RELATED APPLICATIONS

[0001] This application claims priority from the United States provisional application with serial number 61/969231 filed on March 23, 2014. The disclosure of that provisional application is incorporated herein as if set out in full.

BACKGROUND OF THE DISCLOSURE

TECHNICAL FIELD OF THE DISCLOSURE

[0002] The present embodiment relates in general to cell processing. More specifically, the present disclosure relates to a method and system for washing cells suspended in a liquid medium utilizing centrifugation techniques and/or providing cell medium exchange in cell manufacturing.

DESCRIPTION OF THE RELATED ART

[0003] Cell manufacturing is the process of multiplying cells in vitro to produce large numbers of cells from small numbers of starting cells ("cell expansion") and/or the genetic or chemical modification of such cells to provide them with specific characteristics for use in fields such as cell therapy, pharmaceutical drug discovery, or biomedical research. Cells that undergo expansion in a container may be adherent or non-adherent to the surface of the container. Cells that adhere to the surface of the container, as well as the additional cells resulting from the expansion process require different expansion processing than non-adherent cells. Non-adherent cells may settle on the surface of the container but do not adhere and easily distribute through the expansion medium with motion.

[0004] A typical cell expansion process of an adherent stem cell, like the mesenchymal stem cell (MSC) involves multiple rounds of seeding low numbers of cells in culture vessels, allowing the cells to adhere to the plastic surface of the vessel and multiply in the presence of liquid media that contains growth factors, harvesting the cells from the vessels with the use of detachment agents that overcome the adherence of the cells, such as the enzyme trypsin (through a process called "trypsinization"), seeding the cells harvested from one vessel into multiple new vessels, harvesting again following the cells' expansion, and repeating this sequence until the desired quantity of cells is obtained. A single cycle of seeding, expansion, and harvesting is termed a "passage." Depending on the cell type and their intended application, five, ten, or even more passages may be required to produce the desired number of cells.

[0005] In recent years, bone marrow aspirate, placental cord blood, peripheral blood and other human tissue are sources of cells used for the production of a variety of different cell types, such as bone cells, cartilage cells, muscle cells, immune cells neuronal cells and retinal cells. Thus, a method for efficiently collecting, washing, separating and amplifying the cells is required. The method for accomplishing this requires a complicated procedure in which cells are repeatedly washed in mediums using a centrifuge for separating the cells from the liquid and then returned to a container in which the expansion continues..

[0006] Harvesting of trypsinized cells usually requires centrifugation. During this process, the liquid cell suspension is transferred to a centrifuge tube, bottle, or similar simple container. The container is centrifuged to pellet the cells at the bottom of the container. The supernatant fluid above the pelleted cells is removed by aspiration (usually manually by a trained technician carefully wielding a hypodermic needle connected by tubing to a vacuum source to suck off the supernatant fluid), and then the cells are deposited in a fresh medium. This process of removing the cells from one medium and transferring them to a fresh medium is termed "washing." This medium may be constituted solely to wash the cells of the previous medium or it may also contain growth factors to nourish the next round of cell expansion. This manual aspiration and transfer procedure requires considerable training to achieve the manual dexterity and careful attention needed to avoid lost or damaged cells caused by careless technique. Additionally, such manual methods involve the use of open containers, which pose the risk of microbial contamination of the harvested cells, rendering them useless. In addition, this method is accompanied by the risk of damage to cells due to the centrifugation process itself. [0007] A conventional means for solving these drawbacks is to use functionally closed instruments for washing cells, such as the COBE 2991 Cell Processor. However, such traditional systems do not provide efficient washing and processing of cells. A published evaluation (K. Goltry, B. Hampson, N. Venturi, R. Bartel, "Emerging Technology Platforms for Stem Cells. " John Wiley & Sons, Inc., Chapter 9: 153-168 (2009)) of the centrifuge-based COBE 2991 Cell Processor in a stem cell manufacturing process reported a viable cell recovery efficiency of only 73%. The same publication found that a competing system, the Baxter Cytomate®, yielded a viable cell recovery efficiency of 61%. Therefore, there exists a need to raise cell recovery efficiency to increase the number of viable cells for use by patients requiring cell therapy to alleviate various medical conditions.

