Device for isolating or separating cells or cell group from tissue

Abstract

The utility model relates to equipment for separating or separating cells or cell populations from tissues. The apparatus comprises a cell separation chamber (CIC) detachably associated with a cell delivery unit, the CIC adapted to receive tissue to be mechanically manipulated and / or enzymatically manipulated to separate cells or cell populations therefrom, the apparatus is configured to allow cells isolated from the tissue in the CIC to be collected in the delivery unit under gravity, where the delivery unit is configured for delivering the isolated cells in vivo or in vitro. In addition, the utility model generally provides equipment and a method for separating cells from tissues and using the cells in vivo or in vitro.

Description

Technical Field

[0001] The present invention generally contemplates systems and methods for isolating cell populations from various tissues.

[0002] Utility Model Background

[0003] The mesenchymal cells of culture are referred to as mesenchymal stem cells (MSC) to represent their multipotent differentiation potential. The gold standard for separating stromal cells from fat includes decomposing cells by collagenase enzymes, followed by centrifugation, which separates stromal vascular fraction (SVF) precipitation from adipose tissue, and due to its high fat content, stromal vascular fraction precipitation is retained in the upper fluid layer. SVF is separated as a particle that can be separated. After the separation of SVF cells, SVF cells can be active immediately after their separation or used as MSC for clinical purposes after their tissue culture amplification.

[0004] Traditionally, SVF isolation involves manually introducing adipose tissue into a device containing enzymes or mechanical devices for disrupting cells, followed by manual removal of the cell pellet from the device. Storage for later use is limited because storage requires further manipulation and transfer of the cells to an additional container suitable for cryogenic storage. A major obstacle to the storage and later use of SVF cells or the large-scale production of SVF cells is the lack of an apparatus and method to effectively and efficiently isolate, preserve and store SVF cells, as well as other similarly produced cells in highly sterile forms, that does not introduce any sources of contamination and can be carried over from samples extracted directly from the human body and the cells returned directly to the human body in a single methodologically clinically acceptable step.

[0005] The nature of living cells produced by enzymatic or mechanical techniques makes it challenging to develop highly sterile and long-term storage cell samples. Publication [1] attempts to provide at least a partial solution to this problem by providing a system for enriching cell populations from mammalian adipose tissue, the system utilizing an apparatus configured to receive adipose tissue directly from a subject and manipulate the tissue in a single sterile container. The container is suitable for receiving, manipulating and allowing separation of the enriched cell population. The container has a closure lid configured to allow attachment of an electric actuator; a mechanical tissue disruption device that is detachably connected to the electric actuator and is located within the container assembly, the mechanical tissue disruption device being adjacent to but not in contact with the base of the container; and a vacuum pump that is used to activate the aspiration of adipose tissue into the container via an inlet in the closure lid of the container assembly via an aspiration tube. However, the system requires the enriched portion to be separated from its sterile environment and possibly placed in a different container that can be used for storage.

[0006] Background announcement

[0007] [1] WO 2016 / 199149.

[0008] General Description

[0009] The emerging fields of tissue engineering, tissue regeneration, and immunomodulation will make great progress in a large number of clinical protocols in both research and clinical settings. Therefore, the use of isolated cell fractions, in general, or SVF fractions in particular, requires immediate access to such fractions in a live and sterile form in a clinical setting where the cells are produced, manipulated to separate enriched fractions, and used or stored while minimizing sources of contamination that could compromise cell viability and future use. Therefore, what is needed in the art is an improved method and system for separating, processing, and storing cells under conditions routinely required at the point of use.

[0010] The inventors of the technology disclosed herein have developed a unique device and method for separating or separating cells or cell groups from tissues and enabling the cells or cell groups to be further used and stored without resorting to the use of multiple containers or steps. In the most general terms, the inventors provide a device having multiple detachable parts that can be assembled into a single device and operated to obtain a tissue sample through a first opening of the device and deliver the separated cell part directly to the subject's body from another opening, so that all processing steps from tissue to enriched cell groups can be achieved in a single closed and sterile container assembly. The device can also be configured to maintain and contain cell parts under conditions that allow long-term storage (e.g., cryogenic storage, conditions in preparation for future use).

[0011] The novel device of the utility model provides for the first time a high sterility tool that can adapt to all processing steps: cell separation, cell enrichment, cell part collection, cell part separation, cell part storage and cell part clinical application, while maintaining the sterility, viability and functionality of the cells.

[0012] More specifically, the device of the utility model includes two or more detachable parts, which can be assembled and disassembled during use to improve the separation of cells, and also enable cells to be stored or used immediately under highly sterile conditions. Whether used for clinical purposes immediately after the separation of cells, or used as mesenchymal stem cells (MSC) after tissue culture amplification, the separated cells do not need to be manipulated or further processed before use. The device can include multiple parts, two of which are (i) cell separation unit and (ii) cell delivery unit. The cells separated in one unit of the device are collected in another unit of the device under gravity, and then the unit is disassembled and can be used as a delivery unit, as further explained herein. The various configurations of the device of the utility model can also include a cell filtration unit, a culture medium reservoir, an analysis sampling module, etc.

[0013] Therefore, in a first aspect of the technology, a device is provided comprising a cell separation chamber (or unit) (referred to herein as CIC), which is detachably associated with a cell delivery unit, the CIC being suitable for receiving tissue to be mechanically manipulated and / or enzymatically manipulated to separate cells or cell populations therefrom, wherein the device is configured to allow cells separated from the tissue in the cell separation chamber to be collected in a detachable cell delivery unit under the action of gravity, wherein the delivery unit is configured for delivering the separated cells in vivo or in vitro.

