BIOREACTOR FOR BIOLOGICAL MATERIAL

Abstract

A method and system of and for handling, preserving, separating, filtering, collecting, manipulating, and/or culturing ex vivo biological material including red blood cells, white blood cells, and blood plasma within a bioreactor having a gas permeable membrane that allows for passive ventilation.

Claims

1. A closed system of and for collecting and incubating living cells or other biological material comprising: a housing configured to house living cells or other biological material; a gas permeable membrane for passive ventilation of the housing; a frame support configured to hold the gas permeable membrane on the exterior surface of the housing, wherein the housing allows for multi-day to multi-month incubation and manipulation of the living cells or other biological material within the interior of the housing without the use of pumps or mechanically induced motion.

2. The system of claim 1, wherein the gas permeable membrane is located on an exterior surface of the housing, wherein the gas permeable membrane fully surrounds the frame support, so as to encompass an exterior surface of the frame support.

3. The system of claim 1, wherein the frame support is porous, wherein the frame support is attached to the housing by at least one of the group consisting of friction or interference fit, friction or interference fit with a sealing agent, a screw, a gasket, sonic welding, laser welding, and combinations thereof.

4. The system of claim 1, wherein the housing includes a sidewall, wherein the sidewall includes a lip, wherein the frame support fits within the lip to secure the frame support to the housing, wherein the gas permeable membrane is configured to extend beyond the lip in the sidewall of the housing, the gas permeable membrane configured to seal the system such that all gases entering or existing the system must pass through the gas permeable membrane, and wherein the housing includes a flange, the flange configured to allow a user to hold the system.

5. The system of claim 1, wherein the housing includes supports, wherein the supports are configured so that the housing is stackable on a second housing.

6. The system of claim 1, wherein the system is designed and intended to be inserted into a centrifuge.

7. The system of claim 1, wherein the system allows for gas exchange such that carbon dioxide is exchanged into or out of the system and oxygen passes into or out of the system.

8. The system of claim 1, wherein the system is a closed system which allows for selected gases to pass in and out of the system, and wherein the gas pressure within the system is greater than atmospheric pressure.

9. The system of claim 1, wherein the housing further includes gas adjusted culture media located within the interior of the housing, the gas adjusted culture media configured to support the living cells or other biological material, wherein the culture media may over fill or under fill the housing within the system, and wherein the interior of the housing is sterile and maintains sterility during system by use of at least one sterile connection or functionally closed sterile docking port.

10. The system of claim 1, further comprising at least one input and/or output port configured with pre-attached sterile and dockable tubing to maintain sterility of the system.

11. The system of claim 1, wherein an interior surface of the housing is at least partially hydrophilic, and wherein an agent introduced to the interior of the housing adheres to at least a portion of the housing interior surface, wherein the agent is selected from the group including an antigen, an antibody, a major histocompatibility immune complex (MHC), a retronectin reagent, and combinations thereof, wherein the agent adheres to the housing via a charge related to the internal surface of the housing.

12. The system of any of claim 1, wherein the interior surface of the housing includes a microstructure, wherein the microstructure is selected from the group including a coating solution, a gel coating solution, a retronectin reagent, a cytomatrix, microbeads, larger or macro polystyrene beads, and combinations thereof.

13. The system of claim 1, wherein the gas permeable membrane is selected from the group including a non-Class Vl polydimethylsiloxane, a medical grade Class Vl polydimethylsiloxane or a similar gas permeable silicon, a fluoro-ethylene-propylene, a natural gas permeable exchange membrane, and combinations thereof.

14. The system of claim 1, wherein the housing includes an antigen adhering capability to a gravitational bottom surface of the interior of the housing for selectively collecting subsets of various types of blood cells, wherein a specific blood cell antibody identifies the specific antigen for selectively collecting subsets of various types of cells within the interior of the housing.

15. The system of any of claim 1, wherein the gas permeable membrane is located at or near the top of the housing to facilitate cell incubation for a period of days to weeks to months via selective gas exchange.

16. The system of claim 1, wherein the housing is configured to provide for the growth, expansion, and/or maturation of specific cells coming from a patient via a growth factor and/or a cell culture media, wherein specific cells are sterilely introduced into the system, and the selected cells grown, expanded, and/or matured and then taken from the system and reinfused to the patient and/or cryopreserved.