[0008] Several semi-automated systems for washing the cells are currently available in market. However, these systems provide less cell recovery efficiency. One existing cell processing system describes a cell washing method and apparatus, which utilizes centrifugation to separate blood components in a flexible bag. The less dense separated components, e.g., supernatant, is expressed from the bag by centrifugal force acting on a plate adjacent the bag, and the more dense component, e.g., RBCs, remains in the bag. However, the process of collecting and separating blood components involves many steps and frequent human intervention, which leads to the contamination of cells. The arrangement of the bag set does not permit the process to be easily automated since it takes time to correctly load these bag sets into the dedicated devices and to ready the system to process the cells. Moreover, these systems include multiple bag sets and complicated connecting tubing attached thereto.

[0009] Another existing apparatus describes a system for collecting red blood cells (RBCs) and other blood components and reduces the need for human intervention. A disposable set is provided, having a port, an RBC container, a centrifuge rotor having a variable total volume, and a filter, along with tubing connecting the port, the container, the rotor and the filter. A control unit is also provided and includes a spinner in which the rotor may be held, a flow-control arrangement for controlling flow among the various components of the disposable set, and an electronic controller. The spinner rotates the rotor so as to separate the whole blood into plasma and RBCs. After the plasma has been removed from the rotor, the RBCs are urged from the rotor through the filter so that white blood cells (WBCs) are caught in the filter and RBCs pass through the filter to an RBC container. However, such a system includes a single use, disposable bag set linked together with substantial tubing which needs to be manufactured again for another processing of the cells. These prior art bag sets are complex and costly to manufacture.

[00010] Yet another existing closed cell processing system includes a method for washing platelets by introducing an anticoagulant into a platelet product container, drawing anticoagulated platelet product from the platelet product container, and introducing it into a centrifuge bowl. The centrifuge bowl separates the platelets from the supernatant in which they are suspended. The method then washes the platelets by introducing a wash solution into the centrifuge bowl. As the wash solution is introduced into the bowl, it displaces the supernatant from the bowl and into a waste container. The method then introduces a platelet additive solution into the centrifuge bowl, which displaces the wash solution from the centrifuge bowl and into the waste container and further washes the platelets. The method then repeatedly accelerates and decelerates the centrifuge bowl to re-suspend the platelets in the platelet additive solution. However, such a system cannot optically track the migration of the cell populations for each individual blood sample and then deplete certain cell types by diverting the cells into different compartments within the same container during centrifugation.

[00011] There is thus a need for an improved automated cell washing system and method that provides an optimum environment for washing cells suspended in a liquid medium, while maintaining the sterility of the environment and viability of the cells. Such an apparatus and method would include closed containers and provide an efficient cell medium exchange under completely sterile conditions. Such a system would include a container having one or more compartments to receive the cell solution and diluent. Such an apparatus and method would include one or more detectors that optically track the migration of the cell populations for each individual blood sample and then deplete certain cell types by diverting the cells into different compartments within the same container during centrifugation. Such an apparatus and method would include valves that are configured to mechanically close certain tube paths, thereby redirecting the fluid via the tube to appropriate compartments of the container. Such an apparatus and method would include an automated or semi-automated means for directing different sub-volumes of the fluid contained within or outside the container to the compartments. This apparatus would include a computer interface for monitoring and reporting the process and for automatically controlling the valves to control the flow of the fluid into the containers. Finally, this apparatus and method would provide an all-in-one automated workstation in which all cell processing occurs and in which all components related to cell stratification and depletion are located at the completion of the centrifugation.

SUMMARY OF THE DISCLOSURE

[00012] To minimize the limitations found in the prior art and to minimize other limitations that will be apparent upon the reading of the specification, the preferred embodiment of the present invention provides a method and apparatus for washing cells such as hematopoietic stem and progenitor cells and their progeny, including immune cells, as well as mesenchymal stem and progenitor cells and their progeny and endothelial progenitor cells found in normal blood, placental/cord blood, bone marrow or the other fractional cells suspended in a liquid medium utilizing centrifugation techniques and/or providing cell medium exchange in cell manufacturing under a sterile environment.