[0014] The utility model also provides a device, which includes:

[0015] a cell isolation chamber or unit (CIC) configured for separating / isolating cells or cell populations from a tissue containing the cells or cell populations, the CIC being adapted to contain the tissue and a culture medium optionally comprising an enzyme;

[0016] an optional tissue disruption element for mixing or mechanically disrupting the tissue to obtain cells or cell populations (wherein, when the container unit contains an enzyme, the tissue disruption element may or may not be present);

[0017] a delivery unit detachably connected to the CIC via a connector associated with the valve assembly, the delivery unit being used to effect material communication with the CIC to receive a quantity of CIC contents (e.g., separated / isolated cells or cell populations) into the delivery unit, for example, wherein optionally, the CIC contents (separated / isolated cells or cell populations) are in the form of particles;

[0018] The delivery unit has a main body (e.g., a barrel) and a reciprocating unit or member or pump, which is configured to discharge the contents of the delivery unit (i.e., the separated / isolated cells or cell populations or particles thereof).

[0019] A cell separation chamber (CIC) is also provided, which is used to separate / isolate cells or cell groups from tissue containing cells or cell groups, the CIC being suitable for accommodating tissue and a culture medium optionally containing enzymes; wherein the CIC optionally includes a tissue disruption element for mixing or mechanically disrupting the tissue to obtain cells or cell groups (wherein, when the CIC contains enzymes, the tissue disruption element may or may not be present); and wherein the CIC is configured to be detachably associated or connected to a delivery unit via a valve assembly that allows material communication between the CIC and a delivery unit, the delivery unit being configured to receive at least a portion of the contents (e.g., separated / isolated cells or cell groups or culture medium) from the CIC, wherein optionally, the contents (i.e., separated / isolated cells or cell groups) are in solid or granular form; wherein the delivery unit has a main body (e.g., a barrel) and a unit or component for discharging the contents from the delivery unit, such as a reciprocating pump.

[0020] The CIC can have any shape and volume and can be suitable for separating cells from tissue. Since the CIC is configured and operable to process a large amount of adipose tissue, the CIC is usually not a test tube or any other experimental small volume or small size container. Typically, the CIC is conical or has a conical interior or a conical inner base, wherein a detachable delivery unit is directly or indirectly connected or associated to the end of the conical inner end, such as connected or associated to the tip of the conical interior. The CIC is also configured to receive or is suitable for receiving or is equipped with a crushing element, which is suitable for tissue crushing, which maintains cell viability. Such elements can be known in the art, i.e., rotatable blades and / or blade groups rotating in the same or opposite directions, vibrating rotors, grinding units, the grinding units include balls or beads or any other crushing elements, or are composed of balls or beads or any other crushing elements. In the case where the element is operated, for example, the rotor can be connected to an actuator.

[0021] In some embodiments, the CIC may be provided with an element or component configured to induce vortexing, shaking, or strong agitation, wherein the component is not a blade or set of blades.

[0022] In some embodiments, the CIC may be provided with rotating or vibrating elements or components other than blades.

[0023] In some configurations, the CIC is configured to or adapted to receive or hold a mechanical tissue fragmentation unit, which is controlled by an electric actuator outside the CIC and detachably connected to the CIC (optionally through a closure cover of the CIC). The mechanical tissue fragmentation unit can be a rotor or a blade or a member selected to affect the rotational force. The fragmentation unit can be configured to be lowered and retracted to the concave bottom of the CIC or lowered and retracted from the concave bottom, and locked in place during tissue processing. The rotor speed (RPM) can be suitable for maintaining cell viability, maintaining cell integrity and preventing foam and protein denaturation. In some embodiments, the rotor can be selected to rotate at a speed between about 500RPM and about 8000RPM, or between about 1000RPM and 6000RPM, or between about 1100RPM and 1500RPM, or between 1200RPM and 2200RPM.

[0024] In addition to tissue, the CIC is also adapted to receive a liquid or culture medium, which may or may not include enzymes or chemicals that help break down or digest the tissue to release the cells. The liquid or culture medium can be received via one or more inlet members, which are typically located at the top portion of the CIC unit.

[0025] One or more inlet members can be shaped as one or more openings for introducing a tissue sample from which cells are to be separated into the CIC, for example, by suction or by any other means. The inlet members can each be equipped with a tubing or elongated conduit that allows association to a suction unit, a filtration unit, a fat collection unit, or any other fat or tissue processing unit, or directly to an area of ​​the subject's tissue. The suction tube can be used to collect tissue directly from the subject's body or any containing container (e.g., a container for receiving adipose tissue during liposuction). The inlet element can be in the form of an opening, thereby allowing the introduction of a separately obtained tissue sample in the time period before the cells are separated.

[0026] Where one or more inlet members are equipped or adapted with a suction tube, the suction tube may be a cannula having a hollow tube that is blunted at the end and having at least one opening (hole) at the end of the cannula. The cannula may be reusable (to allow it to be used multiple times). The hollow tube of the cannula may be configured to allow a mixture comprising liquid and adipose tissue components suctioned from the subject's body to enter the CIC through an inlet closure cap that may be integrally provided with the cannula or the CIC.

[0027] Alternatively or additionally, one or more or all of the inlet members may be provided with a closure cap adapted to be removed or punctured or penetrated to receive therethrough a tube which may be connected to any auxiliary equipment or container to allow aseptic and efficient delivery of a tissue sample or liquid medium or any other solid or liquid component into the CIC.

[0028] The inlet member as any potential outlet member provided on the body of the CIC may be adapted to the valve member. These valves, other than the valve assembly provided in the connector associating the CIC with the delivery unit, may be configured for association to any shape and form of tubing or material conduit, as further described herein.

