High flow filter and method of use thereof

Abstract

A filter is disclosed. The filter includes: a flexible container defining an inlet port toward an inlet end of the container and an outlet port toward an outlet end of the container, the container having one or more walls connecting the inlet end and the outlet end; and a first filter unit and a second filter unit spaced apart within the container, where: the first filter unit is coupled to the container to define an inlet chamber in fluid communication with the inlet port; a second filter unit coupled to the container to define an outlet chamber in fluid communication with the outlet port; the first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter fluid flowing from the intermediate chamber to the outlet chamber; wherein the inlet chamber comprises a first slot relative to the first peak; wherein the intermediate chamber comprises a second slot relative to the second peak; and wherein the outlet port and the second filter unit are configured to be spaced apart when fluid flows through the second filter unit into the outlet chamber.

Description

[0001] Priority Application

[0002] This application claims priority to U.S. Provisional Application No. 63 / 365,393 filed on May 26, 2022 and U.S. Provisional Application No. 63 / 482,768 filed on February 1, 2023. Technical Field

[0003] The present disclosure relates to devices for preparing cell compositions having reduced particles and / or cell aggregates and methods of using the same.Some embodiments of the present disclosure relate to a filtration device configured to maintain a relatively high flow rate of a filtered cell composition exiting the filtration device. Background Art

[0004] Several cell therapy products for regenerative or immunotherapy applications have entered clinical evaluation and market authorization. The manufacturing process for such products typically involves culturing cells in the presence of non-autologous serum and harvesting the cells by trypsinization.

[0005] At several stages during the manufacturing process, cells are exposed to external materials. At these stages, there is a risk that cells may be contaminated by one or more particles, such as cotton fibers, cellulose, salt crystals, rubber, plastics, glass, etc. If particles are incorporated into the final product, such particles are potentially harmful to cells and / or recipients of the resulting cell therapy. Before the final composition provided for treatment, it is generally necessary to filter to remove particles. However, in view of one or more factors, such as the complex nature of cell culture medium and the trend of cell aggregation, especially in the context of large-scale cell culture, it is not simple to filter cell compositions. Obviously, there is an unmet demand in the art for preparing cell compositions, especially in the context of therapeutic cell compositions.

[0006] Any discussion of documents, acts, materials, devices, articles of manufacture or the like included in this specification is not to be taken as an admission that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present disclosure as they existed before the priority date of each of the appended claims.

[0007] Throughout this specification, the word "comprise" or variations such as "comprising" or "including" will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps. Summary of the invention

[0008] Some embodiments relate to a filter comprising:

[0009] a flexible container defining an inlet port toward an inlet end of the container and an outlet port toward an outlet end of the container, the container having one or more walls connecting the inlet end and the outlet end; and

[0010] a first filter unit and a second filter unit, the first filter unit and the second filter unit being spaced apart within the container, wherein:

[0011] the first filter unit being coupled to the container to define an inlet chamber in fluid communication with the inlet port;

[0012] the second filter unit is coupled to the container to define an outlet chamber in fluid communication with the outlet port; and

[0013] The first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter the fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter the fluid flowing from the intermediate chamber to the outlet chamber;

[0014] wherein the inlet chamber comprises a first trough relative to the first peak;

[0015] wherein the intermediate chamber comprises a second trough relative to the second peak; and

[0016] Wherein the outlet port and the second filter unit are configured to be spaced apart when the fluid flows through the second filter unit into the outlet chamber.

[0017] Some embodiments relate to a filter comprising:

[0018] a container defining an inlet port toward an inlet end of the container and an outlet port toward an outlet end of the container, the container having a flexible front wall and a flexible rear wall connecting the inlet end and the outlet end; and

[0019] a first filter unit and a second filter unit, the first filter unit and the second filter unit being spaced apart within the container, wherein:

[0020] the first filter unit being coupled to the front wall and the rear wall of the container to define an inlet chamber in fluid communication with the inlet port;

[0021] the second filter unit is coupled to the front wall and the rear wall of the container to define an outlet chamber in fluid communication with the outlet port; and

[0022] The first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter fluid flowing from the intermediate chamber to the outlet chamber;

[0023] wherein the first filter unit is coupled to the front wall along a first front seam, and the second filter unit is coupled to the front wall along a second front seam;

[0024] wherein the first filter unit is coupled to the rear wall at a first rear seam and the second filter unit is coupled to the rear wall at a second rear seam;

[0025] wherein a distance between the first front seam and the second front seam is less than a distance between the first back seam and the second back seam;

[0026] Wherein the outlet port and the second filter unit are configured to be spaced apart when the fluid flows through the second filter unit into the outlet chamber.

[0027] The container may be a flexible bag. The wall may be flexible. The distance between the first front seam and the second front seam may be 1 inch or less. The second rear seam may angle the second filter unit and the outlet port away from each other. The front wall may be configured to protrude away from the second filter unit.

[0028] The outlet port and the second filter unit may be configured to be separated by a spacer system. The spacer system may be configured to separate the outlet port and the second filter unit and at least one of: (i) the front wall and the first filter unit; (ii) the rear wall and the first filter unit; and (iii) the rear wall and the second filter unit.

[0029] The spacer system may include a separator. The spacer system may include a clip. The spacer system may include a magnet system. The spacer system may include a bracket.

[0030] The first filter unit may be connected to the front wall of the container at an acute angle to define an inlet chamber slot. The first filter unit may be connected to the rear wall of the container at an obtuse angle. The first filter unit may include a mesh defining pores, the pores having an average pore size of 130 μm to 170 μm. The first filter unit pores may have an average pore size of 150 μm. The first filter unit may be substantially flat.

[0031] The second filter unit may be connected to the rear wall of the container at an acute angle to define the intermediate chamber slot. The second filter unit may be connected to the front wall of the container at an acute angle. The second filter unit may include a mesh defining pores, the pores having an average pore size of 20 μm to 60 μm. The second filter unit pores may have an average pore size of 40 μm. The second filter unit may be substantially flat.

[0032] The volume of the inlet chamber may be about 0.9 to 3.2 times the surface area of ​​the first filter unit. The intermediate chamber may have: (i) a volume of about 1.9 to 4.0 times the surface area of ​​the first filter unit; or (ii) a volume of about 2.7 to 5.6 times the surface area of ​​the second filter unit. The volume of the outlet chamber may be about 1.6 to 4.5 times the surface area of ​​the second filter unit. The volume ratio of the inlet chamber to the intermediate chamber to the outlet chamber may be about (i) 1:1:1; or (ii) 1:2:1; or (iii) 1:2:2; or (iv) 2:2:1; or (v) 1:3:2.

[0033] At least one of the chambers may have a substantially triangular or substantially trapezoidal cross-section when viewed parallel to the direction of fluid flow from the inlet port to the outlet port.

[0034] Some embodiments relate to a filter comprising:

[0035] a flexible bag defining an inlet port toward an inlet end of the bag and an outlet port toward an outlet end of the bag, the bag having opposing front and rear walls connecting the inlet and outlet ends;

[0036] a first filter unit and a second filter unit, the first filter unit and the second filter unit being spaced apart within the bag, wherein:

[0037] the first filter unit being coupled to the bag to define an inlet chamber in fluid communication with the inlet port;

[0038] the second filter unit coupled to the bag to define an outlet chamber in fluid communication with the outlet port;

[0039] The first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter fluid flowing from the intermediate chamber to the outlet chamber; and

[0040] Wherein the outlet port and the second filter unit are configured to be spaced apart when the fluid flows through the second filter unit into the outlet chamber.

[0041] The first filter unit and the second filter unit may not be parallel.

[0042] Some embodiments relate to a kit for filtering cells, the kit comprising:

[0043] Filters as described above;

[0044] an inlet conduit, the inlet conduit comprising:

[0045] a first inlet tube and a second inlet tube, the first inlet tube and the second inlet tube being fluidly connected to a manifold;

[0046] a fill tube in fluid communication with the manifold and adapted to receive fluid from at least one of the first inlet tube and the second inlet tube; and

[0047] an inlet coupling adapted to connect the fill tube to the inlet port of the filter and to allow fluid communication with the inlet chamber;

[0048] An outlet conduit, the outlet conduit comprising:

[0049] Drains; and

[0050] An outlet coupling is adapted to connect the drain to the outlet port of the filter and to allow fluid communication with the outlet chamber and the drain.

[0051] The inlet tube and the fill tube may form a Y-shape or a T-shape. The inlet tube may have an inner diameter of 1 / 8 inch and an outlet diameter of 1 / 4 inch. The fill tube may have an inner diameter of 1 / 8 inch and an outlet diameter of 1 / 4 inch. The drain tube may have an inner diameter of 1 / 8 inch and an outlet diameter of 1 / 4 inch. The kit may also include an outlet valve or a clamp configured to control fluid flow through the drain tube.

[0052] One or more or all of the container, bag, filter unit 120, 130 or tube may be made of DMSO (dimethyl sulfoxide) compatible plastic, preferably made of one or more plastics including polyethylene terephthalate (PET), high-density polyethylene (HDPE), low-density polyethylene (LDPE), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), thermoplastic elastomer (TPE) and polypropylene (PP).

[0053] Some embodiments relate to a filter comprising:

[0054] a container defining an inlet port toward an inlet end of the container and an outlet port toward an outlet end of the container, the container having one or more walls connecting the inlet end and the outlet end; and

[0055] a first filter unit and a second filter unit, the first filter unit and the second filter unit being spaced apart within the container, wherein:

[0056] the first filter unit being coupled to the container to define an inlet chamber in fluid communication with the inlet port;

[0057] the second filter unit is coupled to the container to define an outlet chamber in fluid communication with the outlet port; and

[0058] The first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter the fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter the fluid flowing from the intermediate chamber to the outlet chamber;

[0059] wherein the inlet chamber comprises a first trough relative to the first peak; and

[0060] The intermediate chamber includes a second groove opposite to the second peak.

[0061] Some embodiments relate to a filter comprising:

[0062] a container defining an inlet port toward an inlet end of the container and an outlet port toward an outlet end of the container, the container having opposing front and rear walls connecting the inlet and outlet ends; and

[0063] a first filter unit and a second filter unit, the first filter unit and the second filter unit being spaced apart within the container, wherein:

[0064] the first filter unit being coupled to the front wall and the rear wall of the container to define an inlet chamber in fluid communication with the inlet port;

[0065] the second filter unit is coupled to the front wall and the rear wall of the container to define an outlet chamber in fluid communication with the outlet port; and

[0066] The first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter fluid flowing from the intermediate chamber to the outlet chamber;

[0067] wherein the first filter unit is coupled to the front wall along a first front seam, and the second filter unit is coupled to the front wall along a second front seam;

[0068] wherein the first filter unit is coupled to the rear wall at a first rear seam and the second filter unit is coupled to the rear wall at a second rear seam; and

[0069] Wherein a distance between the first front seam and the second front seam is smaller than a distance between the first rear seam and the second rear seam.

[0070] Some embodiments relate to a filter comprising:

[0071] a flexible bag defining an inlet port toward an inlet end of the bag and an outlet port toward an outlet end of the bag, the bag having opposing front and rear walls connecting the inlet and outlet ends;

[0072] a first filter unit and a second filter unit, the first filter unit and the second filter unit being spaced apart within the bag, wherein:

[0073] the first filter unit being coupled to the bag to define an inlet chamber in fluid communication with the inlet port;

[0074] the second filter unit is coupled to the bag to define an outlet chamber in fluid communication with the outlet port; and

[0075] The first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter fluid flowing from the intermediate chamber to the outlet chamber.

[0076] A filter or kit as described above can be used to filter stem cell culture medium.

[0077] Some embodiments relate to a method of filtering a stem cell culture medium, the method comprising using a filter or kit as described above.

[0078] Some embodiments relate to a method of purifying a cell composition comprising passing cultured cells through a double mesh filter to reduce visible particles and / or cell aggregates, wherein the double mesh filter comprises a first filter having an average pore size of 130 μm to 170 μm and a second filter having an average pore size of 20 μm to 60 μm.

[0079] The cultured cells may be provided in a serum-free cell culture medium. The cells may be culture expanded. The cells may be mesenchymal lineage precursor or stem cells (MLPSC).

[0080] After passing the cells through the double mesh filter, the recovery of viable cell concentration is (i) 60% to 100%; or (ii) 70% to 90%.

[0081] A purified cell composition may exhibit a D90 of less than 150 μm, preferably less than 100 μm, more preferably less than 50 μm. A purified cell composition may be substantially free of visible particles.