[00013] The apparatus comprises at least one sterile, functionally closed container, an input tubing means, at least one cell suspension bag, at least one sterile wash medium bag, containing a medium that may be solely for washing cells but also may contain growth factors for growing the cells, and at least one waste container, an output tubing means, an automated workstation, a centrifuging means, an unloading means and a re-suspending means. The sterile, functionally closed container comprises at least one functionally isolated compartment configured to hold fluid containing harvested cell suspension and medium. The input tubing means having a luer connection filter and a first air filter, is connected to at least one middle tubing and at least one harvest tubing on the at least one sterile, functionally closed container. The input tubing means is configured for loading the harvested cell suspension and medium into the at least one functionally isolated compartment. The at least one cell suspension bag is connected to a means of determining weight or fluid flow so that the reduction of weight allows the precise determination of the volume of cell suspension released and is connected to the input tubing means via a sterile weldable tubing. The at least one cell suspension bag is configured for holding the harvested cell solution, which may be suspended in at least one cell dissociation reagent. Preferably, the at least one cell dissociation reagent is trypsin. The at least one sterile wash medium bag containing medium is connected to the input tubing means via the sterile weldable tubing. The at least one sterile wash medium bag containing medium is configured for holding medium for washing or for washing and with growth factors for further cell expansion. The at least one waste container is configured for collecting the at least one cell dissociation reagent without the presence of cells after the first concentration of cells or at least one waste medium in which the growth factors have been consumed and the exudate from the expanded cells. The output tubing means is connected to a luer lock fitting on the at least one sterile, functionally closed container. The output tubing means is configured to transfer the at least one cell dissociation reagent or at least one waste medium in which the growth factors have been consumed and the exudate from the expanded cells, without the presence of cells, from the at least one functionally isolated compartment to the at least one waste container.

[00014] The at least one sterile, functionally closed container comprises the at least one functionally isolated compartment configured to hold fluid containing harvested cell suspension and medium, at least one detector for detecting the presence or absence of cells in the harvested cell suspension and in different sub-volumes of the fluid within the at least one sterile, functionally closed container, and a means to direct sub-volumes of the fluid in the at least one sterile, functionally closed container to the at least one functionally isolated compartment based on outputs of the at least one detector. The at least one sterile, functionally closed container is removably positioned on the at least one upper platform of the automated workstation. The at least one upper platform is capable of providing programmable rotary motion for mixing the contents of the at least one sterile, functionally closed container during the second concentration of cells.

[00015] The automated workstation comprises at least one upper platform, a vertical support member, a plurality of pinch valves, at least one pump connection, at least one first receptacle, at least one second receptacle and at least one third receptacle; preferably all attached to an upper support member. The automated workstation further comprises a control panel and instructions for use mounted on a lower support member. The at least one upper platform having at least one locking means configured to removably hold the at least one sterile, functionally closed container. The vertical support member includes a first holding means for hanging the at least one cell suspension bag, and precisely measuring the weight of the bag during the release of cell suspension so that the volume of cell suspension can be calculated, and a second holding means for hanging the at least one sterile medium bag and precisely measuring the weight of the bag during the release of the medium so that the volume of medium released can be calculated. The plurality of pinch valves is configured to pinch the input tubing means at selective branch pathways, thereby controlling the flow of the fluid into the at least one sterile, functionally closed container. The at least one pump connection having a pump connection tubing connected to a second air filter on the at least one sterile, functionally closed container. The second air filter is a 0.2-micron filter that is closed with a detachable filter cap and configured to provide pressure/vacuum to the fluid contained in the at least one sterile, functionally closed container. The at least one pump connection is configured to provide positive and negative air pressure via the pump connection tubing to the fluid contained in the at least one sterile, functionally closed container from at least one pump. The at least one first receptacle is adapted to hold the input tubing means to the at least one sterile, functionally closed container. The at least one second receptacle is adapted to hold the pump connection tubing when not in use. The at least one third receptacle is adaptable for holding the output tubing means to the at least one waste container when not in use.

[00016] The fluid and air movement through the input tubing means is based on both gravity and pressure differentials. In the preferred embodiment, at least one cell suspension bag and the at least one sterile wash medium bag containing wash medium are hanging above the at least one sterile, functionally closed container. The movement of the cell suspension or medium fluids can be precisely measured by sensors and can occur solely as a result of gravity or can be supplemented by the addition of positive or negative air pressure to the pump connection tubing from at least one pump through the at least one pump connection. The at least one pump may be an air compressor/vacuum pump that can be attached to the at least one pump connection for applying pressure/vacuum to the fluid. Thus, the positive and negative air pressure along with the gravitational force provide the movement of the fluid through the input tubing means to the at least one sterile, functionally closed container. The second air filter is a 0.2- micron filter that is closed with the detachable filter cap and configured to provide pressure/vacuum to the fluid contained in the at least one sterile, functionally closed container. The output tubing means is configured to transfer the at least one functionally isolated compartment to the at least one waste container.