[0029] The separated cells may settle to the bottom of the CIC without further processing, or may be forced to collect and settle via the use of a centrifuge or vacuum. Subsequently, the cells may be collected in a delivery unit under gravity, for example with or without the assistance of a vacuum or pressure gradient. The cells or cell particles may be washed in the CIC or after they are transferred to the delivery unit. In order to provide effective washing, the CIC may be provided with a removable or fixed mixing member that is actuated to stir the culture medium contained in the CIC. The mixing member may be a mechanical disruption unit as described herein or any other member that allows mixing of the contents of the CIC. Such a mixing member may operate as an axial flow impeller or a radial flow impeller.

[0030] Once cells or particles or any portion of the contents of a CIC (including, for example, culture medium, fat, unprocessed tissue, fibers, etc.) are collected in a delivery unit, the delivery unit containing the cells or cell particles can be separated or disassembled from the CIC and used to deliver the cells or cell particles based on the intended use, or stored for any period of time under any storage conditions in the delivery unit, which can then be used to deliver the contents (e.g., cells or cell particles or tissue) without resorting to post-processing or manual handling.

[0031] Cells separated in the CIC can be filtered to separate from other components of the tissue or culture medium, or can be enriched according to size before entering the delivery unit or after storage in the delivery unit before use. Filtering or separation between cell populations can be achieved by any filtering or separation or enrichment means suitable for use in the art.

[0032] In some embodiments, cells or particles or tissues are further processed into specific desired cell populations, for example using fluorescence activated cell sorting (FACS), gel gravity separation, column-based techniques, or any other suitable cell enrichment method known to the skilled artisan.

[0033] In some embodiments, the cells or cell particles can be used clinically for: for example, immunomodulation for the treatment or prevention of autoimmune diseases, immune disorders (such as graft-versus-host disease), COVID-19, acute respiratory distress syndrome, etc.; tissue regeneration, such as fat transplantation, hair growth, bone regeneration, nerve regeneration, skin regeneration, wound healing, etc.; treatment or prevention of stroke, perianal fistula, rheumatic diseases, systemic arthritis, osteoarthritis, etc.

[0034] The delivery unit can be shaped and operated as a storage unit or a syringe, so that systemic delivery of cells can be achieved, for example, via intravenous or intra-arterial, intramuscular or intraperitoneal delivery; or local delivery of cells can be achieved by intra-articular delivery to the spine, scalp (hair growth), face / breast with or without fat grafts, scaffolds (bone regeneration, breast degradable scaffolds) or by other means. In other words, the delivery unit can be used as a delivery tool for medical or therapeutic or cosmetic or aesthetic purposes. The delivery unit can be similarly used for research purposes.

[0035] In order to achieve variable use, the delivery unit in the device of the utility model can have a main body shaped as a barrel or an elongated tubular structure, which includes a unit, such as a reciprocating pump, or a plunger and a plunger rod (syringe), which is configured to move linearly in the barrel or elongated tubular structure. The barrel can have at least two openings: one of the openings is positioned along a path connected to the CIC material, and the other opening is located at an end of the barrel or structure opposite to the plunger path, and cells can be discharged from the delivery unit through the other opening. Therefore, the delivery unit can be shaped and operable as a syringe with an opening for being associated with the CIC. The opening can be adapted to a valve assembly or fitting that can be directly attached to and removed from the CIC. The opening can be positioned in the middle of the length of the barrel or at any distance from the opening of the barrel. Typically, the opening enables the container unit to be vertically attached to the delivery unit.

[0036] The delivery unit can be configured as an elongated tubular structure including a reciprocating pump unit or plunger and a plunger rod (syringe), the plunger rod being configured to move linearly within the barrel or elongated tubular structure. The length of the plunger rod can end at the entire length of the lumen of the structure or substantially the entire length, so that the end of the plunger rod contacts the opposite end of the barrel. Alternatively, the plunger rod can extend only a portion of the internal length of the structure.

[0037] In some embodiments, the tubular structure is provided with a membrane or filter which is arranged perpendicular to the main axis (or flow axis, i.e., the direction of material flow) of the structure and is configured to selectively discharge material passing through the membrane or filter from the structure and / or collect larger sized particulate matter or fibers that do not pass through the membrane or filter and are collected on the membrane or filter.

[0038] As described herein, the delivery unit can be connected to the distal end of the CIC or the main body of the device or the tapered container by any connector member or fitting known in the art. In order to safely assemble the CIC and the delivery unit, the CIC and the delivery unit are each provided with a connector member, which can be a male or female member with a threaded end or a non-threaded end; a male or female barbed connector; a coupling adapter member; a dual threaded bushing adapter; and other connector members.

[0039] One or both connector members may be associated with the valve member to form a valve assembly, such that, as defined, once both the CIC and the delivery unit are securely assembled via the connector members respectively provided on each of the CIC and the delivery unit, the valve assembly may be operated to controllably allow liquid communication from the CIC to the delivery unit. The valve assembly may include one or more valve members that are together designed to ensure liquid communication from the CIC to the delivery unit as desired and in a controllably manner.

[0040] In some embodiments, the valve member can be a one way-flap member that allows flow in the direction from the CIC to the delivery unit and blocks flow in the direction of origin.

[0041] In some embodiments, the valve member may be an electric valve operated by a motor or a manually operated mechanical valve member.

[0042] In some embodiments, one or more valve members are manual valves. The manual valve member may be in the form of a lever, a pedal, a manual knob or a wheel to rotate a spirally rotating valve stem.

[0043] In some embodiments, the valve member can be a Luer valve.

[0044] In some embodiments, the valve assembly is or comprises a single valve component that is disposed in a connector component of the CIC unit or in a connector component of the delivery unit.

[0045] In some embodiments, the valve assembly is or includes a double valve having two valve members, one valve member being associated with the CIC unit and the other valve member being associated with the delivery unit.