[0082] Some embodiments relate to a filter comprising:

[0083] a flexible container defining an inlet port toward an inlet end of the container and an outlet port toward an outlet end of the container, the container having one or more walls connecting the inlet end and the outlet end;

[0084] a first filter unit, the first filter unit comprising a first filter screen; and

[0085] a second filter unit, the second filter unit comprising a second filter screen;

[0086] wherein the first filter unit and the second filter unit are spaced apart within the container;

[0087] wherein the first filter unit is coupled to the container to define an inlet chamber in fluid communication with the inlet port;

[0088] wherein the second filter unit is coupled to the container to define an outlet chamber in fluid communication with the outlet port; and

[0089] wherein the first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter a fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter a fluid flowing from the intermediate chamber to the outlet chamber;

[0090] wherein the inlet chamber comprises a first trough relative to the first peak;

[0091] wherein the intermediate chamber comprises a second trough relative to the second peak; and

[0092] Wherein at least one of the first filter screen and the second filter screen is configured to be spaced apart from the one or more walls when the fluid flows through the filter.

[0093] Some embodiments relate to a filter comprising:

[0094] a container defining an inlet port toward an inlet end of the container and an outlet port toward an outlet end of the container, the container having a flexible front wall and a flexible rear wall connecting the inlet end and the outlet end;

[0095] a first filter unit, the first filter unit comprising a first filter screen; and

[0096] a second filter unit, the second filter unit comprising a second filter screen;

[0097] wherein the first filter unit and the second filter unit are spaced apart within the container, wherein:

[0098] the first filter unit being coupled to the front wall and the rear wall of the container to define an inlet chamber in fluid communication with the inlet port;

[0099] the second filter unit is coupled to the front wall and the rear wall of the container to define an outlet chamber in fluid communication with the outlet port; and

[0100] The first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter fluid flowing from the intermediate chamber to the outlet chamber;

[0101] wherein the first filter unit is coupled to the front wall along a first front seam, and the second filter unit is coupled to the front wall along a second front seam;

[0102] wherein the first filter unit is coupled to the rear wall at a first rear seam and the second filter unit is coupled to the rear wall at a second rear seam;

[0103] wherein a distance between the first front seam and the second front seam is less than a distance between the first back seam and the second back seam;

[0104] wherein the outlet port and the second filter unit are configured to be spaced apart when the fluid flows through the second filter unit into the outlet chamber; and

[0105] At least one of the first filter screen and the second filter screen is configured to be spaced apart from the front wall or the rear wall when the fluid flows through the filter. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 is a perspective view of a filtration device according to some embodiments;

[0107] FIG. 2A to FIG. 2D is in the filled configuration Figure 1 A side view of a filtering device;

[0108] FIG. 3A to FIG. 3B Observed along line 3-3 Figure 1 A cross-sectional view of a filtering device;

[0109] Figure 4 According to some embodiments, Figure 1 A perspective view of a kit of a filter device;

[0110] Figure 5 According to some embodiments, the Figure 1 an inlet conduit of a kit of filter devices; and

[0111] Figure 6 According to some embodiments, the Figure 1 The outlet conduit of the filter device kit. DETAILED DESCRIPTION

[0112] The present disclosure relates to devices for preparing cellular compositions having reduced particles and / or cell aggregates and methods of using the same.

[0113] In particular, some embodiments of the present disclosure relate to a filter device, which is suitable for filtering fluid materials (such as cell compositions or cell culture media or resuspension culture media including the fluid materials). In an example, the cell composition includes a mesenchymal precursor lineage or a stem cell. Some embodiments of the present disclosure relate to a filter device, which is configured to maintain a relatively high flow rate of the filtered cell composition leaving the filter device. The device may be referred to as a "double mesh filter". The double mesh filter includes a first filter unit and a second filter unit. In some embodiments, the filter unit includes a screen or sheet made of a mesh material (therefore referred to as a "double mesh filter"). Compared with other filters, the double mesh filter is not easy to clog when filtering viscous fluids. Other embodiments of the filter may include a folded filter or a depth filter.

[0114] In some embodiments, the filter devices can be sufficiently flexible so that they can be deformed to fit in tight or irregularly shaped locations. In certain applications, the use of flexible filter devices may be particularly advantageous, particularly relative to more rigid structures, because the flexible filter device can be easily positioned around more rigid components of an overall cell culture and / or cell purification system that includes rigid structures (such as brackets, supports, pumps, etc.). A filter device having such flexibility may include a flexible portion. In some embodiments, the filter device is a flexible bag similar to a saline bag.

[0115] The filter unit is arranged so that during operation, the fluid passing through the device flows through both the first filter unit and the second filter unit before leaving the device. The filter unit may include a net with a mean pore size, which may be selected according to the size of the particle to be filtered from the fluid material. For example, the filter unit including a net with a mean pore size of 150 μm will theoretically prevent particles greater than 150 μm from flowing through the filter unit. When the fluid flows through a series of filters, the pore size of the continuous filter may be reduced to provide gradual filtration. This may help reduce the clogging of the filter. For example, the first filter unit may have a mean pore size of 150 μm, and the second filter unit may have a mean pore size of 40 μm.

[0116] The inventors have recognized that in some cases, one or both of the mesh filters may sag or wrinkle. As fluid flows through the mesh filter under gravity, the mesh filter may sag or wrinkle. In some cases, the fluid may enter the filter device faster than it passes through each of the filter units therein, causing the fluid to accumulate on the inlet side of the filter unit.

[0117] The mesh filter may not sag or wrinkle immediately, but only after a certain amount of fluid has accumulated, causing the mesh filter to sag under the weight of the accumulated fluid. Sagging or wrinkling of the mesh filter may cause a portion of the mesh filter to approach or contact a wall or interior surface of the filter device, thereby obstructing (by restricting or blocking) the flow of fluid through that portion of the mesh filter. Obstructing fluid flow in this manner is more likely to occur in flexible or bag-like filter devices, because the reduced rigidity (or lack of rigidity) in such filter devices means that the filter and the wall can easily move closer to each other.

[0118] The inventors have developed embodiments of devices and techniques for reducing or preventing relative movement of filters and walls of filtration devices.

[0119] The inventors have developed embodiments of devices and techniques for reducing or preventing relative movement of a filter and a wall of a filtration device while maintaining flexibility of the filtration device.

[0120] Advantageously, by providing the filtering device of the embodiment, the filtering device (such as a flexible bag) can be hindered or prevented from collapsing under gravity. In some embodiments, the filtering device of the embodiment can hinder or prevent the filter from leaning against the wall of the filtering device (such as a flexible bag), thereby alleviating the restriction to the flow of fluid.

[0121] The disclosed embodiments can result in a more stable fluid flow through the filter device. Compared to other filter devices that do not use the disclosed embodiments, the disclosed embodiments can result in a fluid flow through the filter device having a stable higher flow rate. FIG. 2A to FIG. 2D as well as Figure 3A and Figure 3B The disclosed embodiments are discussed in greater detail.

[0122] Filtration device

[0123] Figure 1 The embodiment of the filter device 100 for filtering a fluid such as a cell composition is shown. The filter device 100 may alternatively be referred to as a filter. The filter 100 includes a container 102 and a first filter unit 120 (partially shown in dotted lines) and a second filter unit 130 (partially shown in dotted lines) disposed in the container 102. The container 102 is suitable for receiving a fluid, and the first filter unit 120 and the second filter unit 130 are configured to filter the fluid passing through the container 102. The filter units 120, 130 may each include a filter screen having holes, and the size of the holes is designed to allow a material of a specific size / type to flow through the filter units 120, 130. The filter screen in the first filter unit 120 may have holes of the same or different sizes as the filter screen in the second filter unit 130.

[0124] The pores are defined by a pore-defining structure (such as the body of the net). The net body and the container 102 can be made of a material that is compatible (e.g., tolerant) with dimethyl sulfoxide (DMSO). Examples of DMSO-compatible materials include DMSO-compatible polymers. In some embodiments, the filter units 120, 130 comply with USP 788, which is a particulate matter test for the count and size of subvisible particles in quantified parenteral drugs. The USP 788 test involves using a light-blocking particle counter and counting particles on the filter through a microscope.

[0125] In some embodiments, the average pore size of the first filter unit 120 is 130 μm to 170 μm or 140 μm to 160 μm. In some embodiments, the first filter unit 120 has an average pore size of about 150 μm.

[0126] In some embodiments, the average pore size of the second filter unit 130 is 20 μm to 60 μm or 30 μm to 50 μm. In some embodiments, the second filter unit 130 has an average pore size of about 40 μm.

[0127] In some embodiments, the container 102 is a hollow body. The body may have a regular shape, such as being substantially square (cube) or rectangular (cuboid), such as a carton. The container 102 may be rigid enough to support its own weight, or may be flexible enough to allow it to be positioned in a tight or irregularly shaped position. The container 102 may include a combination of a rigid portion and a flexible portion. In some embodiments, the container 102 is a flexible bag similar to a saline bag.

[0128] The container 102 has an inlet end 104 and an outlet end 106. The container 102 defines an inlet port 108 toward the inlet end 104 and defines an outlet port 110 toward the outlet end 106. In some embodiments, the inlet port 108 and the outlet port 110 are arranged generally relative to each other so that when the filter device 100 is in use, the fluid can flow from the inlet port 108 toward the outlet port 110 by gravity. In some embodiments, the container 102 includes a flange 112. The flange 112 can be arranged at the inlet end 104. The flange 112 can define an orifice 114 or a connector through which the container 102 can be suspended on a hook (not shown). The orifice 114 allows the container 102 to be suspended or supported in an upright position so that when the filter device 100 is in use, the fluid can flow from the inlet port 108 toward the outlet port 110 by gravity.

[0129] FIG. 2A to FIG. 2D 1 is a side view of the filter device 100. The container 102 includes one or more walls 200 connecting the inlet end 104 and the outlet end 106. The one or more walls 200 may include a front wall 202 and a rear wall 204. The one or more walls 200 may also include a side wall, a side portion or a side end (not shown) that connects the front wall 202 and the rear wall 204 to each other to define the body of the container 102. In some embodiments, the front wall 202 and the rear wall 204 are relative (arranged relatively). Some embodiments of the container 102 may also include a top connecting portion 210 and a bottom connecting portion 212. The top connecting portion 210 and the bottom connecting portion 212 may be a seam connecting the front wall 202 and the rear wall 204. The seam 210 and the flange 112 may be connected to each other; for example, the flange 112 may extend from the seam 210. In some embodiments, the inlet end 104, the outlet end 106 and the side end include at least one seam or a sterile welded / bonded edge connecting the one or more walls 200.

[0130] The first filter unit 120 and the second filter unit 130 are spaced apart within the container 102 and connected to the wall 200 of the container 102. By spacing the first filter unit 120 and the second filter unit 130 apart, more fluid can pass through the first filter unit 120 before passing through the second filter unit 130. The spaced apart filter units 120, 130 can reduce the likelihood of filter clogging, such as caused by the first filter unit 120 and the second filter unit 130 contacting in a manner where the pores of one filter unit are blocked by the pore-defining structure of the other filter unit. If the fluid being filtered is viscous or particularly viscous, the filter units 120, 130 may also stick together.

[0131] When the filter device 100 is suspended to allow fluid to flow through the filter device 100 by gravity, the first filter unit 120 and the second filter unit 130 can be relatively taut so that they do not generally sag toward either of the walls 200. The taut configurations of the first filter unit 120 and the second filter unit 130 are labeled 120-1 and 130-1, respectively.

[0132] As discussed above, when fluid flows through the first filter unit 120 toward the outlet port 110, the weight of the fluid on the first filter unit 120 can cause at least a portion of the first filter unit 120 to sag and move away from the inlet end 104. This drooping configuration of the first filter unit 120 is labeled 120-2. In the drooping configuration 120-2, at least a portion of the first filter unit 120 can approach or contact the wall 200 of the container 102, thereby hindering or blocking the flow of fluid through the portion of the first filter unit 120. For example, in the drooping configuration 120-2, at least a portion of the first filter unit 120 can contact the back wall 204. This can reduce the surface area of ​​the first filter unit 120 that is available for filtering the fluid.

[0133] Similarly, when the fluid flows through the second filter unit 130 toward the outlet port 110, the weight of the fluid on the second filter unit 130 may cause at least a portion of the second filter unit 130 to sag and move away from the inlet end 104. This drooping configuration of the second filter unit 130 is labeled 130-2. In the drooping configuration 130-2, at least a portion of the second filter unit 130 may approach or contact the wall 200 of the container 102, thereby hindering or blocking the flow of fluid through the portion of the second filter unit 130. For example, in the drooping configuration 130-2, at least a portion of the second filter unit 130 may contact the front wall 202. In some cases, at least a portion of the second filter unit 130 may contact the outlet port 110. This may reduce the surface area of ​​the second filter unit 130 that is available for filtering the fluid.

[0134] The above-described reduction in the filtering surface area of ​​the first filter unit 120 and the second filter unit 130 may significantly affect the rate at which the filtered fluid passes through the outlet port 110 .