[00017] In one embodiment, a method for washing cells suspended in a liquid medium is described. As a first step in the method, the at least one sterile, functionally closed container is placed on the at least one upper platform, such that the at least one sterile, functionally closed container is locked on the at least one upper platform utilizing the at least one locking means. Then, the input tubing means is connected between the at least one cell suspension bag, the at least one sterile diluent bag and the at least one sterile, functionally closed container. After that, the pump connection tubing is connected to the second air filter on the at least one sterile, functionally closed container. Then, the harvested cell suspension is loaded into the main rigid compartment of the at least one sterile, functionally closed container. The medium is loaded into the second rigid harvest compartment of the at least one sterile, functionally closed container when the luer connection filter attached to the sterile weldable tubing is opened. In order to accelerate the fluid through the input tubing means and into the at least one sterile, functionally closed container, the pressure/vacuum is applied to the at least one sterile, functionally closed container to assist the flow. The at least one pump connection is configured to provide the positive and negative air pressure via the pump connection tubing to the fluid from the at least one pump.

[00018] Once the contents of the at least one cell suspension bag have been substantially equally divided into each of two sterile, functionally closed containers, the sterile weldable tubing, connecting the at least one cell suspension bag, and the input tubing means, connecting two sterile, functionally closed containers, are sealed and separated. After the wash medium is transferred to the two sterile, functionally closed containers, the sterile weldable tubing of the at least one sterile diluent bag is removed from the luer connection filter.

[00019] The at least one sterile, functionally closed container along with the input tubing means are transferred to the centrifuging means for the first concentration of the cells. The contents in the at least one sterile, functionally closed container are centrifuged, and sedimented cells are directed into the at least one functionally isolated compartment of the at least one sterile, functionally closed container based on the output of the at least one detector. The centrifugation occurs for an appropriate amount of time and the appropriate gravitational force (G force) level (greater than 1) to cause the sedimenting of cells within the two containers. The at least one detector is configured to optically track the migration of the cells for each individual sample suspension of cells and to then deplete certain cell types by diverting them into the first and second compartments of the at least one sterile, functionally closed container during centrifugation. As described in that document and based on the outputs of the one or more detectors within each sterile, functionally closed container, the sedimenting cells are directed by the automated means into one or more of the functionally isolated compartments during centrifugation.

[00020] Then, the sedimented cells are unloaded, substantially free of supernatant, from their respective functionally isolated compartments. This is accomplished by employing one or more functionally closed, sterile devices to remove the cells of interest from the functionally isolated compartments of the at least one sterile, functionally closed container. The functionally closed, sterile devices may be integral to, or separate from, the at least one sterile, functionally closed container and preferably utilize positive pressure along with gravity to move the liquid. In one embodiment, the at least one pump is utilized for providing the positive pressure. The pressure and gravitational force may also be utilized to remove the at least one cell dissociation reagent, or at least one waste medium in which the growth factors have been consumed and the exudate from the expanded cells, from the main rigid compartments of each of the sterile, functionally closed containers to the at least one waste container. In one embodiment: the sterile, functionally closed container is rotated to one side at least 30 degrees and at most approximately 90 degrees in order to assist the removal of the at least one cell dissociation reagent. This is accomplished by the programmable rotary motion of the at least one upper platform. The cells of interest are then re-suspended in a fresh medium at an appropriate cell density. After the final centrifugation, the at least one harvest tubing, which is pressed into tubing holders positioned on the containers, is released for sterile connection to a syringe or to other equipment for further processing. [00021] In one embodiment, the apparatus comprises a computer that is embedded in or connected to the automated workstation for carrying out the methods described herein and includes a processor which executes program instructions stored in a memory and a data input element for inputting program instructions for controlling the flow of the fluid to the at least one sterile, functionally closed container. The program instructions are stored on an electronic apparatus-readable medium for implementing the steps of the methods described herein.

[00022] A first objective of the present invention is to provide an improved automated cell washing system and method that provides an optimum environment for washing cells suspended in a liquid medium, while maintaining the sterility of the environment and viability of the cells.

