than is typically found in adipose tissue transplantation and other similar stem cell-based therapies. Concentrated stem cells may be administered in the form of a composition comprising adipose-derived stem cells and / or endothelial precursor cells that are substantially free of mature adipocytes and connective tissue, or, as another example, concentrated stem cells may be administered in the form of a composition comprising one unit of adipose tissue (with an increased number of stem cells).

[0121] The pharmaceutical components of the present invention can be formulated according to a specific chosen administration method. For example, the pharmaceutical components can be formulated into a liquid dosage form (e.g., as a suspension) and injected into the subject requiring treatment. Illustrative (non-limiting) examples include formulating the pharmaceutical components of the present invention with a sterile suspension and a pharmaceutically acceptable carrier (such as a saline solution, phosphate-buffered saline (PBS), or any other suitable pharmaceutically acceptable carrier) for preferably administration to a subject (e.g., in the human body) via intravenous, intraperitoneal, subcutaneous, etc., but other routes of administration are also possible.

[0122] According to the present invention, the drug components can be administered to subjects in need using conventional methods. In one particular embodiment, the drug components can be administered intravenously to subjects in need using devices such as syringes, catheters, cannulas, or septa. In any case, any suitable equipment, apparatus, and devices known to those skilled in the art can be used to administer the drug components of the present invention. In a preferred embodiment, the device for administering the drug components is provided as part of the aforementioned kit, or is adapted for use with the aforementioned kit, to facilitate the transfer of the drug components to the subject without contamination.

[0123] In this invention, fat grafts or resuspended cell mixtures can be prepared using fat materials, namely, ex vivo adipose tissue (i.e., adipose tissue / fat aspirate taken from the human body or a donor) or fat cultured in vitro. Once prepared, they can be applied, such as to patients for treatment or cosmetic purposes.

[0124] For illustrative and descriptive purposes, the above description of preferred embodiments of the invention has been provided. These embodiments are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Numerous embodiments have been selected and described to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the various embodiments of the invention, as well as various modifications suitable for the particular intended use. The scope of the invention is intended to be defined by the claims and their equivalents. Those skilled in the art will recognize the possible scope of modifications to the various embodiments described above. Therefore, the invention is defined by the claims and their equivalents. The invention also relates to the following.

[0125] 1. A tissue processing apparatus, the tissue processing apparatus comprising:

[0126] A first centrifuge tube, configured to receive and process biological material, includes an upper cylindrical portion and a lower conical portion, a sterile tissue inlet connector, at least one sterile processing fluid inlet connector, a sterile aspiration connector, and at least one sterile extraction port connected to a first extraction tube.

[0127] The first centrifuge tube further includes an internal space comprising a sieve and a filter located therein. The sieve is configured to provide support for biological material, and the filter is located below the sieve in the lower conical portion of the first centrifuge tube.

[0128] A second centrifuge tube, configured to receive and further process biological material from the first centrifuge tube, the second centrifuge tube having at least one sterile connector, wherein the second centrifuge tube is releasably connected to one of the at least one sterile extraction ports of the first centrifuge tube via the at least one sterile connector.

[0129] 2. The tissue processing apparatus according to claim 1, wherein the sieve divides the internal space into two halves; and

[0130] The first centrifuge tube has a top and a apex, wherein the sterile tissue inlet connector, the at least one sterile processing fluid inlet connector, the sterile aspiration connector, and the at least one sterile extraction port are located near the top of the first centrifuge tube.

[0131] 3. The tissue processing apparatus according to 2, wherein the first extraction tube extends a certain distance from the extraction port to the apex within the first centrifuge tube.

[0132] 4. The tissue processing apparatus according to claim 2, wherein the at least one sterile processing fluid inlet connector, the sterile aspiration connector and the at least one sterile extraction port are disposed within a cap fixed to the top of the first centrifuge tube.

[0133] 5. The tissue processing apparatus according to claim 1 further includes a vacuum device fixed to the sterile suction connector.

[0134] 6. The tissue processing apparatus according to claim 1 further includes a mixing ring located within the upper cylindrical portion of the first centrifuge tube.

[0135] 7. The tissue processing apparatus according to claim 1, wherein the filter comprises pores of about 100 micrometers.

[0136] 8. The tissue processing apparatus according to claim 1, wherein the apparatus is configured for processing fat graft formulations and for processing cell-grade harvests.