[0135] The inventors have recognized that in some embodiments, the rate of filtered fluid passing through outlet port 110 may be more affected by the movement of second filter unit 130 toward wall 202 than by the movement of first filter unit 120 toward wall 204. The inventors believe that this may be because the average pore size of second filter unit 130 is smaller than the average pore size of first filter unit 120.

[0136] The inventors have recognized that where the filtration device 100 is or resembles a saline bag, the geometry of the bag may affect the flow of fluid through the second filter unit 130. This is particularly true in e.g. Figure 2A , which shows a container 102 that is narrower at the ends 104, 106 than at the mid-portion between the ends. Thus, when the second filter 130 is in the drooped configuration 130-2, the second filter unit 130 has a smaller travel distance to contact the wall 202 of the container 102. In contrast, due to Figure 2A The container 102 in FIG. 1 is wider in its middle portion, so when the first filter unit 120 is in its pendent configuration 120 - 2 , it has a greater distance to travel to contact the wall 204 of the container 102 .

[0137] In some embodiments, the outlet port 110 and the second filter unit 130 are configured to be spaced apart from each other. The outlet port 110 and the second filter unit 130 can be configured to be spaced apart from each other when the fluid flows through the second filter unit into the outlet chamber. In embodiments where the container 102 is flexible (such as a saline bag), the distance between the outlet port 110 and the second filter unit 130 can vary, such that in some configurations, the outlet port 110 and the second filter unit 130 are in contact, while in other configurations, the outlet port 110 and the second filter unit 130 are spaced apart.

[0138] refer to FIG. 2A to FIG. 2D , the filtration device 100 may include a spacer system 220. The spacer system 220 is configured to separate the outlet port 110 and the second filter unit 130. The spacer system 220 can enable the container 102 to move between the collapsed state and the expanded state, so that the outlet port 110 and the second filter unit 130 can contact and move apart.

[0139] By spacing the second filter unit 130 from the front wall 202 , the spacer system 220 enables the pores of the second filter unit 130 to remain unobstructed or relatively open and allow fluid to continue to pass through the second filter unit 130 and into the outlet chamber 320 .

[0140] The spacer system 220 can stabilize (or minimize) the flow rate of the fluid through the second filter unit 130 regardless of whether the second filter unit 130 is in the taut configuration 130 - 1 or the drooped configuration 130 - 2 .

[0141] Figure 2A An embodiment of a spacer system 220 is shown in the form of a separator 222. The separator 222 includes a separator body 224. The separator 222 is configured to be connected to the outlet port 110 inside the container 102. The separator 222 and the outlet port 110 can thereby clamp the front wall 202.

[0142] When the second filter unit 130 is in the pendulous configuration 130-2, the separator 222 reduces movement of the second filter unit 130 toward the outlet port 110. The separator 222 can limit the surface area of ​​the second filter unit 130 that can contact the front wall 202. Figure 2A Includes items marked as plug-in Figure 1 and Figure 2, which respectively show embodiments of the separator 222. Figure 1 is a side view showing how separator 222 according to some embodiments may limit the surface area of ​​second filter unit 130 that can contact front wall 202. Inset 2 is an end view (from the filter side) of separator 222 according to some embodiments.

[0143] The separator body 224 may be disc-shaped, with a thickness that sets the distance that the second filter unit 130 is separated from the front wall 202. The separator 222 may be rigid. The separator 222 may be made of a similar material as the outlet port 110. The separator body 224 defines a plurality of apertures through which fluid may flow to the outlet port 110.

[0144] The separator 222 may also include at least one ridge or fence 226 extending from the separator body 224 to effectively increase the thickness of the separator 222 and increase the separation of the second filter unit 130 from the front wall 202. In some embodiments, the separator 222 includes at least two of the ridges or fences 226. The fences 226 and the separator body 224 may define apertures or channels 228 between adjacent fences 226. The channels 228 are configured to allow fluid to flow through the channels, such as Figure 2A Indicated by arrow FF on the Figure 1 2). The fences 226 can be configured to direct fluid flowing through the channels 228 toward the outlet port 110. In some embodiments, some of the fences 226 can define apertures or passages to allow fluid to flow between adjacent channels 228 toward the outlet port 110.

[0145] The fence 226 may be configured to allow the outlet port 110 to be unobstructed by the second filter unit 130. When the second filter unit 130 is in the drooped configuration 130-2, the second filter unit 130 is configured to abut the fence 226. The fence 226 is configured to support the second filter unit 130, spacing the second filter unit 130 and the separator body 224. Adjacent fences 226 may be spaced apart from each other so that the second filter unit 130 does not substantially droop between the fences 226 to block the channel 228. Then, the outlet port 110 may not be blocked by the second filter unit 130. Then, the fluid may flow through the channel 228, as shown in FIG. Figure 2A Indicated by arrow FF on the Figure 1 and illustration 2).

[0146] In some embodiments, the fence 226 is configured to direct fluid flowing through the channels 228 toward the outlet port 110. For example, the fence 226 can extend radially from the outlet port 110, which can be located in a central region of the separator body 224. In this configuration, all of the channels 228 can allow direct fluid flow toward the outlet port 110.

[0147] Figure 2B An embodiment of the spacer system 220 is shown in the form of a clip 230. The clip 230 is configured to be connected to the container 102 to hold the outlet port 110 and the second filter 130 apart. In some embodiments, the clip 230 clamps the front wall 202 and the rear wall 204. When the second filter 130 is connected to the front wall 202 and the rear wall 204, holding the walls 202, 204 apart applies tension to the second filter 130 and separates it from the outlet port 110.

[0148] Figure 2C An embodiment of the spacer system 220 in the form of a magnet system 240 is shown. The magnet system 240 includes a first magnetic portion 242 coupled to the front wall 202 and a second magnetic portion 244 coupled to the rear wall 204. The magnetic portions 242, 244 are configured to be connected to the respective walls 202, 204. For example, this can be achieved by an adhesive or stored in a pouch formed by bonding / welding a layer of material to the walls 202, 204. The magnetic portions 242, 244 are separated from the fluid in the container 102 by the walls 202, 204 so that the fluid in the container 102 does not contact the magnetic portions 242, 244 (e.g., to avoid contaminating the cell composition). The magnetic portions 242, 244 are preferably located near the outlet port 110.

[0149] In some embodiments, each of the magnetic portions 242, 244 is a magnet whose respective polarities are arranged such that the magnets 242, 244 repel each other to separate the front wall 202 and the rear wall 204. When the filter device 100 is in use, the magnets 242, 244 can be magnetically attracted to adjacent metal structures 246, 248. The adjacent metal structures 246, 248 can be part of an exoskeleton (not shown) configured to support the filter device 100. When the magnets 242, 244 engage the adjacent metal structures 246, 248, the magnet system 240 keeps the front and rear walls 202, 204 separated.

[0150] Figure 2D An embodiment of the spacer system 220 in the form of a bracket 250 is shown. The bracket 250 is configured to be applied to the second filter unit 130 to support the net. The bracket 250 may include a plurality of rigid members arranged to reduce sagging of the net of the second filter unit 130. The inventors have recognized that certain arrangements of members may hinder the flow of fluid through the second filter unit 130. The rigid members of the bracket 250 may be primarily arranged at or toward the periphery of the second filter unit 130. The rigid members of the bracket 250 may be thin wires.

[0151] In some embodiments, the support 250 includes a raised portion so that the second filter unit 130 is formed into an at least partially raised shape, such as shown by the dashed line 250-1. The raised shape of the second filter unit 130 causes the second filter unit 130 to be arched away from the outlet port 110. This means that when fluid accumulates on the second filter unit 130, the weight of the fluid must first straighten the second filter unit 130 and is less likely to subsequently cause the second filter unit 130 to sag (sag).

[0152] Embodiments of the spacer system 220 can be combined to operate in combination. For example, the separator 222 can be used in combination with the magnet system 240. The spacer system 220 can be used to space the wall 200 and either of the filter units 120, 130, particularly in embodiments where the container 102 is flexible. The spacer system 220 can be configured to space the front wall 202 and the first filter unit 120. The spacer system 220 can be configured to space the front wall 202 and the second filter unit 130. The spacer system 220 can be configured to space the rear wall 204 and the first filter unit 120. The spacer system 220 can be configured to space the rear wall 204 and the second filter unit 130.

[0153] The aforementioned embodiments of the spacer system 220 are FIG. 2A to FIG. 2D2 is described and shown as being applied only to the second filter unit 130. However, the spacer system 220 can be similarly applied to the first filter unit 120. For example, when the first filter unit 120 is in the drooped configuration 120-2, the separator 222 can be placed on the rear wall 204 to separate it from the first filter unit 120. In some embodiments, instead of or in addition to applying the spacer system 220 to the second filter unit 130, the spacer system 220 is applied to the first filter unit 120.

[0154] like FIG. 3A to FIG. 3B As best shown, the first filter unit 120 and the second filter unit 130 are connected to the container 102 and divide the interior of the container 102 into a plurality of different zones or chambers 300. The chambers 300 are configured to be in fluid communication with each other through the first filter unit 120 and the second filter unit 130. The container 102 may include an inlet chamber 310 and an outlet chamber 320, with an intermediate chamber 330 disposed between the inlet chamber 310 and the outlet chamber 320. The spaced-apart arrangement of the first filter unit 120 and the second filter unit 130 defines the intermediate chamber 330 between the inlet chamber 310 and the outlet chamber 320. The first filter unit 120 may be configured to filter a fluid flowing from the inlet chamber 310 to the intermediate chamber 330, and the second filter unit 130 may be configured to filter a fluid flowing from the intermediate chamber 330 to the outlet chamber 320.

[0155] In some embodiments, the first filter unit 120 is coupled to the container 102 to define an inlet chamber 310. The inlet chamber 310 can span from the inlet end 104 to the first filter unit 120, which is connected to the container 102; for example, to at least the front wall 202 and the rear wall 204. The first filter unit 120 can be coupled to the container 102 along a first front seam 340 (such as at the front wall 202), and can be coupled to the container 102 (such as at the rear wall 204) at a first rear seam 350. The first front seam 340 and the first rear seam 350 can extend to meet each other at the side ends 206, 208, such as in embodiments in which the container 102 is similar in configuration to a saline bag.

[0156] The inlet chamber 310 is configured to be in fluid communication with the inlet port 108, so that the fluid flowing into the inlet port 108 is collected in the inlet chamber 310 and passes through the first filter unit 120. The first filter unit 120 is configured to filter a first substance (e.g., particles) contained in the fluid. For example, the first substance may be a particle having a size larger than the pore size of the first filter unit 120. The first filter unit 120 may be configured to filter the first substance from the fluid flowing from the inlet chamber 310 to the intermediate chamber 330. The first substance is retained on the surface of the first filter unit 120, while the rest of the fluid passes through.

[0157] If the first substance is allowed to accumulate on the surface of the first filter unit 120, more and more pores of the first filter unit 120 will be blocked, thereby reducing the filtration efficiency. Therefore, in some embodiments, the inlet chamber 310 may include a first slot 342. In some embodiments, the first slot 342 is a recessed portion of the inlet chamber 310. The first slot 342 is arranged relative to the first peak 352. In some embodiments, the first peak 352 is a protruding, optionally pointed portion of the inlet chamber 310. In some embodiments, the first peak 352 is defined by the first filter unit 120. The first slot 342 can also be defined as the first peak 352 relative to the adjacent intermediate chamber 330 (which is located on the other side of the first filter unit 120). In use, the first slot 342 is the lowest portion of the inlet chamber 310, and the first peak 352 is a portion of the adjacent intermediate chamber 330 that is arranged higher than the first slot 342 (i.e., closer to the inlet end 104). In some embodiments, the inlet chamber 310 can be substantially wedge-shaped. For example, the inlet chamber 310 may taper from at or near the inlet end 104 toward the first slot 342 .

[0158] The first filter unit 120 can be connected to at least one of the container walls 200 at an angle to define a first slot 342 and to another of the container walls 200 at an angle to define a first peak 352. For example, the first slot 342 can be defined by the first filter unit 120 and the front wall 202. The angle defining the first slot 342 (as measured in the inlet chamber 310) can be an acute angle. The first slot 342 can be referred to as an inlet chamber slot. The first peak 352 can be defined by the first filter unit 120 and the rear wall 204. The angle defining the first peak 352 (as measured in the intermediate chamber 330) can be an obtuse angle. When such an embodiment is in use, the first filter unit 120 will be tilted within the container 102.

[0159] In use, the first filter unit 120 can be configured to direct the first substance into the first slot 342. The tilt of the first filter unit 120 means that particles that do not pass through the filter unit 120 can move along the filter screen toward the slot 342 of the inlet chamber 310 and enter the slot. For example, when the slot 342 is located at the lowest point of the inlet chamber 310, gravity may cause these particles to settle in the slot 342 instead of settling on the filter surface. In some embodiments, the fluid in the inlet chamber 310 can wash the first substance along the surface of the first filter unit 120 into the first slot 342.