17. The system of claim 1, wherein the housing is configured to provide for the growth, expansion, and/or maturation of specific cells coming from a patient via a growth factor and/or a cell culture media, wherein the specific cells are sterilely introduced into the system, and the selected cells grown, expanded, and/or matured and then taken from the system and infused to a donee and/or cryopreserved.

18. The system of any of claim 1, wherein the system receives an ex-vivo collection of a patient's living cells, wherein the introduction to and incubation of the collected living cells within the interior of the housing, and the reintroduction of the incubated living cells back to the patient to stimulate, activate, support, enhance, change, down regulate, up regulate, or other manipulations whereby making it possible to recognize and kill cancer cells, regulate or kill autoimmune cells, and other manipulations of the immune system to create a normally functioning immune system.

19. The system of any of claim 1, wherein the system facilitates the ex-vivo manipulation of a donor's cells, the introduction to and incubation of the collected cells within the interior of the housing, and the introduction of the incubated cells to a donee to stimulate, activate, support, enhance, change, deregulate, up regulate, or other manipulation whereby making it possible to recognize and kill cancer cells, regulate or kill autoimmune cells and other manipulations of the immune system to create a normally functioning immune system.

20. The system of claim 1, wherein the system is configured for cell introduction, selection, maturation, and expansion within the system for incubation of the desired cells within the interior of the housing, and the reintroduction of the incubated cells back to the patient for the improvement and/or the enrichment of their immune system.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIG. 1 is a diagram of an embodiment of the present invention in a flat or horizontal configuration and position.

[0088] FIG. 2 is a diagram of an embodiment of the present invention depicting the seal of a top plate or lid to a culture base upper edge.

[0089] FIG. 3 is a diagram of an embodiment of the present invention in a round or spherical configuration culture base.

[0090] FIG. 4A is a diagram of an embodiment of the present invention in a modified spherical or rounded rectangular configuration culture base.

[0091] FIG. 4B is a diagram of an embodiment of the present invention in a modified spherical or rounded rectangular configuration culture base.

[0092] FIG. 4C is a diagram of an embodiment of the present invention in a modified spherical or rounded rectangular configuration culture base.

[0093] FIG. 5A is a diagram of an embodiment of the present invention depicting a culture base support or pedestal.

[0094] FIG. 5B is a diagram of an embodiment of the present invention depicting a culture base support or pedestal.

[0095] FIG. 6 is a diagram of an embodiment of the present invention depicting a round or spherical configuration culture base resting in a culture base support or pedestal.

[0096] FIG. 7 is a diagram of an embodiment of the present invention depicting a modified spherical or rounded rectangular culture base resting in a culture base support or pedestal.

[0097] FIG. 8 is a passive ventilation closed system bioreactor of an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0098] In the following description, numerous details are set forth for the purpose of explanation. However, one of ordinary skill in the art will realize that the invention may be practiced without the use of these specific details. For instance, the techniques described below are described in a specified order, but other embodiments may change the order of the operations while still embodying the current invention.

[0099] The application incorporates by reference the materials listed in the Appendix.

[0100] In certain embodiments, the inventive method and system comprises a base or housing defining a container or cavity.

[0101] In certain embodiments, the inventive method and system comprises a structure with adherence properties.

[0102] In certain embodiments, the inventive method and system comprises a housing with a selective gas permeable material.

[0103] In certain embodiments, the inventive method and system comprises at least one fluid communicating port.

[0104] In certain embodiments, the inventive method and system is capable of and for capturing and incubating singular or multiple blood cells dependent upon blood cluster designations or blood cluster markers using a selected antigen or a plurality of selected antigens, the system including a selectively permeable material allowing for blood cell incubation via passive gas transpiration thus facilitating multi-day or multi-week long manipulation of the cells in a starting culture housing or container without the use or need of pumps or mechanically induced motion.

[0105] As shown in FIG. 1, an exemplary embodiment of a present inventive capture and incubation housing or container (100) is depicted.

[0106] A culture base (110) is provided which at least in part defines a cavity, troth, or reservoir (115) for containing or holding a culture nutrient solution and/or other materials required for the capture and/or incubation of singular or multiple cells and cell types.

[0107] In certain embodiments of the inventive capture and incubation housing or container (100) includes a microstructure (120) which may be suspended within the cavity, troth, or reservoir (115) for and on which cells, antigens, or other biological materials or chemicals may adhere. It is also contemplated in certain embodiments that the microstructure (120) may be a three-dimensional matrix or other type scaffolding onto which cells, antigens, or other biological materials or chemicals may adhere.