[0046] In the double valve unit, the two valves define an upper valve body (associated with the CIC) and a lower valve body (associated with the delivery unit), which may have the same structure and may be coaxially formed. The two ends of the upper valve body and the two ends of the lower valve body are integrally connected to their respective units and to the two connector members.

[0047] In some embodiments, each valve member in the dual valve unit includes a body, an inlet end, and an outlet end. The inlet end of the CIC valve is connected to the body of the CIC, and the outlet end defines a connector member that can be associated with a connector end of a delivery unit, which defines the inlet end of the delivery unit. The outlet end of the delivery unit valve is connected to the body of the delivery unit.

[0048] One or each of the valve assemblies or valve members may be adapted to an optical sensor that operates the valve to control the flow of material into the delivery unit. For example, using a controller, the sensor may set the flow of material into the delivery unit to a specific or predetermined volume threshold. Alternatively, the sensor may report the passage of cells or cell particles and prevent further flow of material.

[0049] The device of the utility model can be assembled so that the CIC unit is located above the delivery unit to be able to collect the CIC contents (e.g., cell particles) in the delivery unit under gravity without external intervention. In some embodiments, a portion of the CIC contents can be connected to the delivery unit via the use of a pump or an evacuated delivery unit. The CIC can be equipped with a base or a stage or a leg assembly that holds the CIC at a distance far enough from the surface on which it is positioned to assemble the delivery unit at the lower portion of the CIC. Therefore, in some embodiments, the body of the device or CIC unit is supported on the legs, and the legs can be adjusted to a distance that is convenient for operation.

[0050] The device of the utility model provides continuous manipulation of tissue samples to maximize the yield of cell populations. Although the method for separating the desired cell population can be performed using enzyme manipulation or mechanical manipulation of tissue samples to produce a cell population with a given component and optimal yield, the device can also implement multiple tissue manipulation steps, which include both enzyme manipulation and mechanical manipulation of the same tissue sample. This maximizes the separation of cell populations.

[0051] Therefore, the method for separating a cell population from a tissue using the device of the utility model may include conditions that can enzymatically treat the tissue or conditions that can mechanically treat the tissue; or a continuous mechanical treatment step, followed by an enzymatic treatment, which provides separation of two separate enriched parts, the combined yield of the two separate enriched parts being greater than the expected yield from each tissue manipulation step alone. Similarly, the method may include repeating the steps of cell separation using the same conditions (e.g., mechanical or enzyme), wherein the initially treated tissue is continuously treated to separate more desired cell populations, so that each separated part is delivered to a different delivery unit.

[0052] Therefore, the utility model provides a method for separating a cell population from a tissue, the method comprising:

[0053] In an apparatus comprising a cell isolation chamber (CIC) and a delivery unit detachably associated with the CIC, the tissue is disrupted or digested using a tissue disruption unit or an enzyme solution present in the CIC to obtain a cell suspension; and

[0054] A portion of the suspension or CIC contents is allowed to settle through the valve assembly into a delivery unit, wherein the delivery unit is configured for in vivo or in vitro use as disclosed herein.

[0055] In some embodiments, different fractions of isolated cells obtained from the same tissue contained in the CIC can be obtained by repeating the disruption or digestion step one or more times on the same tissue contained in the CIC to further separate additional fractions of cells. Each fraction can be delivered to a different delivery unit, whereby between each repetition step, the delivery unit contacting the previous fraction is replaced by another delivery unit.

[0056] The utility model also provides a method for separating different parts of a cell population from a tissue, the method comprising:

[0057] In an apparatus comprising a cell isolation chamber (CIC) and a delivery unit detachably associated with the CIC, the tissue is disrupted or digested using a tissue disruption unit or an enzyme solution present in the CIC to obtain a cell suspension; and

[0058] allowing a portion of the suspension or CIC contents to settle through a valve assembly into the delivery device,

[0059] wherein the disrupting or digesting of the tissue contained in the CIC is repeated one or more times to further separate additional portions of cells into different delivery units.

[0060] Also provided is a method for isolating a cell population from a tissue, the method may include:

[0061] In an apparatus comprising a cell isolation chamber (CIC) and a delivery unit detachably associated with the CIC, disrupting or digesting tissue using a tissue disruption unit or an enzyme solution present in the CIC to obtain a cell suspension;

[0062] optionally washing the suspension of cells within the CIC while the container unit is attached to the delivery unit;

[0063] Optionally, transferring the device comprising the assembled CIC / delivery unit or the CIC unit alone to a centrifuge and centrifuging it to separate the cells or cell particles;

[0064] as well as

[0065] The cells or cell particles are allowed to settle into the delivery unit through a valve assembly that allows material communication with the CIC.

[0066] In some embodiments, the tissue is processed, i.e., enzymatically or mechanically processed, while the delivery unit is detached from the CIC; wherein the delivery unit is attached to the CIC and the valve assembly is positioned to provide material flow into the delivery unit before allowing cells or cell particles to settle into the delivery unit.

[0067] In some embodiments, cells or cell particles or otherwise at least a portion of the contents of the CIC are collected in the delivery unit under the action of gravity.

[0068] In some embodiments, a certain amount of CIC contents, which include or consist of cells or cell particles, is allowed to collect in the delivery unit under the action of gravity.

[0069] In some embodiments, the cells or cell particles or CIC contents are collected in liquid culture medium. The volume of culture medium collected in the delivery unit can vary based on the volume and intended use of the delivery unit.

[0070] In some embodiments, cells or cell populations are collected in one delivery unit that is disassembled and replaced with another delivery unit to receive another portion of cells or to receive another portion of the contents of the CIC, for example in the form of liquid culture medium.