[00023] A second objective of the present invention is to provide an apparatus that includes closed containers that provide an efficient and precisely volume measured cell medium exchange under completely sterile conditions.

[00024] A third objective of the present invention is to provide an apparatus that includes two containers, each having one or more compartments to receive the cell solution and wash medium.

[00025] A fourth objective of the present invention is to provide an apparatus that includes one or more detectors that optically track the migration of the cell populations for each individual cell suspension sample and then deplete certain cell types by diverting the cells into different compartments within the same container during centrifugation.

[00026] A fifth objective of the present invention is to provide valves that are configured to mechanically close certain tube paths thereby redirecting the fluid via the tube to appropriate compartments of the container.

[00027] A sixth objective of the present invention is to provide an automated or semi-automated means for directing different sub-volumes of the fluid contained within or outside the container to the compartments.

[00028] A seventh objective of the present invention is to provide an apparatus and method that is in communication with a computer interface for monitoring and reporting the process and for automatically controlling the valves to control the flow and volume of the fluid into the containers. [00029] An eighth objective of the present invention is to provide an all-in-one automated workstation in which all cell processing occurs and in which all components related to cell stratification and depletion are located at the completion of the centrifugation.

[00030] These and other advantages and features of the present invention are described with specificity so as to make the present invention understandable to one of ordinary skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[00031] The elements in the figures have not necessarily been drawn to scale in order to enhance their clarity and improve understanding of these various elements and embodiments of the invention. Furthermore, elements that are known to be common and well understood to those in the industry are not depicted in order to provide a clear view of the various embodiments of the invention, thus the drawings are generalized in form in the interest of clarity and conciseness.

[00032] FIG. 1 illustrates a front perspective view of an apparatus in accordance with the preferred embodiment of the present invention;

[00033] FIG. 2 illustrates a side perspective view of an automated work station in accordance with the preferred embodiment of the present invention;

[00034] FIG. 3 illustrates a perspective view of at least one sterile, functionally closed container in accordance with the preferred embodiment of the present invention;

[00035] FIGS. 4A-4C illustrate perspective, side and top views of a plurality of pinch valves in accordance with the preferred embodiment of the present invention;

[00036] FIG. 5 illustrates a diagrammatic representation of an input tubing means connected with the at least one sterile, functionally closed container in accordance with the preferred embodiment of the present invention;

[00037] FIG. 6 illustrates the at least one sterile, functionally closed container in operation wherein a harvested cell suspension is being loaded into a main functionally isolated compartment and a diluent is being loaded in a second functionally isolated harvest compartment in accordance with the preferred embodiment of the present invention;

[00038] FIG. 7 illustrates a perspective view of the at least one sterile, functionally closed container being prepared to be transferred to a centrifuging means;

[00039] FIG. 8 illustrates a top down perspective view of the at least one sterile, functionally closed container when placed in the centrifuging means for centrifugation;

[00040] FIG. 9 illustrates a bottom perspective view of the at least one sterile, functionally closed container showing approximately -200 x 10 ⁶ (0.8 mL) of concentrated mesenchymal stem cells harvested after centrifugation;

[00041] FIG. 10 illustrates a front perspective view of the apparatus wherein the apparatus is configured to prepare a second concentration of cells;

[00042] FIG. 11 illustrates a perspective view of the at least one sterile, functionally closed container after the final centrifugation wherein at least one harvest tubing is shown pressed into tubing holders on the at least one sterile, functionally closed container;

[00043] FIG. 12 illustrates a perspective view of the at least one sterile, functionally closed container after the final centrifugation wherein the at least one harvest tubing has been extended for sterile welding;

[00044] FIG. 13 illustrates a plot of a time along the X-axis versus gravitational force along the Y-axis;

[00045] FIG. 14 illustrates a summary of results for washing and concentrating

Mesenchymal Stem Cells (MSCs) derived from the TerumoBCT QuantumR Cell Expansion System, showing a graph of MSC recovery following concentration after each washing step; and

[00046] FIG. 15 illustrates the summary of results for washing and concentrating

Mesenchymal Stem Cells (MSCs) derived from the TerumoBCT QuantumR Cell Expansion System, showing a graph of MSC viability following concentration and washing.