[0108] In certain embodiments, it is contemplated that the culture base (110) and/or microstructure (120) may be manufactured via injection molding or by other means of shaping a Class Vl styrene or similar plastic, and be capable of sterilization via commonly known methods such as but not limited to; gas or liquid ethylene oxide, chlorine dioxide, hydrogen peroxide, gamma ray, or electron beam sterilization.

[0109] If the culture base (110) and/or microstructure (120) comprise material which is non-adherent to cells or other biological material (i.e. hydro and/or lipid phobic); if desired, gamma ray or electron beam sterilization methods may preferably be used to render the culture base (110) and/or microstructure (120) adherent (i.e. hydrophilic and/or lipophilic).

[0110] In certain embodiments of the present invention, the capture and incubation housing or container (100) includes a selective gas permeable material (130).

[0111] In certain embodiments of the present invention, the capture and incubation housing or container (100) comprises a selective gas permeable material (130) preferably capable of allowing desired oxygen and carbon dioxide transfer and/or exchange.

[0112] In certain embodiments of the present invention, the capture and incubation housing or container (100) comprise a selective gas permeable material (130) preferably capable of allowing desired oxygen and carbon dioxide transfer while maintaining interior system sterility.

[0113] In certain embodiments of the present invention, the capture and incubation housing or container (100) includes a lid or frame (FIG. 2, 150) to support, secure, and/or affix the selective gas permeable material (130) above or abutted against the cavity, troth, or reservoir (115).

[0114] In certain embodiments of the present invention, the capture and incubation housing or container (100) culture base (110) includes at least one input and/or output port or receptacle (140) for the introduction or extraction of biological or other material to and from the cavity, troth, or reservoir (115) of the culture base (110).

[0115] In certain embodiments of the present invention, the culture base (110) includes at least one input and/or output port or receptacle (140) for the introduction or extraction of biological or other material to and from the cavity, troth, or reservoir (115) while maintaining interior system sterility.

[0116] In an embodiment of the present invention, the culture base (110) includes at least one heat source (Not Shown) to establish and maintain a desired interior incubation temperature of the cavity, troth, or reservoir (115).

[0117] One dynamic theory of operation of the present embodiment of the cell capture and incubation housing or container (100) may be as follows:

[0118] A capture and incubation housing or container (100) including a culture base (110) defining a cavity, troth, or reservoir (115) a microstructure structure (120) may be “pre-loaded” with a suitable nutrient media and desired adhered capture antigen to “snag” or “hook” a desired biological material such as by way of example and not limitation, white blood cells, dependent upon the cell's cluster designation or cluster marker. When a patient's whole blood is introduced to the cavity, troth, or reservoir (115) of the capture and incubation housing or container (100) via the at least one input and/or output port or receptacle (140), adhered antigens “capture” desired white blood cells. Once a desired quantity of white blood cells is captured, remaining whole blood components may then be removed and replaced with a desirable nutrient media for incubating and/or growing the captured white blood cells over a period of hours to days or weeks. After captured cell incubation, growth, treatment, and/or manipulation; further biological material or chemicals may be introduced to the capture and incubation housing or container (100) to “release” the adhered cells and/or antigens for immediate or eventual reintroduction to the patient or a donee.

[0119] In one embodiment of the present invention, via an additional input and/or output port or receptacle (140′) a patient's whole blood may be purposefully and continuously recirculated through the capture and incubation housing or container (100) if desired.

[0120] In one embodiment of the present invention, the capture and incubation housing or container (100) may be used to capture and/or incubate bacterium, viruses, prions, spores, fungi, and the like.

[0121] In certain embodiments of the invention, a lid or frame (150) supporting securing and/or affixing the selective gas permeable material (130) above or abutted against the cavity, troth, or reservoir system has a series of holes such that CO2 is released out and O2 passes into the system.

[0122] In certain embodiments of the invention, a layer of gas permeable plastic sheeting is affixed to a ventilation scaffold to create a simple, yet effective bioreactor. Furthermore, the system includes tubing at the edges of the bioreactor functional to provide ingress and egress of pre-selected blood cells coming from the use of an apheresis machine, or other devices presently available to the practitioner or researcher. In certain embodiments, the system works with using whole blood cells and cells derived from buffy coats, adipose (fat from the abdomen) derived tissue, cells for MSC selection.