[0071] As discussed herein, material flow or material communication from the CIC to the delivery unit is achieved through a valve assembly (e.g., a dual valve) that typically separates the CIC from the delivery unit and prevents uncontrolled or undesirable or untimed flow of material or contents from the CIC into the delivery unit. When material is to be prevented from being transferred to the delivery unit, for example, during the processing of the tissue, or after cells or cell particles have been collected in the delivery unit, the valve member or valve assembly is automatically, electrically, or manually operated to a "closed" configuration. The "closed" configuration can be achieved by closing one or all valve members provided in the connector assembly. When material flow from the CIC to the contents of the delivery unit is allowed, the valve member or valve assembly is automatically, electrically, or manually operated to an "open" configuration. The open configuration requires that all valve members are in an open configuration to allow flow.

[0072] Thus, in some embodiments, any of the methods disclosed herein may include the step of operating a valve member or valve assembly to allow or prevent the flow of material from the CIC to the delivery unit.

[0073] In some embodiments, when the CIC is associated or detached from the delivery unit, at least one valve member is automatically, electrically, or manually operated to a "closed" configuration.

[0074] In some embodiments, the method comprises detaching a delivery unit from the CIC, wherein the delivery unit comprises a cell particle.

[0075] In some embodiments, the method is used to isolate enriched cell fractions for use in the following methods: immunomodulatory methods for treating or preventing autoimmune diseases, immune disorders (e.g., graft-versus-host disease), COVID-19, acute respiratory distress syndrome, etc.; methods for tissue regeneration (e.g., fat transplantation, hair growth, bone regeneration, nerve regeneration, skin regeneration, wound healing, etc.); or methods for treating or preventing stroke, perianal fistula, rheumatic diseases, systemic arthritis, osteoarthritis, etc.

[0076] In some embodiments, the method is used to isolate SVF cells from adipose.

[0077] In some embodiments, the separation step is an enzymatic or non-enzymatic process step.

[0078] In another aspect, a method for isolating a cell population from a tissue is provided, the method comprising:

[0079] As disclosed herein, in an apparatus comprising a CIC unit and a detachable delivery unit, tissue is disrupted using a tissue disruption unit; the CIC being associated with a first delivery unit;

[0080] optionally washing the suspension of cells within the CIC while the CIC is attached to the delivery unit;

[0081] optionally transferring the CIC to a centrifuge and centrifuging while the CIC is attached to the delivery unit to separate cells or cell particles therefrom;

[0082] allowing a first portion of cells or cell particles to settle into the delivery unit attached to the CIC and in communication with the CIC material;

[0083] disconnecting said detachable first delivery unit comprising the cell particles (after closing one or more valve members, as disclosed herein);

[0084] Treating the disrupted tissue in the CIC with an enzyme solution to digest the tissue in the CIC to separate cells or cell particles of the second portion (wherein the cells or particles of the first portion and the cells or particles of the second portion have the same or different components);

[0085] optionally washing the suspension of cells in the CIC;

[0086] A second delivery unit is attached and the second portion of cells or cell particles is allowed to settle into the second delivery unit.

[0087] In some embodiments, the method comprises transferring the CIC or delivery unit to a centrifuge and centrifuging the CIC or unit to isolate the second cell pellet.

[0088] In some embodiments, the second portion of cells or cell particles are washed to remove excess enzyme.

[0089] In some embodiments, the cellular component of the first portion and the cellular component of the second portion are substantially the same.

[0090] In some embodiments, the first particle or moiety and the second particle or moiety are combined.

[0091] The utility model also provides a method for separating a cell population from a tissue, the method comprising:

[0092] As disclosed herein, in a device comprising a CIC unit and a detachable delivery unit, a tissue disruption unit or an enzyme is used to disrupt or digest tissue, respectively, to separate cells or cell particles from the tissue; the CIC is associated with a first delivery unit;

[0093] optionally washing the suspension of cells within the CIC while the CIC is attached to the delivery unit;

[0094] allowing a first portion of cells or cell particles to settle into the delivery unit attached to the CIC and in communication with the CIC material;

[0095] disconnecting said detachable first delivery unit comprising the cell particles (after closing one or more valve members, as disclosed herein);

[0096] using a tissue disruption unit or enzymes to disrupt or digest tissue remaining in the CIC to separate additional amounts of cells or cell particles from the tissue;

[0097] optionally washing the additional amount of cells within the CIC;

[0098] attaching a second delivery unit and allowing the additional amount of cells or cell particles to settle into the second delivery unit; and

[0099] The method is repeated one or more times to separate multiple amounts of cells, wherein each amount is provided in a different delivery unit.

[0100] In some embodiments, any of the methods disclosed herein further comprises separating the fat or any tissue portion from the CIC into a delivery unit.

[0101] In some embodiments, the methods of the present invention allow for collection of isolated cell populations, cell pellets, adipose, tissue fractions, processing medium, or any combination thereof, as appropriate, in one or more delivery units.

[0102] In some embodiments, once separated into one or more delivery units, the delivery units can be centrifuged to separate the cell particles, for example, from the culture medium. In some embodiments, the cell population present in the CIC or in the CIC containing processed tissues and cells is not centrifuged. In some embodiments, the delivery unit is provided with an inner membrane to enable size-based separation during centrifugation.

[0103] In some embodiments of the systems and methods of the present invention, a delivery unit comprising a cell population or cell particles can be stored for a long time and then can be used as disclosed herein. In order to ensure cell viability during storage, the cells in the delivery unit can be frozen at low temperatures to preserve and protect their properties. Cryopreservation can be achieved by treating the cells or samples in the delivery unit with more reagents that allow cryopreservation. Reagents can include one or more cryoprotectants or freezing culture media. Non-limiting examples of cryoprotectants include dimethyl sulfoxide (DMSO), glycerol, alginate, polyvinyl alcohol, etc. After the contents of the delivery unit are thawed, the cryoprotectant may be washed out or may not be washed out.