DETAILED DESCRIPTION OF THE DRAWINGS

[00047] In the following discussion that addresses a number of embodiments and applications of the present invention, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and changes may be made without departing from the scope of the present invention.

[00048] Various inventive features are described below that can each be used independently of one another or in combination with other features. However, any single inventive feature may not address any of the problems discussed above or only address one of the problems discussed above. Further, one or more of the problems discussed above may not be fully addressed by any of the features described below.

[00049] The present invention is a method and apparatus for washing alone, or washing and then re-suspending the concentrated washed cells in new medium. These cells may be hematopoietic stem and progenitor lineage cells, mesenchymal stem and progenitor lineage cells or endothelial progenitor cells found in normal blood, placental/cord blood, bone marrow or other tissue or other fractional cells suspended in a liquid medium, utilizing centrifugation techniques, and/or providing cell medium exchange in cell manufacturing under a sterile environment.

[00050] Turning first to FIG. 1, a front perspective view of the apparatus 100 in accordance with the preferred embodiment of the present invention is illustrated. The apparatus comprises at least one sterile, functionally closed container 102, an input tubing means 104, at least one cell suspension bag 106, at least one sterile diluent bag 108, at least one waste container 110, an output tubing means 112, an automated workstation 114, all preferably shown in FIG. 1, a centrifuging means 166 (See FIG. 8), an unloading means (not shown) and a re-suspending means (not shown). The at least one sterile, functionally closed container 102 is fully described in U.S. Pat. Application 13/634520 filed September 12, 2012, entitled "System for Purifying Certain Cell Populations in Blood or Bone Marrow by Depleting Others," and assigned to the assignee of the present application, which is incorporated by reference in its entirety and attached hereto as Exhibit A. In the shown preferred embodiment there are two sterile, functionally closed containers 102a, 102b each placed at the automated workstation 114. References made to the at least one sterile, functionally closed container 102 apply to both functionally closed containers 102a, 102b unless specified otherwise. Each sterile, functionally closed container 102a, 102b comprises at least one functionally isolated compartment configured to hold fluid containing harvested cell suspension and diluent. The input tubing means 104 having a luer connection filter 116 and a first air filter 118 is connected to at least one middle tubing 120 and at least one harvest tubing 122 on the at least one sterile, functionally closed container 102. The input tubing means 104 is configured for loading the harvested cell suspension and diluent into the at least one functionally isolated compartment. The at least one cell suspension bag 106 is connected to the input tubing means 104 via a sterile weldable tubing 124. The at least one cell suspension bag 106 is configured for holding the harvested cell solution suspended in at least one cell dissociation reagent, or a solution in which the growth factors have been consumed and replaced with exudate from the expanded cells. Preferably, the at least one cell dissociation reagent is trypsin and the at least one growth factor is IL2. The at least one sterile wash medium bag 108 is connected to the input tubing means 104 via the sterile weldable tubing 124. The at least one sterile wash medium bag 108 is configured for holding a variety of wash mediums, including phosphate buffered saline (PBS), solutions including albumin, and solutions including growth factors, such as IL2.. The at least one waste container 110 is configured for collecting the at least one cell dissociation reagent or at least one waste medium, in which the growth factors have been consumed, and the exudate from the expanded cells, without the presence of cells after first concentration of cells. The output tubing means 112 is connected to a luer lock fitting 126 on the at least one sterile, functionally closed container 102. The output tubing means 112 is configured to transfer the at least one cell dissociation reagent without the presence of cells from the at least one functionally isolated compartment to the at least one waste container 110.