[0123] In certain embodiments of the invention, the system allows for not only the capture of specific cells, but also offers passive gas exchange of the housing or container to allow a multi-day or multi-week manipulation of cells in the starting container without use of mechanical pumps or mechanical motions.

[0124] In certain embodiments of the invention, the interior of the system housing or container (100) interior is coated with a desired antigen for the purpose of “snagging” or to “snag” a certain cell type.

[0125] In certain embodiments of the invention, the system housing or container (100) interior is coated with styrene plastic. In and of itself styrene plastic is hydrophobic, that is nothing aqueous will adhere to its surface. However, by use of gamma irradiation the styrene plastic surface can be made hydrophilic in order to render the housing or container interior adherent to cells, antigens, and the like. Once accomplished, injection via a port and pre-attached tubing of an antigen in liquid form that is specific to a particular TARGET BLOOD CELL whereby it is incubated and or manipulated as desired.

[0126] In certain embodiments of the invention, the system allows for the sterile ingress or egress of fluids or gases via known and FDA approved sterile connection and/or docking techniques.

[0127] In certain embodiments of the invention, the gas permeable membrane (130) comprises Class Vl PDMS or similar gas permeable (preferably and predominately O.sub.2 and CO.sub.2 permeable) silicon, FEP, or other natural gas permeable exchange membranes.

[0128] In certain embodiments of the invention, the system is used to capture a single or multiple blood cells with an antigen to a chosen capability defined by that cells CD marker. The marker could be CD4, CD8, CD14, CD19, and/or CD34 and so on. This is accomplished by conjugation of an antigen to an antibody surface marker on a particular chosen blood cell for ex vivo manipulation. Blood cells have surface markers that have designated antibodies. Science has the ability to manufacture antigens (the bacteria or virus or other”) that certain blood cells are on the constant look out for to prevent or ultimately cure an ailment; think of those ailment fighting cells as having hooks that can only “snag” one particular antigen. At the time that an immune fighting cell detects a foreign bacteria or virus a cascade of events happens. In some people or with certain diseases there is a breakdown of the immune signaling and the disease progresses into problems such as cancer.

[0129] As depicted in FIGS. 3 and 4A, the system capture and incubation housing or container (100) culture base (110) may be of a round, spherical, modified spherical, or rounded rectangular, other shape to provide additional volume or culture depth to the system. The system capture and incubation housing or container (100) is shown having a selective gas permeable material (130) as well as ports into the system.

[0130] As shown in FIG. 4B, filter or vertical separation member (180) is shown as well as bar, which is magnetic (190). Magnetic bar (190) is used for scraping the bottom of the container (100) to encourage cell release for cells stuck to the bottom adherent complex. This would aid in drawing a small portion of the cells to an exit port or scrape the entire bottom for total cell release. The bar (190) may be coated with Teflon® or silicon or other inert material.

[0131] As shown in FIG. 4C, metallic bar scrapper (190′) inside the device is not a magnet, but rather it would be metallic and would be controlled by an external magnet (195). One could position the metallic bar scrapper (190′) by flipping the device over, guide the metallic bar scrapper (190′) over a specific area attracted through the membrane, and then from the bottom using the external magnet (195) on a long skinny handle, drag and scrape the cells to where they want. In certain embodiments, the device includes a strong external magnet (195) that will lift the metallic bar scrapper (190′) to the membrane (130) and by pulling the external magnet (195) away from the metallic bar scrapper (190′), the metallic bar scrapper (190′) will fall to a desired location.

[0132] As depicted in FIG. 5A-7, in certain embodiments of the invention the system capture and incubation housing or container (100) may include a holder or pedestal (160) to position the culture base (110) as desired.

[0133] As depicted in FIG. 8, a passive ventilation closed system bioreactor of an embodiment of the invention is shown. The passive ventilation closed system bioreactor eliminates the centrifuge bucket to hold a device and acts as its own centrifuge bucket. In certain embodiments, the passive ventilation closed system bioreactor has flanges designed into two sides to fit onto the centrifuge rotor. It is specific to centrifugation on its own.

[0134] In certain embodiments, the bottom angled section of FIG. 8 includes a plurality of 2 mm beads. In certain embodiments, a mesh screen is provided within the system of FIG. 8 about 0.3 centimeters or so above the bottom port to trap the beads inside after centrifugation, but to allow dislodged blood cells to escape and be sterile collected via sterile connection.

[0135] In certain embodiments, a sterile connection device is further added to the system of FIG. 8.