[0104] Cryopreservation or frozen storage at temperatures below -50°C, sometimes below -80°C or -100°C, requires that the delivery unit of the present invention be formed of a material that can withstand low temperatures. Such materials can be polymeric materials, glass, and various composite materials known in the art. Thus, while the system of the present invention can generally be formed of any material (including polymers, glass, composite materials, etc.), in the case of frozen storage, the delivery unit can be formed of such a stage material.

[0105] The utility model also provides a kit of parts for assembling a device for separating a cell population from a tissue, the kit comprising:

[0106] a container unit (CIC) for separating a cell population from a tissue containing the cell population, as disclosed herein;

[0107] A detachable delivery unit as disclosed herein; and

[0108] Instructions for assembly and / or use.

[0109] The present invention also provides a kit, which includes: a CIC according to the present invention, the CIC being configured to be attached to a delivery unit according to the present invention; and instructions for assembling the CIC to the delivery unit and using the CIC and the delivery unit.

[0110] A kit is also provided, comprising: a delivery unit according to the present invention, the delivery unit being configured for attachment to a CIC unit according to the present invention; and instructions for assembling the CIC to the delivery unit and using the CIC and the delivery unit.

[0111] In some configurations, the apparatus and methods of the present invention are suitable for separating an enriched cell population from adipose tissue obtained from a subject. The tissue can be any cell and / or tissue portion separated from a subject. The enriched portion can be an isolated stromal cell portion (SVF) known in the art.

[0112] The utility model also relates to the following contents:

[0113] A device for separating or separating cells or cell groups from tissue, characterized in that the device comprises a cell separation chamber CIC, the CIC being detachably associated with a cell delivery unit, the CIC being suitable for receiving tissue to be mechanically and / or enzymatically manipulated to separate cells or cell groups therefrom, wherein the device is configured to allow cells separated from the tissue in the CIC to be collected in the delivery unit under the action of gravity, wherein the delivery unit is configured for delivering the separated cells in vivo or in vitro.

[0114] A device for separating or isolating cells or cell groups from tissue, characterized in that the device comprises:

[0115] a cell separation chamber CIC configured for separating or isolating cells or cell populations from a tissue comprising the cells or cell populations, said CIC being suitable for containing said tissue and a culture medium optionally comprising an enzyme;

[0116] an optional tissue disruption element for mixing or mechanically disrupting the tissue to obtain cells or cell populations;

[0117] a delivery unit removably connectable to the CIC via a connector associated with the valve assembly for receiving a quantity of CIC contents into the delivery unit;

[0118] Therein, the delivery unit has a main body and a reciprocating member configured to discharge the contents of the delivery unit.

[0119] In some embodiments, the CIC is provided with an element or component configured to induce vortex, shaking or agitation, wherein the component is not a blade or a set of blades.

[0120] In some embodiments, the CIC is provided with rotating or vibrating elements or components other than blades.

[0121] In some embodiments, the CIC is configured or adapted to receive or hold a mechanical tissue disruption unit controlled by an electric actuator external to the CIC and detachably connected to the CIC.

[0122] In some embodiments, the CIC is provided with one or more inlet members, the one or more inlet members being located at a top portion of the CIC.

[0123] In some embodiments, the one or more inlet members are formed as one or more openings that are equipped with a tube or an elongated conduit that allows connection to a suction unit, a filtration unit, a fat collection unit, or a fat or tissue processing unit, or directly to an area of ​​the subject's tissue.

[0124] In some embodiments, the one or more inlet members are adapted to mate with a valve member.

[0125] In some embodiments, the delivery unit is shaped and operable as a storage unit or a syringe for delivering cells.

[0126] In some embodiments, the delivery unit has a main body shaped as a barrel or an elongated tubular structure, comprising a reciprocating unit or a plunger and a plunger rod configured for linear movement within the barrel.

[0127] In some embodiments, the barrel has at least two openings, one of the openings being located along a path in communication with the CIC material and the other opening being located at an end of the barrel opposite the plunger path.

[0128] In some embodiments, the delivery unit is shaped and operable as a syringe having an opening for associating with the CIC.

[0129] In some embodiments, the opening for association with the CIC is adapted to accommodate a valve assembly or fitting that can be directly attached to and detached from the CIC.

[0130] In some embodiments, the opening for association with the CIC is positioned midway along the length of the barrel or at any distance from the barrel opening.

[0131] In some embodiments, the delivery unit is provided with a membrane or filter arranged perpendicular to the main axis of the tubular structure of the delivery unit.

[0132] In some embodiments, the delivery unit is connected to the CIC via a connector member.

[0133] In some embodiments, the connector member is a male or female member having a threaded end or a non-threaded end; a male or female barbed connector; a coupling adapter member; or a dual threaded bushing adapter.

[0134] In some embodiments, the connector component is associated with a valve component to form a valve assembly, such that once both the CIC and the delivery unit are securely assembled via the connector component respectively disposed on each of the CIC and the delivery unit, the valve assembly is operated to controllably allow liquid communication from the CIC to the delivery unit.

[0135] In some embodiments, the valve assembly includes one or more valve components designed to ensure fluid communication from the CIC to the delivery unit as desired and in a controllable manner.

[0136] In some embodiments, any of the one or more valve members is a one-way flap member that allows flow in a direction from the CIC to the delivery unit and blocks flow in a direction toward the origin.

[0137] In some embodiments, any of the one or more valve members is an electric valve operated by a motor or a manually operated mechanical valve member.

[0138] In some embodiments, the mechanical valve member is in the form of a lever, a pedal, a manual knob, or a wheel to rotate a helically rotating valve stem.

[0139] In some embodiments, the valve assembly is or comprises a single valve component, the single valve component being disposed in the connector component of the CIC or in the connector component of the delivery unit.