[0137] 9. The tissue processing apparatus according to claim 1, wherein the screen comprises holes with a diameter of about 1 mm.

[0138] 10. The tissue processing apparatus according to claim 1, wherein the screen comprises holes with a diameter of less than 1.2 mm.

[0139] 11. The tissue processing apparatus according to claim 1, wherein the screen comprises pores with a diameter in the range of 350 micrometers to 450 micrometers.

[0140] 12. The tissue processing apparatus according to 11, wherein the surface area of ​​the holes is at least 60% of the total surface area of ​​the sieve.

[0141] 13. The tissue processing apparatus according to 11, wherein the surface area of ​​the holes is at least 80% of the total surface area of ​​the sieve.

[0142] 14. A system for processing tissue in a closed pathway to maintain sterility, the system comprising:

[0143] A first centrifuge tube, the first centrifuge tube having a substantially cylindrical body, the body having a top, an inverted conical apex and an internal volume, the first centrifuge tube further comprising:

[0144] At least two needleless shut-off valves;

[0145] An extraction tube releasably fixed to one of the at least two needleless shut-off valves, the extraction tube extending a certain distance towards the apex within the internal volume; and

[0146] A screen disposed within the internal space of the main body; and

[0147] A second centrifuge tube, comprising a substantially cylindrical body having a top, an inverted conical apex, and an internal volume, further comprising:

[0148] At least one needleless shut-off valve; and

[0149] An extraction tube releasably fixed to at least one needleless sealing valve, the extraction tube extending a certain distance towards the apex within the internal volume.

[0150] The second centrifuge tube is releasably connected to one of the at least two needleless valves of the first centrifuge tube via the at least one needleless shut-off valve.

[0151] 15. The system according to 14, wherein the screen divides the internal space of the first centrifuge tube into an upper part and a lower part, and furthermore, wherein the screen is adapted to dissociate tissue in the lower part.

[0152] 16. The system of claim 14 further includes a vacuum device connected to one of the at least two needleless shut-off valves to aspirate tissue into the internal volume.

[0153] 17. The system according to 14, wherein the at least two needleless shut-off valves are located near the top of the first centrifuge tube.

[0154] 18. The system according to 17, wherein the at least two needleless sealing valves are disposed within a cap fixed to the top of the first centrifuge tube.

[0155] 19. The system according to 14, wherein the extraction tube extends variably a distance from at least two needleless shut-off valves toward the apex within the first centrifuge tube.

[0156] 20. The system of claim 14 further includes a syringe connected to one of the at least two needleless shut-off valves to aspirate tissue into the internal volume.

[0157] 21. A multi-device integrated system for processing tissues in a closed pathway to maintain sterility, the multi-device integrated system comprising:

[0158] Centrifuge tubes having a substantially cylindrical body, the body having a top, an inverted conical apex, and an internal volume, and further comprising:

[0159] At least two needleless shut-off valves;

[0160] An extraction tube releasably fixed to each of the at least two needleless shut-off valves, the extraction tube extending a certain distance toward the apex within the internal volume;

[0161] A screen disposed within the internal space of the main body; and

[0162] The filter located below the screen at the inverted conical apex of the centrifuge tube; and

[0163] A container having at least one needleless shut-off valve,

[0164] The needleless shut-off valve can be releasably connected to one of the at least two needleless shut-off valves on the centrifuge tube to aseptically transfer material contained in the closed system.

[0165] 22. The system of claim 21 further includes a vacuum device connected to one of the at least two needleless shut-off valves to aspirate tissue into the internal volume.

[0166] 23. The system according to 21, wherein the screen is configured to divide the internal volume into two halves.

[0167] 24. A method for preparing fat grafts, the method comprising the following steps:

[0168] (a) Aseptically transferring fat into a centrifuge tube, the centrifuge tube comprising an upper cylindrical portion and a lower conical portion, a sterile tissue inlet connector, at least one sterile processing fluid inlet connector, a sterile aspiration connector, and at least one sterile extraction port connected to a first extraction tube, the centrifuge tube further comprising a sieve and a filter located therein, the filter being located below the sieve in the lower conical portion of the centrifuge tube;

[0169] (b) Wash the fatty substance in the centrifuge tube by adding one or more fluids through at least one sterile processing fluid inlet connector;

[0170] (c) Centrifuge the centrifuge tube containing the fatty substance at low speed to separate contaminants and dense fatty portions, wherein the dense fatty portion is retained at the top of the sieve and the liquid is collected in the lower conical portion;

[0171] (d) Centrifuge the centrifuge tube containing washed dense fat at a higher speed to force the dense fat through the holes of the screen by centrifugal force and accumulate broken fat particles in the compartment between the screen and the filter, wherein the surface of the filter serves as the bottom for accumulating fat particles, and wherein the broken fat and debris are filtered downward through the filter into the lower conical portion.