[0160] Directing the first substance to accumulate in the first slot 342 can keep a larger proportion of the filter unit surface unobstructed / unblocked, thereby improving filtration efficiency and / or reducing the amount of time until the filter unit 120 (or filter device 100) needs to be cleaned or replaced. In contrast, if a horizontally arranged filter unit is used, these "filtered out" particles (first substances) will settle and accumulate on the pores of the horizontally arranged filter unit and block the flow of fluid therethrough. Therefore, by directing the "filtered out" particles into the slot 342, the inclined filter unit 120 reduces the risk of pore blockage and allows a larger amount of fluid to pass through the filter unit 120.

[0161] The fluid is configured to pass through the first filter unit 120 and enter the next chamber, which in some embodiments is the intermediate chamber 330. The top of the intermediate chamber 330 is defined by the first filter unit 120. In some embodiments, the second filter unit 130 is coupled to the container 102 (and is spaced apart from the first filter unit 120) to define the bottom of the intermediate chamber 330. In some embodiments, when the second filter unit 130 is coupled to the container 102, one side of the second filter unit 130 defines the bottom of the intermediate chamber 330, and the opposite side of the second filter unit 130 defines the outlet chamber 320.

[0162] In some embodiments, the intermediate chamber 330 includes a second slot 372. In some embodiments, the second slot 372 is a recessed portion of the intermediate chamber 330. The second slot 372 is disposed relative to the second peak 362. In some embodiments, the second peak 362 is a protruding, optionally pointed portion of the intermediate chamber 330. In some embodiments, the second peak 362 is defined by the second filter unit 130. The second slot 372 may also be defined relative to the second peak 362 of the adjacent outlet chamber 320 (which is located on the other side of the second filter unit 130). In use, the second slot 372 is the lowest portion of the intermediate chamber 330 (i.e., closer to the outlet end 106), while the second peak 362 is a portion of the adjacent outlet chamber 320 disposed higher than the second slot 372 (i.e., closer to the inlet end 104).

[0163] The second filter unit 130 can be connected to at least one of the container walls 200 at an angle to define a second groove 372 and connected to another of the container walls 200 at an angle to define a second peak 362. For example, the second groove 372 can be defined by the second filter unit 130 and the rear wall 204. The angle defining the second groove 372 (as measured in the middle chamber 330) can be an acute angle. The second groove 372 can be referred to as a middle chamber groove. The second peak 362 can be defined by the second filter unit 130 and the front wall 202. The angle defining the second peak 362 (as measured in the middle chamber 330) can be an acute angle. When such an embodiment is in use, the second filter unit 130 will be tilted within the container 102.

[0164] The second filter unit 130 can be configured to filter a second substance from the fluid flowing from the intermediate chamber 330 to the outlet chamber 320. The second substance can be particles of a different size or type than the first substance. The second filter unit 130 can cooperate with the first filter unit 120 to gradually filter the substance from the fluid passing through the container 102. The gradual filtering of the substance can increase the yield of the desired fluid product and can reduce the possibility that the filter units 120, 130 become blocked due to trying to remove too many particles at the same time.

[0165] In some embodiments, the second substance can be particles of the same size or type as the first substance, wherein the second filter unit 130 is configured to capture or trap any particles that are not intended to pass through the first filter unit 120. Similar to the first filter unit 120 and the first slot 342, the captured particles accumulated on the second filter unit 130 can be directed to the second slot 372. The second slot 372 is configured to receive the second substance. For example, the fluid in the intermediate chamber 330 can flush the second substance along the surface of the second filter unit 130 into the second slot 372. Directing the second substance to accumulate in the second slot 372 can keep a larger proportion of the filter unit surface unobstructed / unblocked, thereby improving filtration efficiency and / or reducing the amount of time until the filter unit 130 (or filter device 100) needs to be cleaned or replaced.

[0166] The fluid that passes through the second filter unit 130 enters the outlet chamber 320. The outlet chamber 320 is in fluid communication with the outlet port 110, which allows the fluid in the outlet chamber 320 to exit the filter container 102. The fluid in the outlet chamber 320 contains the yield; that is, the desired fluid product is substantially separated by removing particles by the filter units 120, 130. In some embodiments, some trace amounts of particles may have managed to pass through the filter units 120, 130, but the use of the filter device 100 allows the amount of particles in the filtered fluid to be significantly reduced compared to the unfiltered fluid. Further filtration can be used to reduce the amount of these residual particles.

[0167] The outlet chamber 320 may span from the outlet end 106 to the second filter unit 130, which is coupled to the container 102; for example, to at least the front wall 202 and the rear wall 204. The second filter unit 130 may be coupled to the container 102 along a second front seam 360 (such as at the front wall 202), and may be coupled to the container 102 at a second rear seam 370 (such as at the rear wall 204). The second front seam 360 and the second rear seam 370 may extend to meet each other at the side ends 206, 208, such as in an embodiment in which the container 102 is similar in configuration to a saline bag. In some embodiments, the outlet chamber 320 may be substantially wedge-shaped. For example, the outlet chamber 320 may taper from at or near the outlet end 106 to the second peak 362.

[0168] The geometry of the container 102 can be changed to space the second filter unit 130 and the front wall 202 / outlet port 110 apart, as similarly achieved by the spacer system 220. The spacer system 220 can generally be classified as a structural device that involves mechanically separating the second filter unit 130 and the front wall 202. In contrast, the geometry of the container 102 does not rely on a structural device. However, the spacer system 220 can be used in conjunction with the changed geometry of the container 102.

[0169] Figure 3A and Figure 3B Embodiments of containers 102 having varying geometries are shown.

[0170] The position of the second rear seam 370 may be adjusted to further space the second filter unit 130 from the outlet port 110 . Figure 3A Two example positions of the second rear seam 370 are shown. The first position 370-1 is closer to the outlet end 106 than the second position 370-2. Therefore, at the first position 370-1, the second filter unit 130 is closer to the outlet port 110 than the second position 370-2. When the second rear seam 370 is in the second position 370-2, the indicated position of the second filter unit 130 is shown in dashed lines. The second position 370-2 can be selected so that when in use, the outlet port 110 and the second filter unit 130 are angled further away from each other than in the first position 370-1.

[0171] Figure 3B An embodiment of the container 102 is shown wherein the front wall 202 is configured to protrude away from the second filter unit 130 . Figure 3B The front wall 202 is shown in a convex configuration 202-2. For comparison, the front wall 202 is shown in phantom as being in its "regular" configuration 202-1 (such as Figure 3A). In the protruding configuration 202-2, the front wall 202 may protrude between the second front seam 360 and the second rear seam 370. The front wall 202 may protrude between the second front seam 360 and the bottom seam 212. For example, the amount of material between the second front seam 360 and the second rear seam 370 may be greater than Figure 3A The amount of material in the embodiment shown ("regular" configuration 202-1). This allows the front wall 202 of the container 102 to bulge in a bulging configuration 202-2.

[0172] The outlet port 110 is coupled to the front wall 202. The outlet port 110 has a mass sufficient to deform the front wall 202. For example, when the filter device 100 is arranged so that the fluid passes through the second filter unit 130 under gravity, the gravity pulls the outlet port 110 downward, which in turn pulls the front wall 202 downward and causes it to assume the convex configuration 202-2. The outlet port 110 can be positioned closer to the lower end 106 to depress / pull the front wall 202 in a desired direction. When the fluid passes through the second filter unit 130 and enters the outlet chamber 320, the weight of the fluid may cause the front wall 202 to assume the convex configuration 202-2 downward.

[0173] In the convex configuration 202-2, the front wall 202 (and / or the outlet port 110) moves away from the second filter unit 130. Even when fluid accumulated on the second filter unit 130 causes the second filter unit 130 to sag, the front wall 202 (and / or the outlet port 110) remains spaced apart from the second filter unit 130, thereby leaving the pores of the second filter unit 130 unobstructed or substantially unobstructed and allowing the fluid to continue to pass through the second filter unit 130 and enter the outlet chamber 320.

[0174] Additional outlet ports 110 may be added to provide more locations for the discharge of fluid in outlet chamber 320. In some embodiments, outlet port 110 may be positioned generally perpendicular to the sag of filter unit 130 such that in the sag configuration, filter unit 130 leaves outlet port 110 substantially unobstructed.

[0175] When the first filter unit 120 and the second filter unit 130 are connected to the container 102 in a spaced-apart arrangement, there is a front seam distance between the first front seam 340 and the second front seam 360, and there is a rear seam distance between the first rear seam 350 and the second rear seam 370. The front seam distance and the rear seam distance may be equal or unequal. In some embodiments, the front seam distance and / or the rear seam distance are in the range of 0.5 inches to 5 inches. In some embodiments, the front seam distance and / or the rear seam distance are approximately 1 inch.

[0176] In some embodiments, the filter units 120, 130 are substantially planar (flat), so that when the front seam distance and the rear seam distance are equal, the filter units 120, 130 are parallel. This can result in the intermediate chamber 330 having a square or parallelogram cross-section.

[0177] In some embodiments, the filter units 120, 130 are substantially planar (flat), so that the filter units 120, 130 are not parallel to each other (such as FIG. 2A to FIG. 2D as well as Figure 3A and Figure 3B ). When the filter units 120, 130 are not parallel to each other, this can reduce the likelihood that the filter units 120, 130 will contact each other and become blocked along a large portion of their surface area. For example, if the filter is a flexible bag that is bent into a curved shape, the filter units 120, 130 can be made to contact only at specific points (despite the spacing or non-parallel configuration) rather than across a large portion of their surface area.

[0178] The filter units 120, 130 may be angled or tilted relative to each other in a generally "V" configuration, such as FIG. 2A to FIG. 2D as well as Figure 3A and Figure 3B Filter units 120, 130 may be angled or tilted relative to each other such that the cross-section of intermediate chamber 330 may be substantially similar to a triangle, a trapezoid, or an irregular trapezoid. In some embodiments, the minimum front seam distance is less than the minimum rear seam distance.

[0179] The tilting of the filter units 120, 130 means that the first filter unit 120 and the second filter unit 130 are angled relative to each other, i.e. not parallel (such as FIG. 2A to FIG. 2D as well as Figure 3A and Figure 3B102 is a flexible embodiment of the invention. The invention relates to a filter unit 120, 130 having a plurality of filter surfaces disposed adjacent to each other. The filter unit 120, 130 is preferably a plurality of filter units ...

[0180] In some embodiments, the filter units 120, 130 are conical (or otherwise have a triangular cross-section) with the front seam distance and the rear seam distance being equal. The conical or triangular filter may rise toward the peak portion toward the middle of the inlet chamber 310 and fall toward the container wall 200 to define a first trough 342. Alternatively, the conical filter may define a first trough 342 toward the middle of the inlet chamber 310 and rise toward the container wall 200 to define a first peak 352. Similar configurations may apply relative to the intermediate chamber 330 and the outlet chamber 320 when defining a second peak 362 and a second trough 372.

[0181] To reduce the likelihood of the filter units 120, 130 contacting each other and clogging, the filter units 120, 130 may not be nested; for example, the first filter unit 120 does not extend into or toward the second filter unit 130, or vice versa.

[0182] Systems and Kits

[0183] Figure 4 A system 400 for filtering cells is shown. The system can be provided as a kit, wherein the system 400 is at least partially disassembled. The kit includes Figure 1 To the filter device 100 of FIG. 3, the inlet conduit 500 ( Figure 5 ) and outlet conduit 600 ( Figure 6 ).

[0184] The inlet conduit 500 includes a fill tube 510 adapted to be coupled to the inlet port 108 of the filter device 100 so as to allow fluid communication with the inlet chamber 310. In use, the fill tube 510 can receive fluid from a fluid source 520 and direct the fluid into the inlet chamber 310. The fill tube 510 can be connected to the inlet port 108 via an inlet coupling 530. The fill tube 510 can be in fluid communication with an inlet valve, which can be used to allow, prevent, and / or control the flow of fluid into the inlet chamber 310. In some embodiments, the inlet coupling 530 includes an inlet valve.

[0185] The outlet conduit 600 includes a drain 610 adapted to be coupled to the outlet port 110 of the filtration device 100 so as to allow fluid communication with the outlet chamber 320. In use, the drain 610 can receive the output (filtered fluid), which can be a fluid with the first substance and the second substance removed, and direct the output elsewhere, such as to one or more vials 620. The drain 610 can be connected to the outlet port 110 via an outlet coupling 630. The drain 610 can be in fluid communication with an outlet valve, which can be used to allow, prevent and / or control the flow of filtered fluid from the outlet chamber 320. In some embodiments, the outlet coupling 630 includes an outlet valve.