[0140] In some embodiments, the valve assembly is or comprises a double valve having two valve members, one valve member being associated with the CIC and the other valve member being associated with the delivery unit.

[0141] In some embodiments, the valve member or at least one valve in the valve assembly is a Luer valve.

[0142] In some embodiments, each valve member in the dual valve unit includes a body, an inlet end and an outlet end, wherein the inlet end of the CIC valve is connected to the body of the CIC and the outlet end defines a connector member that can be associated with the connector end of the delivery unit, and the connector end defines the inlet end of the delivery unit. BRIEF DESCRIPTION OF THE DRAWINGS

[0143] In order to better understand the subject matter disclosed herein and to illustrate how it may be implemented in practice, embodiments will now be described, by way of non-limiting examples only, with reference to the accompanying drawings, in which:

[0144] Figure 1 A schematic diagram of the system of the present invention is depicted.

[0145] Figure 2 A top view of the system of the present invention is shown.

[0146] Figure 3 A side view of a system according to some embodiments of the present invention is given.

[0147] Figure 4 A representation of a delivery unit according to the invention is shown.

[0148] Figure 5A and Figure 5B Exemplary valve and connector components according to some embodiments of the present invention are provided. DETAILED DESCRIPTION

[0149] As disclosed herein, the utility model relates to a device comprising a cell separation chamber (or unit) which is detachably associated with a cell delivery unit, the cell separation chamber being suitable for receiving tissue to be manipulated to separate cells or cell groups therefrom, wherein the device is configured to allow cells separated from the tissue in the cell separation chamber to be collected in a detachable cell delivery unit under the action of gravity.

[0150] Figure 1 A schematic diagram of a system according to the present invention is provided. In the most general conditions, the system 100 includes a cell isolation chamber CIC or unit 110 and a delivery unit (DU) 120, which are detachably connected through a connector 130 associated with a valve assembly for affecting material communication from the CIC 110 to receive a certain amount of CIC contents into the delivery unit 120.

[0151] Figure 2 A top view of the system 100 is shown in FIG. 1 , showing the detachably associated CIC unit 130 and delivery unit 140 , as well as the support legs 150 .

[0152] Figure 3 Another embodiment of a system 200 according to the present invention is depicted. As shown, the system includes a CIC unit 210 for separating / isolating cells or cell groups from tissues containing cells or cell groups. The container unit 210 has a conical end 210A, which is associated with a delivery unit 240 via a connector 230. The CIC and the delivery unit are in material communication for receiving the separated / isolated cells or cell groups or tissues into the delivery unit 240. The detachable delivery unit 240 has a main body (e.g., a barrel) and a reciprocating pump unit 250A, which is used to discharge the separated / isolated cells or cell groups (or their particles) or tissues via an outlet 250B.

[0153] exist Figure 4An exemplary structure of a delivery unit is provided in . As shown, unit 400 has a main body (e.g., a barrel) 410 and a reciprocating pump unit 430A, which is used to discharge the separated / separated cells or cell groups (or their particles) or tissues from the reciprocating pump unit through outlet 430B in the direction indicated by the arrow. The unit can be used as a syringe for systemic delivery of cells such as intravenous or intraarterial, intramuscular or intraperitoneal; or for local delivery of cells into the spine, scalp (hair growth), face / breast with or without fat grafts, scaffolds (bone regeneration, breast degradable scaffolds) and by other means through intra-articular access.

[0154] The main body 410 is shaped as a barrel with at least two openings: one opening located opposite the plunger path through which cells can be expelled (outlet 430B), and another opening 420 located along the barrel and allowing fluid communication with the main unit.

[0155] like Figure 3 As further shown in FIG. 2 , the delivery unit 240 can be connected to the distal end of the conical CIC 210 via a connector member or fitting 230. In order to securely assemble the CIC 210 and the delivery unit 240, each of the CIC 210 and the delivery unit 240 is provided with a connector member 501 or 502, which can be a male member or a female member with a threaded end or a non-threaded end; a male or female barbed connector; a coupling adapter member; a double threaded bushing adapter; and other connector members, such as Figure 5A and Figure 5B . One or two connector components may be associated with valve components 510A and 510B or 520A and 520B, respectively, to form valve assemblies 530 and 540, such that once both the CIC and the delivery unit are securely assembled via connector components 501 or 502, respectively, disposed on each of the CIC and the delivery unit, the valve assembly may be operated to controllably allow liquid communication from the CIC to the delivery unit. The valve assembly 530 or 540 may include one or more valve components 510A and 510B, and 520A and 520B, respectively, which together are designed to ensure liquid communication from the CIC 540 or 550 to the delivery unit 560 or 570, respectively, as desired and in a controllable manner.

[0156] Each of the valve members 510A, 510B, 520A, and 520B may be a one-way flap member that allows flow in a direction from the CIC 540 or 550 to the delivery unit 560 or 570 and blocks flow in a direction toward the origin.

[0157] In some embodiments, the valve members 510A, 510B, 520A, 520B may be electric valves operated by a motor or mechanical valve members operated manually.

[0158] In some embodiments, one or more of the valve members 510A, 510B, 520A, and 520B are manual valves. The manual valve member may be in the form of a lever, a pedal, a manual knob, or a wheel to rotate a spirally rotating valve stem.

[0159] In some embodiments, valve assembly 530 or 540 is or includes a single valve component that is disposed in a connector component of a CIC unit or in a connector component of a delivery unit.

[0160] In some embodiments, valve assembly 530 or 540 is or includes a dual valve having two valve members, one valve member being associated with the CIC unit and the other valve member being associated with the delivery unit.

[0161] In such Figure 5A and Figure 5B In the double valve unit depicted in FIG. 1 , two valves 510A and 510B or 520A and 520B define an upper valve body (associated with CIC 540 or 550) and a lower valve body (associated with delivery unit 560 or 570), which may have the same structure and may be coaxially formed. Both ends of the upper valve body and both ends of the lower valve body are integrally connected to their respective units and two connecting members 501 or 502.