[0136] In certain embodiments, the passive ventilation closed system bioreactor includes a fitted cap, preferably made of plastic, to cover the membrane when the device is removed form an incubator in use. This cap, or temporary cap, over the membrane would prevent the change of the internal environment of the bioreactor over a short removal time from the gas controlled incubator. This prevents cells in the system from being negatively affected by the change in environment.

[0137] Washing of Cells

[0138] In certain embodiments, of the invention, the method includes a process and procedure for washing cells prior to introduction of the cells into the bioreactor.

[0139] In certain embodiments, the washing of cells will be accomplished by sterile docking 600 ml transfer bags on one end of the device. The process involves adding saline to the housing to flush the system, so that the other end of the device, the waste (effluent) is captured and centrifuged along with the concentrated cells. The concentrated cells can then be reintroduced into the housing after the washing.

[0140] Cell Growing Procedure

[0141] In certain embodiments, the cell growing procedure is as follows:

[0142] 1. Add apheresis cells to CD14 Ab coated container at 2×10e6 monocytes/cm2;

[0143] 2. Incubate 37 degrees C. for 30-60 min;

[0144] 3. Gently shake (can be mechanized) and wash off cells via sterile docking outlet with a large bore tubing;

[0145] 4. Wash via 2× using sterile docking inlet and sterile docking outlet;

[0146] 5. Add media and Growth Factors (GFs);

[0147] 6. Incubate 3 days;

[0148] 7. Re-feed via sterile docking inlet;

[0149] 8. Day 6 add virus via sterile docking inlet;

[0150] 9. Incubate 2 hours, wash via sterile docking inlet with 3× media exchange and sterile docking pig tail outlet with cell retainer for feeding;

[0151] 10. Incubate with activation as desired or harvest via outlet for cryopreservation. One can concentration using outlet with cell retainer and peristaltic pump.

[0152] This protocol is exemplary and the additional protocols are possible using the device and methods sets forth in this application.

[0153] Device Construction

[0154] In certain embodiments, the device includes three parts, the base, the membrane, and the frame that will go around the perimeter and be sealed by any of the following means—interference fit alone; interference fit with a solvent sealing agent or glue or silicon; screws; a gasket may be employed, sonically welded, or combinations of various means.

[0155] In certain embodiments, the device further includes a gasket.

[0156] In certain embodiments, the base has a slotted interior perimeter as to allow a membrane that has been previously precut to size to fit into the slotted lip and slotted frame.

[0157] In certain embodiments, the membrane will be sealed by any one of the following means: interference fit alone; interference fit with a solvent sealing agent or glue or silicon; screws; a gasket may be employed, sonically welded, or combinations of various means.

[0158] In certain embodiments, the gas permeable membrane is located on gravitational top of the housing.

[0159] In certain embodiments, the gas permeable membrane is located on a side wall of the housing to provide for increase release of cell waste products, as CO.sub.2 is a heavy gas and having the gas permeable membrane located on a side wall of the housing will increase CO.sub.2 release from the housing.

[0160] In certain embodiments, the device is 1 cm tall or taller, depending upon the cells incubated in the device. In certain embodiments, the device is between 4 and 6 cm.

[0161] In certain embodiments, the device is stackable and/or flipable.

[0162] In certain embodiments, the device includes a drip or pump feeding at a very low rate for adding fresh media continuously.

[0163] In certain embodiments, the device includes a vertical separation member in the middle of the device so that various groups of cells can be cultured separately from one another.

[0164] In certain embodiments, pumped gas could be used to assist in emptying the housing and to remove cells and media.

[0165] In certain embodiments, the device includes one or more support posts to support the gas permeable membrane. In certain embodiments, the support posts are small (preferably about 1 cm in diameter) and rounded at the base and top. The gas permeable membrane is supported by the support posts.

[0166] In certain embodiments, the gas permeable membrane will overlap the top frame to the base, the base, and how the membrane will “sandwich” in with the slots. This gives exposure to solvent fixation from polystyrene to polystyrene as a preferred sealing method. The “slot and sandwich” approach goes for all the devices, regardless of shape.

[0167] In certain embodiments, the device is stackable on top of one another and configured to be inserted within a centrifuge. In certain embodiments, two or more devices are stackable on top of one another and configured to be inserted within a centrifuge. In certain embodiments, this configuration enhances cell washing and concentration.