[0186] Some uses of system 400 may involve receiving fluid from multiple sources. In such scenarios, fluid may be the same fluid; For example, in order to ensure continuous supply of fluid by filtering device 100, both of two fluid sources 520 may be connected to system 400, so that when first fluid source 520A is exhausted (for example, tank), second fluid source 520B may supply fluid with minimum / zero interruption. In another scenario, system 400 may receive two (or more) different types of fluids. This may allow two fluids to be mixed immediately when filtering or before filtering. For example, when preparing a cell composition for filtering, first fluid source 520A may contain cells in formulation buffer (for example, 2X formulation buffer), and second fluid source 520B may contain cryoprotectants for protecting filtered cells from damage during cryopreservation. In one example, cryoprotectants may be DMSO, which shows some cytotoxicity. Therefore, cryoprotectants may be added immediately before filtering to minimize cell exposure to DMSO, and allow more time to fill filtered cell fluid into vials for freezing.

[0187] Therefore, in some embodiments, the inlet conduit 500 may also include a first inlet pipe 540 and a second inlet pipe 550. The first inlet pipe 540 is adapted to receive a first fluid from the first fluid source 520A, and the second inlet pipe 550 is adapted to receive a second fluid from the second fluid source 520B, wherein the first fluid and the second fluid are different from each other. In some embodiments, the first inlet pipe 540 may be in fluid communication with a first supply valve, and the second inlet pipe 550 may be in fluid communication with a second supply valve. The first supply valve and the second supply valve may be used to allow, prevent and / or control the flow of fluids from the fluid sources 520A, 520B.

[0188] The inlet tubes 540, 550 are configured to be in fluid communication with the fill tube 510. In some embodiments, the inlet conduit 500 includes a manifold 560. The fill tube 510 is configured to be in fluid communication with the manifold 560 and is adapted to receive a fluid from at least one of the first inlet tube 540 and the second inlet tube 550. The manifold 560 may be a Y-shaped or T-shaped connector through which the first fluid and the second fluid may flow from the inlet tubes 540, 550 to the fill tube 510. Thus, the inlet tubes 540, 550 and the fill tube 510 may form a Y-shape or a T-shape.

[0189] The various tubes 510, 540, 550, 610 may have an inner diameter in the range of 1 / 8 inch to 3 / 4 inch. The various tubes 510, 540, 550, 610 may have an outer diameter in the range of 1 / 4 inch to 1 inch. For example, in some embodiments, at least one of the tubes 510, 540, 550, 610 has an inner diameter of 1 / 8 inch and an outer diameter of 1 / 4 inch. However, other sized tubes may be used depending on the flow properties of the fluid (e.g., viscosity) and / or the desired throughput (flow rate, yield quality) of the system 400.

[0190] The kit may have one or more components (filtration device 100, inlet conduit 500, and outlet conduit 600) made of DMSO compatible plastic. In some embodiments, all components of the kit are made of DMSO compatible plastic. For example, the DMSO compatible plastic can be a plastic comprising one or more of polyethylene terephthalate (PET), high density polyethylene (HDPE), low density polyethylene (LDPE), polytetrafluoroethylene (PTFE), polypropylene (PP), polyvinyl chloride (PVC), and thermoplastic elastomer (TPE).

[0191] The kit may have one or more components that may include aseptic welding parts to connect the filter units 120, 130 to the container 102. The system 400, kit, and / or filter device 100 may comply with USP788.

[0192] The kit may also include a pump that creates pressure in the system 400 to draw fluid through the filter device 100. The pump may be connected to the outlet conduit 600 and used to assist in priming the system 400 (removing air bubbles). When fluid begins to flow into the container 102, the pump may be operated to suck air out of the container 102, thereby creating a pressure differential that forces fluid to flow through the filter units 120, 130 and the chamber 300. Removing air may be preferred to introducing air (via the inlet conduit 500) because introducing air into the system 400 may cause air bubbles to appear in the filtered fluid and reduce the amount / volume of filtered fluid produced.

[0193] operate

[0194] In some embodiments, the fluid can flow through the filtration device 100 by gravity without mechanical assistance. In use, the container 102 is placed in an upright position with the inlet port 108 oriented higher than the outlet port 110. In some embodiments, the container 102 includes a flange 112 at the inlet end 104. The flange 112 can define an orifice 114 or connector through which the container 102 can be hung on a hook to maintain an upright position.

[0195] In the upright position, the first filter unit 120 forms the inclined base of the inlet chamber 310 (the inclined cover of the intermediate chamber 330), while the second filter unit 130 forms the inclined base of the intermediate chamber 330 (the inclined cover of the outlet chamber 320). In the upright position, fluid flow through the filter units 120, 130 is assisted by gravity.

[0196] Prior to use, the system 400 should be "primed". Priming involves removing air bubbles in the system 400, such as in the inlet conduit 500 and the outlet conduit 600. To remove the air bubbles, the inlet conduit 500 and the outlet conduit 600 may be mechanically manipulated by hand. The container 102 may also be selectively squeezed to push air out of the container 102, thereby forcing the air bubbles along the inlet conduit 500 and the outlet conduit 600. During operation, the filtration device 100 may require intermittent priming.

[0197] In some embodiments, the filter device 100 can be connected to a pump that creates pressure in the system 400 to draw fluid through the filter device 100. The pump can be particularly helpful in scenarios where the filter device 100 is used in a configuration where gravity is insufficient or adversely affects the flow of fluid from the inlet port 108 to the outlet port 110. For example, the pump can assist in the flow of fluid when the inlet port 108 and the outlet port 110 are at / near the same level. The pump can be connected to the outlet conduit 600. The pump can assist in priming.

[0198] When the fluid flows into the inlet chamber 310, it contacts the inclined / sloped base of the inlet chamber 310 (the first filter unit 120). The first filter unit 120 blocks the first substance from flowing through, and the first substance may be washed into the first groove 342. The fluid passing through the first filter unit 120 may be referred to as a first filtered fluid.

[0199] The first filtered fluid passes through the first filter unit 120 into the intermediate chamber 330. The first filtered fluid contacts the inclined base of the intermediate chamber 330 (the second filter unit 130) and is filtered by the second filter unit 130. In some embodiments, the second filter unit 130 is inclined at a different angle relative to the first filter unit 120. The second substance is prevented from flowing through the second filter unit 130, and the second substance may be washed into the second groove 372. The fluid passing through the second filter unit 130 may be referred to as the second filtered fluid.

[0200] The second filtered fluid passes through the second filter unit 130 into the outlet chamber 320. The second filtered fluid is collected in the outlet chamber 320 and discharged through the outlet port 110.

[0201] The filtration device 100 and kits containing the same can be used to filter various fluids, such as stem cell culture media, cell compositions, and harvested cells suspended in cell culture media or another suitable buffer.

[0202] Some embodiments of the present disclosure relate to a method of filtering a stem cell culture medium. The method includes using a filtration device 100. The method may also include using a kit, wherein the stem cell culture medium is fed through an inlet conduit 500, passes through the filtration device 100, and flows out through an outlet conduit 600. The outlet conduit 600 can deliver the filtered stem cell culture medium to be distributed to at least one vial 620.

[0203] Some embodiments of the present disclosure relate to a method for filtering harvested cells in a suspension. The harvested cells may be present with enzymes for separation or formulation reagents for filling. The method includes using a filtration device 100. The method may also include using a kit. An outlet conduit 600 may deliver the filtered harvested cells to be distributed to at least one vial 620.

[0204] Some embodiments of the present disclosure relate to a method for purifying a cell composition. The method includes passing cultured cells through a double mesh filter (such as a filter device 100) to reduce visible particles and / or cell aggregates. The kit can be used to perform the method. The outlet conduit 600 can deliver the purified cell composition to be distributed to at least one vial 620.

[0205] The average pore size of the first filter unit 120 of the filter device 100 may be 130 μm to 170 μm, such as 150 μm. The average pore size of the second filter unit 130 of the filter device 100 may be 20 μm to 60 μm, such as 40 μm. The purified cell composition may be substantially free of visible particles. The inspection process may be performed by a trained qualified operator by the naked eye, such as in accordance with USP 790 and USP 1790. A light box with a black and white background may be used to perform the inspection. This inspection does not require magnification or polarized light.

[0206] The cultured cells can be provided in a serum-free cell culture medium. In some embodiments, the cultured cells are provided in a cell culture medium comprising serum such as fetal bovine serum (FBS). As known to those familiar with cell culture, cell culture medium includes various components that support cell growth, which are largely dependent on the cells in culture. Exemplary cell culture medium and the components in the cell culture medium are discussed below.

[0207] In some embodiments of the method, the cells are culture expanded.

[0208] In some embodiments of the method, the cell is a mesenchymal lineage precursor or stem cell (MLPSC).

[0209] After passing the cells through the filtration device 100, the recovery of the viable cell concentration can be between 60% and 100%. For example, a 100% recovery of viable cells means that the yield / filtered fluid contains all of the cells contained in the fluid introduced into the inlet chamber 310. A 60% recovery would indicate that, of the cells contained in the fluid introduced into the inlet chamber 310, 40% of those cells were retained in the filtration device 100; e.g., captured in the first filter unit 120 with the first substance or captured in the second filter unit 130 with the second substance. In some embodiments, the recovery is between 70% and 90%.

[0210] In some embodiments of the method, the purified cell composition exhibits a D90 of less than 150 μm, preferably less than 100 μm, more preferably less than 50 μm.

[0211] Chamber volume and its relationship to filter element surface area

[0212] When determining the size of the chamber 300 and the filter units 120, 130, the relationship between the chamber volume and the filter unit surface area should be considered. If the surface area of ​​the filter unit is too small relative to the volume of the chamber feeding the filter unit, the filter unit is likely to act as a bottleneck or clogging point. This may slow down the overall filtration process, thereby affecting the yield. Bottlenecks may also cause the filter unit to become clogged.

[0213] In some embodiments, the filter unit is tilted within the filter container 102. By tilting the filter unit relative to the wall 200 of the container 102, a filter unit having a larger surface area can fit within the container 102 compared to a configuration where the filter unit is not tilted.

[0214] If the filter unit is not tilted within the container 102, the size of the container 102 may have to be increased to accommodate the filter unit. Increasing the size of the container 102 may affect the chamber volume, which, as described above, must be proportional to the filter surface area in order to reduce the likelihood that the filter unit will become a bottleneck or clogging point. Increasing the size of the container 102 may be difficult if the container 102 is used in a small or irregularly shaped space.

[0215] The relative volumes of the inlet chamber 310, the intermediate chamber 330 and the outlet chamber 320 should also be considered to promote fluid flow. If the volume of the subsequent chamber (e.g., the intermediate chamber 330) is greater than the volume of the previous chamber (e.g., the inlet chamber 310 that feeds the intermediate chamber 330), it is generally not detrimental to fluid flow. However, if the subsequent chamber volume is too small compared to the previous chamber volume, the speed at which the fluid enters the subsequent chamber volume may be faster than the speed at which it leaves. This may cause bottlenecks or choking points. The filter unit pore size and the fluid flow rate via the inlet port 108 and the outlet port 110 also affect the speed at which the fluid moves through the various chambers 300 in the filter, thus affecting the size of the chamber volume.

[0216] In some embodiments, the inlet chamber 310, the intermediate chamber 330, and the outlet chamber 320 all have approximately equal volumes. Thus, the chamber volumes may be expressed in a 1:1:1 ratio.

[0217] In some embodiments, the volumes of two of the inlet chamber 310, the intermediate chamber 330, and the outlet chamber 320 are approximately equal. The volume of the other chamber may be less than the volume of at least one of the other chambers. For example, the volume of the other chamber may be 30% to 70% of the volume of one of the other chambers.

[0218] In some embodiments, the size of the intermediate chamber 330 is twice the size of the inlet chamber 310 and the outlet chamber 320. Therefore, the chamber volumes can be expressed in a ratio of 1:2:1 (inlet:intermediate:outlet).

[0219] In some embodiments, the volumes of the intermediate chamber 330 and the outlet chamber 320 are approximately equal. The inlet chamber 310 may have a smaller volume. The chamber volumes may be expressed in a ratio of 1:2:2 (inlet:intermediate:outlet).

[0220] In some embodiments, the volumes of the inlet chamber 310 and the intermediate chamber 330 are approximately equal. The outlet chamber 320 may have a smaller volume. The chamber volumes may be expressed in a ratio of 2:2:1 (inlet:intermediate:outlet).

[0221] In some embodiments, the volumes of the inlet chamber 310, the intermediate chamber 330, and the outlet chamber 320 are different from each other. For example, the volume of the intermediate chamber 330 may be three times that of the inlet chamber 310, and the volume of the outlet chamber 320 may be three times that of the inlet chamber 310. Therefore, the chamber volumes may be expressed in a ratio of 1:3:2.