[0172] (e) Collect the accumulated fat graft using a syringe and connect the syringe to at least one inlet, allowing the corresponding tubing to lead to the compartment between the filter and the sieve, thus preparing the fat graft; and

[0173] (f) Applying fat grafts to patients for therapeutic or cosmetic purposes.

[0174] 25. The method according to 24 further includes dividing the extracted fat into a first part and a second part;

[0175] The fat transferred to the centrifuge tube in (a) includes the first portion of the extracted fat; and

[0176] Also includes:

[0177] The second portion of the fat is aseptically transferred to the centrifuge tube;

[0178] One or more fluids are added through the at least one sterile processing fluid inlet connector to process the second portion of the fat in the centrifuge tube, the processing including cleaning and dissociating one or more of the fat;

[0179] Centrifuge the centrifuge tube containing the second portion of the processed fat to concentrate the cell cluster containing the enriched stem cell fraction;

[0180] The cell clusters are resuspended in a fluid to obtain a cell mixture richer in stem cells than the original second portion of the fat, which is called a resuspended cell mixture.

[0181] 26. The method of 25, further comprising, after treating a second portion of the liposuction agent in a centrifuge tube, transferring the second portion of the treated fat to a second centrifuge tube for further treatment of the second portion of the fat, the second centrifuge comprising a centrifuge tube with at least one sterile fitting, wherein the second centrifuge tube is releasably connected to one of at least one sterile extraction port of the first centrifuge tube via at least one sterile fitting.

[0182] 27. The method according to 25, wherein the fat graft is removed from the centrifuge tube before the second portion of the fat is aseptically transferred to the centrifuge tube.

[0183] 28. The method according to 27, wherein the resuspended cell mixture and the fat graft can be used as a composition.

[0184] 29. The method of 28, wherein applying the resuspended cell mixture and the fat graft includes 3D printing using the resuspended cell mixture and the fat graft.

[0185] 30. The method of 29, wherein 3D printing includes dispensing the fat graft suspended in bio-ink using a first nozzle in the 3D printer, and dispensing the resuspended cell mixture in a second nozzle of the 3D printer.

[0186] 31. The method according to 24, wherein the centrifuge tube comprises a first centrifuge tube;

[0187] It also includes dividing the extracted fat into a first part and a second part;

[0188] The fat material transferred to the first centrifuge tube in (a) includes the first portion of the extracted fat material;

[0189] Also includes:

[0190] The process involves aseptically transferring the second portion of the fat to a second centrifuge tube, the second centrifuge tube being configured in the same way as the first centrifuge tube, including the upper cylindrical portion and the lower conical portion, the aseptic tissue inlet connector, the at least one aseptic processing fluid inlet connector, the aseptic aspiration connector, and the at least one aseptic extraction port connected to the first extraction tube. The second centrifuge tube also includes the sieve and the filter located therein, the filter being located below the sieve and in the lower conical portion of the second centrifuge tube.

[0191] One or more fluids are added through the at least one sterile processing fluid inlet connector to process the second portion of the fat in the second centrifuge tube, the processing including cleaning and dissociating one or more of the fat;

[0192] Centrifuge the second centrifuge tube containing the second portion of the processed fat to concentrate a cell cluster containing enriched stem cell fractions;

[0193] The cell clusters are resuspended in a fluid to obtain a cell mixture with a higher stem cell concentration than the original second portion of the fat, which is called a resuspended cell mixture.

[0194] 32. The method according to 31, wherein the fat graft produced by the first centrifuge tube and the resuspended cell mixture produced by the second centrifuge tube can be used as a composition.

[0195] 33. The method of 32, wherein applying the resuspended cell mixture and the fat graft includes 3D printing using the resuspended cell mixture and the fat graft.