[0162] The apparatus of the present invention can be assembled so that the CIC unit is located above the delivery unit to enable the CIC contents to be collected by gravity without external intervention. Figure 2 The base or stage or leg assembly shown holds the CIC at a distance far enough from the surface on which it is positioned to fit a delivery unit at the lower portion of the CIC. Thus, in some embodiments, the body of the device or CIC unit is supported on legs that can be adjusted to a distance that facilitates operation.

Claims

1. A device for separating or isolating cells or cell groups from tissues, characterized in that: The device comprises a cell separation chamber CIC, which is detachably associated with a cell delivery unit, and the CIC is suitable for receiving a tissue to be mechanically and / or enzymatically manipulated to separate cells or cell groups therefrom, wherein the device is configured to allow cells separated from the tissue in the CIC to be collected in the delivery unit under the action of gravity, wherein the delivery unit is configured for delivering the separated cells in vivo or in vitro.

2. A device for separating or isolating cells or cell groups from tissues, characterized in that: The device comprises: a cell separation chamber CIC configured for separating or isolating cells or cell populations from a tissue comprising the cells or cell populations, said CIC being suitable for containing said tissue and a culture medium optionally comprising an enzyme; an optional tissue disruption element for mixing or mechanically disrupting the tissue to obtain cells or cell populations; a delivery unit removably connectable to the CIC via a connector associated with the valve assembly for receiving a quantity of CIC contents into the delivery unit; Therein, the delivery unit has a main body and a reciprocating member configured to discharge the contents of the delivery unit.

3. The device according to any one of claims 1 to 2, wherein: The CIC is provided with an element or member configured to induce vortex, shaking or stirring, wherein the member is not a blade or a blade set.

4. The device according to any one of claims 1 to 2, wherein: The CIC is provided with rotating or vibrating elements or components other than blades.

5. The device according to any one of claims 1 to 2, wherein: The CIC is configured or adapted to receive or hold a mechanical tissue disruption unit controlled by an electric actuator external to the CIC and detachably connected to the CIC.

6. Apparatus according to any one of the preceding claims, wherein: The CIC is provided with one or more inlet members located at a top portion of the CIC.

7. The device according to claim 6, wherein: The one or more inlet members are formed as one or more openings equipped with a tube or an elongated conduit allowing connection to a suction unit, a filtration unit, a fat collection unit or a fat or tissue processing unit, or directly to a region of the subject's tissue.

8. The device according to claim 7, wherein: The one or more inlet members are adapted to mate with the valve member.

9. Apparatus according to any one of the preceding claims, wherein: The delivery unit is shaped and operated as a storage unit or a syringe for delivering cells.

10. Apparatus according to any one of the preceding claims, wherein: The delivery unit has a main body shaped as a barrel or an elongated tubular structure comprising a reciprocating unit or a plunger and a plunger rod configured for linear movement within the barrel.

11. The device according to claim 10, wherein: The barrel has at least two openings, one of the openings being located along a path in communication with the CIC material and the other opening being located at an end of the barrel opposite the plunger path.

12. The device according to claim 11, wherein The delivery unit is shaped and operable as a syringe having an opening for associating with the CIC.

13. The device according to claim 12, wherein: The opening for association with the CIC is adapted to accommodate a valve assembly or fitting that can be directly attached to and detached from the CIC.

14. The device according to claim 13, wherein: The opening for association with the CIC is located midway along the length of the barrel or at any distance from the barrel opening.

15. Apparatus according to any one of the preceding claims, wherein: The delivery unit is provided with a membrane or filter arranged perpendicularly to the main axis of the tubular structure of the delivery unit.

16. Apparatus according to any one of the preceding claims, wherein: The delivery unit is connected to the CIC via a connector member.

17. The device according to claim 16, wherein: The connector member is a male or female member having a threaded end or a non-threaded end; a male or female barbed connector; a coupling adapter member; or a dual threaded bushing adapter.

18. The apparatus according to claim 16 or 17, wherein: The connector member is associated with a valve member to form a valve assembly, so that once both the CIC and the delivery unit are securely assembled via the connector member respectively disposed on each of the CIC and the delivery unit, the valve assembly is operated to controllably allow liquid communication from the CIC to the delivery unit.

19. The apparatus according to claim 18, wherein: The valve assembly includes one or more valve components designed to ensure fluid communication from the CIC to the delivery unit as desired and in a controlled manner.

20. The apparatus of claim 19, wherein: Any of the one or more valve members is a one-way flap member that allows flow in a direction from the CIC to the delivery unit and blocks flow in a direction toward the origin.

21. The apparatus of claim 19, wherein: Any of the one or more valve members is an electric valve operated by a motor or a manually operated mechanical valve member.

22. The apparatus according to claim 21, wherein The mechanical valve member is in the form of a lever, a pedal, a manual knob or a wheel to rotate a spirally rotating valve stem.

23. The apparatus of claim 19, wherein: The valve assembly is or comprises a single valve member, which is arranged in the connector member of the CIC or in the connector member of the delivery unit.

24. The apparatus of claim 19, wherein: The valve assembly is or comprises a double valve having two valve members, one valve member being associated with the CIC and the other valve member being associated with the delivery unit.

25. The apparatus of claim 18 or 19, wherein: The valve member or at least one valve in the valve assembly is a Luer valve.

26. The apparatus of claim 19, wherein: Each valve member in the double valve unit comprises a body, an inlet end and an outlet end, wherein the inlet end of the CIC valve is connected to the body of the CIC and the outlet end defines a connector member that can be associated to a connector end of the delivery unit, the connector end defining the inlet end of the delivery unit.

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