[0222] In some embodiments, the volume of the middle chamber 330 is the largest of all the chambers. By making the middle chamber 330 the chamber with the largest volume, the fluid flowing through the first filter unit 120 can accumulate in the middle chamber 330 before passing through the second filter unit 130, thereby promoting smooth fluid flow through the filtration device 100. In some embodiments, the container 102 has a total volume of 1.5 liters, and this can allow approximately 2 liters of fluid to pass through while minimizing scaling or clogging of the filter units 120, 130. In some embodiments, the container 102 has a total volume of 2.0 liters.

[0223] In embodiments where outlet chamber 320 is smaller than intermediate chamber 330, the rate of fluid flow discharged via outlet port 110 can be controlled so that excess fluid does not accumulate in outlet chamber 320. This reduces the risk of excess fluid in outlet chamber 320 detrimental to fluid flow from intermediate chamber 330.

[0224] method

[0225] In one embodiment, the present disclosure relates to a method for filtering a cell culture medium including cells, the method comprising passing the cells through a filter as described herein. For example, the cells may pass through a mesh filter as discussed above. When using the method of the present disclosure, the cell type is not particularly limited, and in some instances, the method is associated with both differentiated and undifferentiated cells. In one example, the cell is a mesenchymal lineage or precursor cell (MLPSC). In this example, the method of the present disclosure can be used to filter a stem cell culture medium. For example, MLPSC can be cultured according to the method discussed below, and then filtered by passing the cells through a filter as described herein. In one example, cells are provided in a stem cell culture medium. In one example, before passing the resulting composition including the cells through a filter as described herein, the stem cell culture medium is purified or partially purified. For example, the stem cell culture medium including the cells may be centrifuged to remove the culture medium from the cells. Then, the cells may be resuspended in a suitable buffer or resuspension medium. In this example, multiple rounds of centrifugation and resuspension may be performed to the cells to rinse the cells. Then, the centrifuged cells may be resuspended in a suitable buffer or resuspension medium and passed through a filter disclosed herein.

[0226] In certain embodiments, cells passing through the filter disclosed herein may be referred to as purified cell compositions. In one example, the purified cell composition is characterized by certain structural features that can be specifically identified via visual inspection or other analytical methods. For example, the purified cell composition may be substantially free of visible particles. In another example, the purified cell composition is characterized by the size of the aggregates. As used herein, "aggregation" means the sum of multiple individual cells in a cluster grouped by one or more adhesion properties (including aggregation, cohesion and agglutination). As used herein, "aggregation" means the tendency of cells to aggregate. In one example, 90% of the cell populations / aggregates (e.g., stem cell populations / aggregates) have a diameter (D90) of less than 150 μm. For example, the purified stem cell compositions encompassed by the present disclosure may have a D90 of less than 150 μm. 90 In another example, D 90 Less than 100 μm. In another example, D 90 Less than 50 μm. For example, D 90 In another example, D 90 Available in 50 μm and 100 μm.

[0227] In certain embodiments, the cell or cell culture medium or resuspended culture medium including the cell is passed through a filter as described herein to provide a viable cell concentration of 60% to 100%. In some embodiments, the recovery rate of viable cells is 70% to 90%. In one example, the viable cell number in the composition before the composition passes through the filter as described herein is determined to determine the recovery rate percentage of viable cells. In another example, the percentage is determined relative to the total number of cells in the purified cell composition (i.e., after filtration). Various conventional methods for determining cell viability are known in the art. In one example, automatic cell counting is used. Such methods can use techniques and equipment based on Coulter counting or flow cytometry. These systems generally rely on counting the number of particles of a specific size by electrical or optical detection, and in some cases dyes (or the like) can also be incorporated to distinguish dead cells from live cells. In another example, semi-automatic or manual cell counting can be used to determine cell viability. Such methods generally involve using a well-known substance such as trypan blue or propidium iodide (PI) to mark dead cells, and then count the number of dead cells relative to live cells in the sample. As will be appreciated by those skilled in the art, cell viability is typically assessed in a sample that is representative of a larger composition, with results from the representative sample extrapolated to provide an overall level of cell viability for the larger composition.

[0228] Mesenchymal precursor lineage or stem cells

[0229] As used herein, the term "mesenchymal lineage precursor or stem cell (MLPSC)" refers to an undifferentiated pluripotent cell that has the ability to self-renew while maintaining pluripotency and the ability to differentiate into a number of cell types of mesenchymal origin (e.g., osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons) or non-mesodermal origin (e.g., hepatocytes, neural cells, and epithelial cells). For the avoidance of doubt, "mesenchymal lineage precursor cells" refer to cells that can differentiate into mesenchymal cells (such as bone, cartilage, muscle, and adipocytes) and fibrous connective tissue.

[0230] The term "mesenchymal lineage precursor or stem cell" includes both parental cells and their undifferentiated progeny. The term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells (MSC), perivascular mesenchymal precursor cells, and their undifferentiated progeny.

[0231] Mesenchymal lineage precursors or stem cells can be autologous, allogeneic, xenogeneic, syngeneic or isogenic. Autologous cells are isolated from the same individual into which they are to be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0232] In one example, the mesenchymal lineage precursors or stem cells are allogeneic. In one example, the allogeneic mesenchymal lineage precursors or stem cells are culture expanded and cryopreserved.

[0233] Mesenchymal lineage precursors or stem cells mainly reside in the bone marrow, but have also been shown to be present in a variety of host tissues, including, for example, umbilical cord blood and umbilical cord, adult peripheral blood, adipose tissue, trabecular bone and dental pulp. They are also present in skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestines, lungs, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis and periosteum; and can be differentiated into reproductive system such as mesoderm and / or endoderm and / or ectoderm. Therefore, mesenchymal lineage precursors or stem cells can be differentiated into a large number of cell types, including but not limited to adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue and fibrous connective tissue. The specific lineage-stereotype and differentiation pathways that these cells enter depend on the various effects from mechanical influences and / or endogenous bioactive factors (such as growth factors, cytokines and / or local microenvironment conditions established by host tissues).

[0234] The term "enriched", "enrichment" or its variant is used herein to describe such a cell colony, in which the ratio of a specific cell type or the ratio of many specific cell types increases when compared to an untreated cell colony (e.g., cells in their native environment). In an example, the colony enriched for mesenchymal lineage precursors or stem cells comprises at least about 0.1%, or 0.5%, or 1%, or 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 50%, or 75% mesenchymal lineage precursors or stem cells. In this regard, the term "cell colony enriched for mesenchymal lineage precursors or stem cells" will be considered as the term "cell colony comprising X% mesenchymal lineage precursors or stem cells" provides clear support, wherein X% is a percentage as listed herein. In some examples, mesenchymal lineage precursors or stem cells can form clonogenic colonies, such as CFU-F (fibroblasts) or a subset thereof (e.g., 50%, or 60%, or 70%, or 70%, or 90%, or 95%) can have such activity.

[0235] In one example of the present disclosure, the mesenchymal lineage precursor or stem cell is a mesenchymal stem cell (MSC). MSC can be a homogenous composition or can be a mixed cell population enriched with MSC. Homogeneous MSC compositions can be obtained by culturing adherent bone marrow or periosteal cells, and MSC can be identified by specific cell surface markers identified with unique monoclonal antibodies. The method for obtaining a cell population enriched with MSC is described in, for example, U.S. Patent No. 5,486,359. Alternative sources of MSC include, but are not limited to, blood, skin, cord blood, muscle, fat, bone, and perichondrium. In one example, MSC is allogenic. In one example, MSC is cryopreserved. In one example, MSC is cultured, amplified, and cryopreserved.

[0236] In another example, the mesenchymal lineage precursor or stem cell is a CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSC.

[0237] In one example, mesenchymal lineage precursors or stem cells are cultured and expanded from MSC colonies expressing markers (including CD73, CD90, CD105 and CD166) and lacking hematopoietic cell surface antigens (such as CD45 and CD31). For example, mesenchymal lineage precursors or stem cells can be cultured and expanded from MSC colonies (CD73+, CD90+, CD105+, CD166+, CD45- and CD31-). In one example, the colony of MSC is further characterized by low-level major histocompatibility complex (MHC) class I. In another example, MSC is negative for major histocompatibility complex class II molecules, and is negative for costimulatory molecules CD40, CD80 and CD86. In one example, culture expansion includes 5 generations.

[0238] The isolated or enriched mesenchymal lineage precursors or stem cells can be expanded in vitro by culture.The isolated or enriched mesenchymal lineage precursors or stem cells can be cryopreserved, thawed and subsequently expanded in vitro by culture.

[0239] In one example, the isolated or enriched mesenchymal lineage precursors or stem cells are cultured at 50,000 viable cells / cm 2 The cells are plated in culture medium (serum-free or serum-supplemented), e.g., alpha minimum essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, and allowed to adhere to the culture vessels overnight at 37° C., 20% O 2. The culture medium is then replaced and / or changed as needed, and the cells are cultured at 37° C., 5% O 2 for an additional 68 to 72 hours.

[0240] As will be appreciated by those skilled in the art, cultured mesenchymal lineage precursors or stem cells are phenotypically different from in vivo cells. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal lineage precursors or stem cells are also biologically different from in vivo cells, having a higher proliferation rate than most non-circulating (quiescent) cells in vivo.

[0241] In one example, a cell population is enriched from a cell preparation comprising STRO-1+ cells in a selectable form. In this regard, the term "selectable form" is understood to mean that the cell expresses a marker (e.g., a cell surface marker) that allows the selection of STRO-1+ cells. The marker may be STRO-1, but not necessarily. For example, as described and / or illustrated herein, cells (e.g., mesenchymal precursor cells) expressing STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (and may be STRO-1bright). Therefore, the indication that the cell is STRO-1+ does not mean that the cell is selected only by STRO-1 expression. In one example, cells are selected based on at least STRO-3 expression, for example, they are STRO-3+ (TNAP+).

[0242] Reference to the selection of cells or their populations does not necessarily require selection from a specific tissue source. As described herein, STRO-1+ cells can be selected from or isolated from or enriched from a variety of sources. That is, in some instances, these terms provide support for selection from any tissue or vascularized tissue containing STRO-1+ cells (e.g., mesenchymal precursor cells) or tissue containing pericytes (e.g., STRO-1+ pericytes) or any one or more of the tissues listed herein.

[0243] In one example, the cells used in the present disclosure express one or more markers, alone or collectively, selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0244] "Individually" means that the disclosure individually encompasses the recited marker or marker groups, and although individual markers or marker groups may not be individually listed herein, the appended claims may define such markers or marker groups individually and separately from each other.

[0245] "Collectively" means that the present disclosure encompasses any number or combination of the listed markers or marker groups, and although such number or combination of markers or marker groups may not be specifically listed herein, the appended claims may define such combinations or sub-combinations individually and separately from any other combination of markers or marker groups.

[0246] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue nonspecific alkaline phosphatase. For example, the term encompasses liver isoforms (LAP), bone isoforms (BAP), and kidney isoforms (KAP). In one example, TNAP is BAP. In one example, TNAP as used herein refers to a molecule that can bind to a STRO-3 antibody produced by a hybridoma cell line deposited with the ATCC on December 19, 2005 under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0247] Furthermore, in one example, STRO-1+ cells were able to generate clonogenic CFU-F.

[0248] In one example, a significant proportion of STRO-1+ cells are able to differentiate into at least two different germ lineages. Non-limiting examples of lineages to which STRO-1+ cells can be committed include bone precursor cells; hepatocyte progenitor cells, which are multipotent for bile duct epithelial cells and hepatocytes; neural restricted cells, which can generate glial precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; myocardium and precursors of cardiomyocytes; pancreatic beta cell lineage that secretes glucose-responsive insulin. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as precursors of the following cells: retinal pigment epithelial cells, fibroblasts, skin cells (such as keratinocytes), dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiomyocytes, smooth muscle cells, skeletal muscle cells, pericytes, vascular cells, epithelial cells, glial cells, neurons, astrocytes, and oligodendrocytes.

[0249] In one example, mesenchymal lineage precursors or stem cells are obtained from a single donor or multiple donors, wherein the donor samples or mesenchymal lineage precursors or stem cells are subsequently pooled and then expanded in culture.

[0250] Mesenchymal lineage precursors or stem cells encompassed by the present disclosure may also be cryopreserved prior to administration to a subject. In one example, the mesenchymal lineage precursors or stem cells are culture expanded and cryopreserved prior to administration to a subject.