[0168] In certain embodiments, the device is configured to lay flat, on its end and placed into a centrifuge.

[0169] In certain embodiments, the device eliminates the needs for magnetic beads within the housing that separate processing of cells.

[0170] In certain embodiments, the gas permeable membrane suspends over the top of the housing and is a lattice type framework to allow oxygen and carbon dioxide gas transpiration into and out of the housing.

[0171] In certain embodiments, the device allows for positive and/or negative selection of blood and/or blood components such as stem cells, leukocyte subsets, or other pre-determined cell or tissue lines to be introduced into the housing of the device.

[0172] These components are sterilely introduced into the housing, specifically adhered within the housing, and captured within the interior of the housing. Positive selection means the desired cell or tissue is biologically attached within the invention, conversely, negative selection occurs when unwanted cells are attached within the invention allowing wanted cells to be retrieved from the invention by the use of the pre-attached tubing.

[0173] In certain embodiments, the interior of the housing is made of polystyrene or other materials with a suitable charge, is manufactured to provide acceptable adherent properties (hydrophilic) either at the GMP manufacturing facility or at the end users site under GMP conditions (for example, by gamma irradiation) to facilitate attachment of targeted cellular receptors or affinity reagents.

[0174] In certain embodiments, the housing has or develops an adherent surface that allows this coating step within the invention. The coating will be such as a monoclonal antibody, or other, that adheres to the interior surface of the invention.

[0175] In certain embodiments, the cellular introduction and subsequent attachment of a desired cell's surface receptor to the already attached monoclonal antibody or targeted receptor within the housing, thereby causing the desired membrane ligand to bind with the monoclonal antibody/receptor within the housing (positive selection). The housing is placed in an incubator for two-hours (can be variable) that allows for an antibody/receptor interaction with the membrane ligand.

[0176] Following incubation, the unwanted cells are removed by various FDA accepted means such as buffered saline or trypsin via pre-attached PVC tubing. After removal of unwanted cells, a fourth phase occurs for further manipulation of the desired cells or tissues, this is achieved within the invention by introduction of cell culture media and cytokine growth factors. Specifically, the present invention relates to systems and methods to handle, preserve viability, separate, filter where appropriate, collect, manipulate, and/or culture ex vivo various hematological materials including red blood cells, white blood cells, tissues, and blood plasma such that, depending upon at least in part, the desired hematological CD target. Selected cells in the invention are matured, expanded in numbers, and viability retained within the invention.

[0177] In certain embodiments, the present invention may also be used for collection and manipulation of other biological materials and organisms including viruses, prions, spores, fungi, and the like. The invention is designed with legs at the four curved corners allowing for even temperature distribution to surround the invention in the incubator. The legs also allow for stacking of multiple devices within an incubator and for storage. In certain embodiments, the legs are stackable legs that extend at the bottom and the top of the device.

[0178] In certain embodiments, the device includes magnetic bars that are used for scraping the bottom of the housing to encourage cell release for cells stuck to the bottom adherent complex. This would aid in drawing a small portion of the cells to an exit port or scrape the entire bottom for total cell release. The bar would be coated with Teflon or silicon or other inert material.

[0179] In certain embodiments, the scrapper inside the device would not be a magnet, rather it would be metallic and would be controlled by an external magnet. One could position the scrapper by flipping the device over, guide the scrapper over a specific area attracted through the membrane, and then from the bottom using the magnet on a long skinny handle, drag and scrape the cells to where they want.

[0180] In certain embodiments, the device includes a strong magnet that will lift the metallic scrapper to the membrane and by pulling away from the metallic scrapper the scrapper will fall to a desired location.

[0181] In certain embodiments, the housing includes a 2 to 4-micron mesh or other filter type within the device to prevent cell loss when emptying. In certain embodiments, the mesh is positioned away from the exit ports. In certain embodiments, the mesh has a curved shape towards the inside that is positioned to capture cells.

[0182] In certain embodiments, the system is a closed system, port or multiple ports if a client wants to add a sterile filter (words are there to cover that option), an internal cytomatrix, designed for centrifugation, long duration incubation, removing some cells (material) and leaving some cells (material) behind for extending the incubation.

[0183] Having thus described several embodiments for practicing the inventive method, its advantages and objectives can be easily understood. Variations from the description above may and can be made by one skilled in the art without departing from the scope of the invention.

[0184] Accordingly, this invention is not to be limited by the embodiments as described, which are given by way of example only and not by way of limitation.