[0196] 34. The method of 33, wherein 3D printing includes dispensing the fat graft suspended in bio-ink using a first nozzle in the 3D printer, and dispensing the resuspended cell mixture in a second nozzle of the 3D printer.

[0197] 35. The method according to 24 further includes, after step (e), transferring the fat graft to a second centrifuge tube for further washing and concentration, and collecting the fat graft by means of a needle passing through a valve in the second centrifuge tube.

[0198] 36. The method according to 35, wherein the higher speed is in the range of 200g to 400g.

[0199] 37. The method according to 36, wherein centrifugation is performed on a machine including a plurality of second speed settings, in which a higher speed is selected for the corresponding rheological properties of the fat graft.

[0200] 38. The method of 24, wherein the screen is configured to prepare micronized fat compatible with the nozzle of a 3D printer for bioprinting using the fat graft.

[0201] 39. The method of claim 38, wherein the screen comprises pores with a diameter in the range of 350 micrometers to 450 micrometers.

[0202] 40. A method for preparing fat-derived cell fractions, the method comprising the following steps:

[0203] (a) The fat is aseptically transferred into the first centrifuge tube of the device;

[0204] (b) Adding one or more fluids through the at least one sterile processing fluid inlet connector to process the fatty substances in the first centrifuge tube, the processing including cleaning and dissociating one or more of the fatty substances;

[0205] (c) Centrifuge the first centrifuge tube containing the processed fat to concentrate the cell cluster containing the stem cell fraction;

[0206] (d) Aseptically transfer the concentrated cell cluster containing stem cell fractions into the second centrifuge tube of the device;

[0207] (c) Add one or more fluids through the at least one sterile processing fluid inlet connector to wash the cell fractions in the second centrifuge tube;

[0208] (f) Centrifuge the second centrifuge tube containing cell fractions to concentrate the cell clusters containing stem cell-rich fractions;

[0209] (g) Resuspending the cell clusters in a fluid to obtain a cell mixture richer in stem cells relative to the stem cell concentration of the original adipose material; and

[0210] (h) Administering a cell mixture to a patient for therapeutic or cosmetic purposes.

[0211] 41. The method according to 40, wherein the sieve of the first centrifuge tube is configured to prepare micronized fat compatible with the nozzle of a 3D printer for bioprinting using the resuspended cell mixture.

[0212] 42. The method according to 41, wherein the screen comprises pores with a diameter in the range of 350 micrometers to 450 micrometers.

Claims

1. A system for processing tissue in a closed path to maintain sterility, the system comprising: a first centrifuge tube having a substantially cylindrical body with a top, an inverted conical apex, and an interior volume, the first centrifuge tube further comprising: at least two needleless containment valves, the at least two needleless containment valves comprising at least one extraction port; an extraction tube releasably secured to one of the at least two needleless containment valves, the extraction tube extending a distance within the interior volume toward the apex; and a screen disposed within the interior space of the body; the screen dividing the interior space of the first centrifuge tube into an upper portion and a lower portion, and further wherein the screen is adapted to dissociate tissue in the lower portion and provide support for liquid / non-liquid fractions of the tissue; the surface of the screen having uniformly distributed a plurality of pores having a diameter no greater than 1.2 mm; and a filter located in the lower conical portion of the interior volume below the screen, the filter comprising 100 micron pores; and a mixing ring disposed within the interior space of the body, the mixing ring being located above the screen; and and a second centrifuge tube comprising a substantially cylindrical body with a top, an inverted conical apex, and an interior volume, the second centrifuge tube further comprising: at least one needleless containment valve; and an extraction tube releasably secured to the at least one needleless containment valve, the extraction tube extending a distance within the interior volume toward the apex, wherein the second centrifuge tube is releasably connected to one of the extraction ports of the at least two needleless containment valves of the first centrifuge tube via the at least one needleless containment valve; and and a vacuum device connected to one of the at least two needleless containment valves to draw tissue into the interior volume; and a syringe connected to one of the at least two needleless containment valves to draw tissue into the interior volume.

2. The system of claim 1, wherein the at least two needleless containment valves are located proximate the top of the first centrifuge tube.

3. The system of claim 2, wherein the at least two needleless containment valves are disposed within a cap secured to the top of the first centrifuge tube.

4. The system of claim 1, wherein the extraction tube variably extends a distance within the first centrifuge tube from the at least two needleless containment valves toward the apex.

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