[0251] Cell culture and expansion

[0252] In one example, mesenchymal lineage precursors or stem cells are cultured and expanded. The difference between "cultured and expanded" mesenchymal lineage precursors or stem cell culture medium and freshly separated cells is that they have been cultured and passed down (i.e., passage culture) in cell culture medium. In one example, mesenchymal lineage precursors or stem cells cultured and expanded are passed down for about 4-10 times. In one example, mesenchymal lineage precursors or stem cells are cultured and expanded for at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages. For example, mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5 passages. In one example, mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5-10 passages. In one example, mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5-8 passages. In one example, mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5-7 passages. In one example, mesenchymal lineage precursors or stem cells can be cultured and expanded for more than 10 passages. In another example, mesenchymal lineage precursors or stem cells can be cultured and expanded for more than 7 passages. In these examples, stem cells can be cultured and expanded before being cryopreserved to provide an intermediate cryopreserved MLPSC population. In one example, the composition of the present disclosure is prepared by an intermediate cryopreserved MLPSC population. For example, the MLPSC population cryopreserved in the middle can be further cultured and expanded before administration. Therefore, in one example, mesenchymal lineage precursors or stem cells are cultured, expanded and cryopreserved. In the embodiments of these examples, mesenchymal lineage precursors or stem cells can be obtained from a single donor or multiple donors, wherein these donor samples or mesenchymal lineage precursors or stem cells are subsequently collected, and then cultured and expanded. In one example, the culture expansion process includes:

[0253] - i. expanding said number of viable cells by passage expansion to provide a preparation of at least about 1 billion viable cells, wherein the passage expansion comprises establishing a primary culture of the isolated mesenchymal lineage precursors or stem cells, and then successively establishing a first non-primary (P1) culture of the isolated mesenchymal lineage precursors or stem cells from the previous culture;

[0254] - ii. expanding the P1 culture of the isolated mesenchymal lineage precursors or stem cells into a second non-primary (P2) culture of the mesenchymal lineage precursors or stem cells by subculture expansion; and,

[0255] - iii. preparing and cryopreserving an intermediate mesenchymal lineage precursor or stem cell preparation obtained from a P2 culture of mesenchymal lineage precursor or stem cells; and,

[0256] - iv. thawing the cryopreserved in-process intermediate mesenchymal lineage precursor or stem cell preparation and expanding the in-process intermediate mesenchymal lineage precursor or stem cell preparation by passage expansion.

[0257] In one example, the expanded mesenchymal lineage precursor or stem cell preparation has an antigenic profile and an activity profile comprising:

[0258] - i. Less than approximately 0.75% CD45+ cells;

[0259] - ii. at least about 95% CD105+ cells;

[0260] - iii. At least about 95% CD166+ cells.

[0261] In one example, the expanded mesenchymal lineage precursor or stem cell preparation is capable of inhibiting IL2Ra expression by CD3 / CD28 activated PBMCs by at least about 30% relative to a control.

[0262] In one example, the culture-expanded mesenchymal lineage precursor or stem cell is culture-expanded for about 4-10 passages, wherein the mesenchymal lineage precursor or stem cell has been cryopreserved after at least 2 passages or 3 passages before further culture expansion. In one example, the mesenchymal lineage precursor or stem cell is culture-expanded for at least 1, at least 2, at least 3, at least 4, at least 5 generations, cryopreserved and then further culture-expanded for at least 1, at least 2, at least 3, at least 4, at least 5 generations before administration or further cryopreservation.

[0263] In one example, most of the mesenchymal lineage precursors or stem cells in the composition of the present disclosure have approximately the same number of generations (that is, they are within about 1 or about 2 or about 3 or about 4 cell doublings of each other). In one example, the average number of cell doublings in the composition of the present invention is about 20 to about 25 doublings. In one example, the average number of cell doublings in the composition of the present invention is about 9 to about 13 (e.g., about 11 or about 11.2) doublings from the primary culture, plus about 1, about 2, about 3 or about 4 doublings / generation (e.g., about 2.5 doublings / generation). When produced by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 and about 10 generations respectively, the exemplary average cell doubling in the composition of the present invention is about 13.5, about 16, about 18.5, about 21, about 23.5, about 26, about 28.5, about 31, about 33.5 and about 36. Any one of.

[0264] Any equipment and cell processing methods known in the art can be used to separate and amplify mesenchymal lineage precursors or stem cells in vitro. Various culture expansion embodiments disclosed herein use steps that require manipulation of cells, such as inoculation, feeding, dissociation of adherent cultures, or washing steps. Any step of manipulating cells is likely to damage the cells. Although mesenchymal lineage precursors or stem cells can generally tolerate a certain amount of damage during preparation, it is preferred to manipulate cells by fully performing one or more given steps of the operating procedures and / or equipment while minimizing damage to the cells.

[0265] In one example, mesenchymal lineage precursors or stem cells are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculating wash bag, a rotating membrane filter having an inlet port and an outlet port, a filtrate bag, a mixing zone, a final product bag for the washed cells, and appropriate tubing, e.g., as described in US 6,251,295, which is hereby incorporated by reference.

[0266] In one example, according to the mesenchymal lineage precursor or stem cell composition disclosed herein, it is 95% homogeneous with respect to CD105 positive and CD166 positive and CD45 negative. In one example, this homogeneity persists by ex vivo amplification (i.e., by multiple population doublings). In one example, compositions comprises at least one mesenchymal lineage precursor or stem cell of therapeutic dose, and the mesenchymal lineage precursor or stem cell comprises less than about 1.25% CD45+ cell, at least about 95%CD105+ cell and at least about 95%CD166+ cell. In one example, this homogeneity persists after low temperature storage and thawing, and wherein cell also generally has about 70% or higher vitality.

[0267] Cell culture medium

[0268] Mesenchymal lineage precursors or stem cells disclosed herein can be cultured and expanded in various suitable culture media. The term "culture medium (medium or media)" as used in the context of the present disclosure includes components of the environment surrounding the cell. Culture medium helps and / or provides conditions suitable for allowing cell growth. Culture medium can be a solid, liquid, gas, or a mixture of phases and materials. Culture medium can include liquid growth medium and a liquid culture medium that does not maintain cell growth. Culture medium also includes gelatinous culture media such as agar, agarose, gelatin and collagen matrix. Exemplary gas culture medium includes the gas phase to which cells grown on culture dishes or other solid or semi-solid supports are exposed.

[0269] The cell culture medium used for culture expansion contains all essential amino acids and may also contain non-essential amino acids. Generally, amino acids are classified into essential amino acids (Thr, Met, Val, Leu, Ile, Phe, Trp, Lys, His) and non-essential amino acids (Gly, Ala, Ser, Cys, Gln, Asn, Asp, Tyr, Arg, Pro).

[0270] Those skilled in the art will appreciate that, in order to obtain optimal results, the basal medium must be appropriate for the cell line of interest. For example, if it is found that the glucose (or other energy source) in the basal medium is depleted and therefore restricts growth, it may be necessary to increase the glucose (or other energy source) level in the basal medium, or to add glucose (or other energy source) during the culture process. In one example, the dissolved oxygen (DO) level may also be controlled.

[0271] In one example, the cell culture medium contains additives of human origin. For example, human serum and human platelet cell lysate can be added to the cell culture medium. For example, the culture medium can contain 2% to 10% human serum. In one example, the culture medium contains 3% v / v human serum.

[0272] In one example, the cell culture medium contains only additives of human origin. Thus, in one example, the cell culture medium is free of exogenous components. For the avoidance of doubt, in these examples, the culture medium does not contain animal protein.

[0273] In one example, the culture medium comprises serum. In other examples, the culture medium is a culture medium without fetal bovine serum that contains growth factors that promote proliferation of mesenchymal lineage precursors or stem cells. In one embodiment, the culture medium is a serum-free stem cell culture medium. In one example, the cell culture medium comprises:

[0274] Basal culture medium;

[0275] Platelet-derived growth factor (PDGF);

[0276] Fibroblast growth factor 2 (FGF2).

[0277] In one example, the culture medium comprises platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2), wherein the level of FGF2 is less than 5 ng / ml. For example, the level of FGF2 may be 1 ng / ml to 2 ng / ml.

[0278] In one example, the PDGF is PDGF-BB. In one example, the level of PDGF-BB is about 1 ng / ml to 150 ng / ml. In another example, the level of PDGF-BB is about 7.5 ng / ml to 20 ng / ml. In another example, the level of PDGF-BB is at least 10 ng / ml.

[0279] In other examples, additional factors can be added to the cell culture medium. In one example, the culture medium further comprises EGF. EGFEGF is a growth factor that stimulates cell proliferation by binding to its receptor EGFR. In one example, the level of EGF is 0.1 ng / ml to 7 ng / ml. For example, the level of EGF can be at least 5 ng / ml. In one example, the level of EGF is about 2 ng / ml to 5 ng / ml.

[0280] In the above examples, a basal medium such as αMEM or StemSpan™ can be supplemented with a reference amount of growth factors.

[0281] In other examples, additional factors can be added to the cell culture medium. For example, the cell culture medium can be supplemented with one or more stimulatory factors selected from the group consisting of epidermal growth factor (EGF), 1α,25-dihydroxyvitamin D3 (1,25D), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and stromal derived factor 1α (SDF-1α).

[0282] In another example, the cell culture medium promotes stem cell proliferation while maintaining the stem cells in an undifferentiated state. When stem cells have not yet been stereotyped into a specific differentiation lineage, they are considered to be undifferentiated. As discussed above, stem cells exhibit morphological characteristics that distinguish them from differentiated cells. In addition, undifferentiated stem cells express genes that can be used as markers for detecting differentiation states. Polypeptide products can also be used as markers for detecting differentiation states. Therefore, those skilled in the art can easily determine whether the methods disclosed herein maintain stem cells in an undifferentiated state using conventional morphological analysis, genetic analysis, and / or proteomic analysis.

[0283] Prototype and Experimental Data

[0284] The inventor tested Figure 1 3, wherein the total volume of the container 102 is in the range of 1.5 liters to 2 liters. The first filter unit 120 has a pore size of 150 μm and a surface area of ​​approximately 258 cm2. The second filter unit 130 has a pore size of 40 μm and a surface area of ​​180 cm2. The chamber volumes are summarized in Table 1 below.

[0285] Table 1

[0286] Given the minimum and maximum chamber volumes in Table 1, the ratio of chamber volume to filter element surface area is therefore:

[0287] Entrance Room:

[0288] • Minimum inlet chamber volume = 250 cm3

[0289] • Maximum inlet chamber volume = 800 cm3

[0290] • Therefore, the inlet chamber volume is in the range of about 0.9 (250 / 258) to about 3.2 (800 / 258) times the surface area of ​​the first filter element.

[0291] Intermediate Chamber:

[0292] • Minimum intermediate chamber volume = 500 cm3

[0293] • Maximum intermediate chamber volume = 1000 cm3

[0294] • Therefore, the intermediate chamber volume is in the range of about 1.9 (500 / 258) to about 4.0 (1000 / 258) times the surface area of ​​the first filter element.

[0295] • Minimum intermediate chamber volume = 500 cm3

[0296] • Maximum intermediate chamber volume = 1000 cm3

[0297] • Therefore, the intermediate chamber volume is in the range of about 2.7 (500 / 180) to about 5.6 (1000 / 180) times the surface area of ​​the second filter element.

[0298] Exit Room:

[0299] • Minimum outlet chamber volume = 300 cm3

[0300] • Maximum outlet chamber volume = 800 cm3

[0301] • Therefore, the outlet chamber volume is in the range of about 1.6 (300 / 180) to about 4.5 (800 / 180) times the surface area of ​​the second filter element.

[0302] The filter units 120, 130 are inclined relative to each other and connected to the container wall 200 to form a "V" configuration (such as FIG. 2A to FIG. 2D as well as Figure 3A and Figure 3B The distance between the front wall seams 340, 360 (where the first filter unit 120 and the second filter unit 130 are respectively coupled to the front wall 202 of the container 102) is approximately 1 inch.

[0303] In order to determine the filtration efficiency of the filter device 100, the inventors used beads to represent particles greater than 40 μm in size, and used other beads to represent cells. The beads representing particles greater than 40 μm are Cospheric Red colored beads Cat # UVPMS-BR-1.20 45-53 μm (density is 1.20g / cc, and the particle size range is 45-53 μm). The beads used to represent cells are Cospheric Blue colored beads Cat # BLPMS-1.08 20-27 μm (density is 1.08g / cc, and the particle size range is 20-27 μm). The two groups of beads are mixed in a matrix (MES Stop of Lonza Singapore), and the density of the matrix is ​​the same as the density of the matrix containing 10% DMSO (BloodStor 100) in αMEM cell culture medium.

[0304] Filter device 100 was also tested using only Cospheric Red colored beads 45-53 μm (representative of particles greater than 40 μm in size in the BloodStor 100 matrix).

[0305] Some embodiments of the filtration device 100 were tested using cells in a BloodStor 100 matrix representing a quenched cell suspension (1 L trypLE + 4 L of v2.2 medium).

[0306] Some embodiments of the filtration device 100 were tested using cells concentrated by centrifugation at 400 xg for 8 minutes at 2-8° C. The cells were resuspended in 400 mL of modified αMEM (SGTS-10533, Lonza) to represent a concentrated cell suspension.

[0307] Some embodiments of the filtration device 100 were tested using cells concentrated by centrifugation at 400 xg for 8 minutes at 2-8° C. The cells were resuspended in 300 mL of cryogenic medium containing Plasmalyte A, 5% human serum albumin, and 10% DMSO to represent a 1× drug product.

[0308] To determine the amount of cells filtered from the fluid (including the beads in the matrix), two 1 mL samples were taken before and after filtration. The number of two types of beads in the 10 μL aliquots was counted using a glass hemocytometer or using trypan blue exclusion. The amount of beads measured before and after filtration was compared to determine the filtration efficiency. The weight of the fluid was measured before and after filtration to determine the amount of fluid retained in the filtration device 100.

[0309] The inventors found that if Figure 1 The filtration device 100 shown in FIG. 3 successfully filters out beads representing particles larger than 40 μm in size without impeding the flow of beads representing cells (20-27 μm). Although some red beads (representing particles larger than 40 μm) were found in the filtered sample, the size of these beads was similar to that of 20-27 μm beads. Therefore, the counts of 45-53 μm beads were considered "zero". Particle and cell removal performance did not appear to be affected by the various matrices tested, especially those containing 10% DMSO.

[0310] To provide a performance comparison, the inventors also tested a commercially available filtration device (Haemonetics SQ40S) with a 40 μm filter unit / filter screen. The filtration device successfully filtered out beads representing particles larger than 40 μm in size. However, the flow of beads representing cells (20-27 μm) was hampered, resulting in a 21.9% loss of smaller beads.

[0311] The inventor observes that, in some embodiments, some of 20-27 μm beads (representing cells) remain attached to the inside of chamber 300. Matrix (including beads / cells) flows through filter unit 120, 130 without too much resistance. The inventor observes that the amount of residual beads can be reduced by allowing filter device 100 to accumulate about 1 / 3 or 1 / 2 of the volume capacity (~ 300-500 mL). In order to enable filter device 100 to accumulate in this way, a fixture or valve is used to temporarily prevent fluid from flowing out of filter device 100 (such as a fixture or valve in fluid communication with drain pipe 610). Once filter device 100 is full of about 1 / 3 to 1 / 2, outlet fixture / valve is removed. In some embodiments, it is observed that this allows fluid to flow through filter device 100 better. The inventor observes in some embodiments that the accumulated fluid reduces the possibility of the wall 200 of filter device 100 contacting and acting as a bottleneck, thereby causing residual beads to accumulate. In some embodiments, the accumulated fluid is observed to allow the control of purification process to be improved from a processing perspective.

[0312] It will be appreciated by those skilled in the art that many changes and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure. Therefore, the embodiments of the present invention are to be considered in all aspects as illustrative and not restrictive.

Claims

1. A filter comprising: a flexible container defining an inlet port toward an inlet end of the container and an outlet port toward an outlet end of the container, the container having one or more walls connecting the inlet end and the outlet end; as well as a first filter unit and a second filter unit, the first filter unit and the second filter unit being spaced apart within the container, wherein: the first filter unit being coupled to the container to define an inlet chamber in fluid communication with the inlet port; the second filter unit being coupled to the container to define an outlet chamber in fluid communication with the outlet port; and The first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter the fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter the fluid flowing from the intermediate chamber to the outlet chamber; wherein the inlet chamber comprises a first trough relative to the first peak; wherein the intermediate chamber comprises a second trough relative to the second peak; and Wherein the outlet port and the second filter unit are configured to be spaced apart when the fluid flows through the second filter unit into the outlet chamber.

2. The filter of claim 1, wherein the outlet port and the second filter unit are configured to be separated by a spacer system.

3. A filter comprising: a container defining an inlet port toward an inlet end of the container and an outlet port toward an outlet end of the container, the container having a flexible front wall and a flexible rear wall connecting the inlet end and the outlet end; as well as a first filter unit and a second filter unit, the first filter unit and the second filter unit being spaced apart within the container, wherein: the first filter unit being coupled to the front wall and the rear wall of the container to define an inlet chamber in fluid communication with the inlet port; the second filter unit is coupled to the front wall and the rear wall of the container to define an outlet chamber in fluid communication with the outlet port; and The first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter fluid flowing from the intermediate chamber to the outlet chamber; wherein the first filter unit is coupled to the front wall along a first front seam, and the second filter unit is coupled to the front wall along a second front seam; wherein the first filter unit is coupled to the rear wall at a first rear seam and the second filter unit is coupled to the rear wall at a second rear seam; wherein a distance between the first front seam and the second front seam is less than a distance between the first back seam and the second back seam; and Wherein the outlet port and the second filter unit are configured to be spaced apart when the fluid flows through the second filter unit into the outlet chamber.

4. The filter of claim 3, further comprising a spacer system configured to separate the outlet port from the second filter unit and at least one of: (i) the front wall and the first filter unit; (ii) the rear wall and the first filter unit; as well as (iii) the rear wall and the second filter unit.

5. A filter as claimed in claim 3 or claim 4, wherein the first filter unit is connected to the front wall of the container at an acute angle to define an inlet chamber slot.

6. The filter of claim 5, wherein the first filter unit is connected to the rear wall of the container at an obtuse angle.

7. The filter according to any one of claims 3 to 6, wherein the second filter unit is connected to the rear wall of the container at an acute angle to define an intermediate chamber slot.

8. The filter of claim 7, wherein the second filter unit is connected to the front wall of the container at an acute angle.

9. The filter of any one of claims 1 to 8, wherein the first filter unit comprises a mesh defining pores having an average pore size of 130 µm to 170 µm.

10. The filter of claim 9, wherein the first filter unit pores have an average pore size of 150 μm.

11. The filter according to any one of claims 1 to 10, wherein the second filter unit comprises a mesh defining pores having an average pore size of 20 µm to 60 µm.

12. The filter of claim 11, wherein the second filter unit pores have an average pore size of 40 μm.

13. The filter of any one of claims 1 to 12, wherein the first filter element is substantially flat.

14. The filter of any one of claims 1 to 13, wherein the second filter element is substantially flat.

15. A filter as claimed in claim 4 or any one of claims 5 to 14 when dependent on claim 3, wherein the spacer system comprises a separator.

16. A filter as claimed in claim 4 or any one of claims 5 to 14 when dependent on claim 3, wherein the spacer system comprises a clip.

17. A filter as claimed in claim 4 or any one of claims 5 to 14 when dependent on claim 3, wherein the spacer system comprises a magnet system.

18. A filter as claimed in claim 4 or any one of claims 5 to 14 when dependent on claim 3, wherein the spacer system comprises a stent.

19. The filter of any one of claims 3 to 18, wherein the second rear seam angles the second filter element and the outlet port away from each other.

20. The filter of any one of claims 3 to 18, wherein the front wall is configured to protrude away from the second filter unit.

21. A filter comprising: a flexible bag defining an inlet port toward an inlet end of the bag and an outlet port toward an outlet end of the bag, the bag having opposing front and rear walls connecting the inlet and outlet ends; a first filter unit and a second filter unit, the first filter unit and the second filter unit being spaced apart within the bag, wherein: the first filter unit being coupled to the bag to define an inlet chamber in fluid communication with the inlet port; the second filter unit coupled to the bag to define an outlet chamber in fluid communication with the outlet port; The first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter fluid flowing from the intermediate chamber to the outlet chamber; and Wherein the outlet port and the second filter unit are configured to be spaced apart when the fluid flows through the second filter unit into the outlet chamber.

22. The filter of claim 21, wherein the first filter unit and the second filter unit are non-parallel.

23. A kit for filtering cells, the kit comprising: The filter according to any one of claims 1 to 22; an inlet conduit, the inlet conduit comprising: a first inlet tube and a second inlet tube, the first inlet tube and the second inlet tube being fluidly connected to a manifold; a fill tube in fluid communication with the manifold and adapted to receive fluid from at least one of the first inlet tube and the second inlet tube; and an inlet coupling adapted to connect the fill tube to the inlet port of the filter and to allow fluid communication with the inlet chamber; an outlet conduit, the outlet conduit comprising: Drains; and An outlet coupling is adapted to connect the drain to the outlet port of the filter and to allow fluid communication with the outlet chamber and the drain.

24. The kit of claim 23, wherein the inlet tube and the fill tube form a Y-shape or a T-shape.

25. A kit as claimed in claim 23 or claim 24, wherein the inlet tube has an inner diameter of 1 / 8 inch and an outlet diameter of 1 / 4 inch.

26. A kit as claimed in any one of claims 23 to 25, wherein: (i) the fill tube has an inner diameter of 1 / 8 inch and an outlet diameter of 1 / 4 inch; or (ii) the drain tube has an inner diameter of 1 / 8 inch and an outlet diameter of 1 / 4 inch.

27. The kit of any one of claims 23 to 26, further comprising an outlet valve or clamp configured to control fluid flow through the drain.

28. The filter or kit of any one of claims 1 to 27, wherein one or more or all of the container, the bag, the filter unit 120, 130 or the tube are made of a DMSO compatible plastic, preferably a plastic comprising one or more of polyethylene terephthalate (PET), high density polyethylene (HDPE), low density polyethylene (LDPE), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), thermoplastic elastomer (TPE) and polypropylene (PP).

29. The filter or kit of any one of claims 1 to 28 when used to filter a stem cell culture medium.

30. A method of filtering a stem cell culture medium, the method comprising using the filter of any one of claims 1 to 22 or the kit of any one of claims 23 to 28.

31. A method for purifying a cell composition, comprising passing cultured cells through a double mesh filter to reduce visible particles and / or cell aggregates, wherein the double mesh filter comprises a first filter having an average pore size of 130 μm to 170 μm and a second filter having an average pore size of 20 μm to 60 μm.

32. The method of claim 31, wherein the cultured cells are provided in a serum-free cell culture medium.

33. The kit of any one of claims 23 to 28 or the method of claim 31 or claim 32, wherein the cells are culture expanded.

34. The kit or method of claim 33, wherein the cells are mesenchymal lineage precursor or stem cells (MLPSC).

35. The method of any one of claims 31 to 34, wherein after passing the cells through the double mesh filter, the recovery of viable cell concentration is (i) 60% to 100%; or (ii) 70% to 90%.

36. The method of any one of claims 31 to 35, wherein the purified cell composition exhibits a D value of less than 150 μm, preferably less than 100 μm, more preferably less than 50 μm. 90 .

37. The method of any one of claims 31 to 36, wherein the purified cell composition is substantially free of visible particles.

38. A filter comprising: a flexible container defining an inlet port toward an inlet end of the container and an outlet port toward an outlet end of the container, the container having one or more walls connecting the inlet end and the outlet end; a first filter unit, the first filter unit comprising a first filter screen; and a second filter unit, the second filter unit comprising a second filter screen; wherein the first filter unit and the second filter unit are spaced apart within the container; wherein the first filter unit is coupled to the container to define an inlet chamber in fluid communication with the inlet port; wherein the second filter unit is coupled to the container to define an outlet chamber in fluid communication with the outlet port; and wherein the first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter a fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter a fluid flowing from the intermediate chamber to the outlet chamber; wherein the inlet chamber comprises a first trough relative to the first peak; wherein the intermediate chamber comprises a second trough relative to the second peak; and Wherein at least one of the first filter screen and the second filter screen is configured to be spaced apart from the one or more walls when the fluid flows through the filter.

39. A filter comprising: a container defining an inlet port toward an inlet end of the container and an outlet port toward an outlet end of the container, the container having a flexible front wall and a flexible rear wall connecting the inlet end and the outlet end; a first filter unit, the first filter unit comprising a first filter screen; and a second filter unit, the second filter unit comprising a second filter screen; wherein the first filter unit and the second filter unit are spaced apart within the container, wherein: the first filter unit being coupled to the front wall and the rear wall of the container to define an inlet chamber in fluid communication with the inlet port; the second filter unit is coupled to the front wall and the rear wall of the container to define an outlet chamber in fluid communication with the outlet port; and The first filter unit and the second filter unit define an intermediate chamber between the inlet chamber and the outlet chamber, the first filter unit is configured to filter fluid flowing from the inlet chamber to the intermediate chamber, and the second filter unit is configured to filter fluid flowing from the intermediate chamber to the outlet chamber; wherein the first filter unit is coupled to the front wall along a first front seam, and the second filter unit is coupled to the front wall along a second front seam; wherein the first filter unit is coupled to the rear wall at a first rear seam and the second filter unit is coupled to the rear wall at a second rear seam; wherein a distance between the first front seam and the second front seam is less than a distance between the first back seam and the second back seam; wherein the outlet port and the second filter unit are configured to be spaced apart when the fluid flows through the second filter unit into the outlet chamber; and At least one of the first filter screen and the second filter screen is configured to be spaced apart from the front wall or the rear wall when the fluid flows through the filter.

40. The steps, features, integers, compositions and / or compounds disclosed herein or referred to in the specification of this application, individually or collectively, and any and all combinations of two or more of said steps or features.

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