[00051] As shown in FIGS. 1 and 2, the automated workstation 114 (not labeled in this Figure) comprises at least one upper platform 128, a vertical support member 130, a plurality of pinch valves 132, at least one pump connection 134, at least one first receptacle 136, at least one second receptacle 138 and at least one third receptacle 140, preferably all attached to an upper support member 142. The automated workstation 114 further comprises a control panel 144 and instructions for use 146 mounted on a lower support member 148. The at least one upper platform 128 having at least one locking means 150 configured to removably hold the at least one sterile, functionally closed container 102 as shown in FIG. 1. The at least one locking means 150 may be a plurality of locking tabs or any sort of fastener that allows a component inserted therein to click, snap, or latch into place. The vertical support member 130 includes a first holding means 152 for hanging and weighing the at least one cell suspension bag 106 and a second holding means 154 for hanging and weighing the at least one sterile wash medium bag 108. The plurality of pinch valves 132 is configured to pinch the input tubing means 104 at selective branch pathways, thereby controlling the flow of the fluid into the at least one sterile, functionally closed container 102. The at least one pump connection 134 having a pump connection tubing 156 connected to a second air filter 158 on the at least one sterile, functionally closed container 102. The second air filter 158 is a 0.2-micron filter that is closed with a detachable filter cap 164 (Fig. 3) and configured to provide pressure/vacuum to the fluid contained in the at least one sterile, functionally closed container 102. The at least one pump connection 134 is configured to provide positive and negative air pressure via the pump connection tubing 156 to the fluid contained in the at least one sterile, functionally closed container 102 from at least one pump (not shown). The at least one first receptacle 136 is adapted to hold the input tubing means 104 to the at least one sterile, functionally closed container 102. The at least one second receptacle 138 is adapted to hold the pump connection tubing 156 when not in use. The at least one third receptacle 140 is adapted to hold the output tubing means 112 to the at least one waste container 110 when not in use. In one embodiment, the automated workstation 114 further comprises at least one clamp member that securely holds the input tubing means 104 during the transfer of the fluid to the at least one sterile, functionally closed container 102.

[00052] Turning briefly to FIG. 3, in the preferred embodiment, the apparatus

100 is configured to accept prior art disposable cartridges used in the automated depleting, purifying and harvesting of target cell populations and described in more detail in U.S. Patent 8,747,289 B2, granted June 10, 2014, which is incorporated herein by reference as if set out in full. The at least one sterile, functionally closed container 102 comprises the at least one functionally isolated compartment configured to hold fluid containing harvested cell suspension and wash medium, at least one detector for detecting the presence or absence of cells in the harvested cell suspension and in different sub-volumes of the fluid within the at least one sterile, functionally closed container, and a means to direct sub-volumes of the fluid in the at least one sterile, functionally closed container to the at least one functionally isolated compartment based on outputs of the at least one detector. As explained in the U.S. Patent 8,747,289 B2, the at least one sterile, functionally closed container comprises a main rigid compartment, a large first rigid compartment and a small second rigid harvest compartment. The at least one sterile, functionally closed container 102 within is removably positioned on the at least one upper platform 128 of the automated workstation 114 as shown best in FIGS. 1, 6 and 10. The at least one upper platform 128 is capable of providing programmable rotary motion for suspending and evenly distributing cells in the cell solution media in the at least one sterile, functionally closed container 102 during second concentration of cells as shown in FIG. 10.

[00053] FIGS. 4A-4C illustrate perspective, side and top views of the plurality of pinch valves 132 in accordance with the preferred embodiment of the present invention. Preferably each valve 132 includes a compact design with functions of soft opening and closing capabilities, state sensing, tube detection, self-monitoring usage and diagnostics. The plurality of pinch valves 132 is configured to mechanically close certain tube paths of the input tubing means 104, thereby redirecting the fluid via the input tubing means 104 to the appropriate location according to the intended application. The plurality of pinch valves 132 either opens the tubing (in the normally closed configuration) or closes the tubing (in the normally open configuration). The plurality of pinch valves 132 may be a 3 -way valve having both a normally open and a normally closed flow path, which are operated simultaneously. The amount of fluid passing through the input tubing means 104 to the at least one functionally isolated compartment of the at least one sterile, functionally closed container 102 is controlled by the opening and closing of the plurality of pinch valves 132. The opening and closing of the plurality of pinch valves 132 are controlled by a signal received from the computer that is in communication with the apparatus 100.

[00054] FIG. 5 illustrates a diagrammatic representation of the input tubing means 104 connected with the at least one sterile, functionally closed container 102 in accordance with the preferred embodiment of the present invention. The input tubing means 104 is functionally closed and is connected between the at least one cell suspension bag 106, the at least one sterile wash medium bag 108 and the at least one sterile, functionally closed container 102. The luer connection filter 116 attached to the input tubing means 104 is adaptable to filter the medium as it flows from the at least one sterile medium bag 108 to the at least one sterile, functionally closed container 102. Preferably, the luer connection filter 116 has a pore size of 0.2 microns and is closed with a detachable filter cap 160. The first air filter 118 attached to the input tubing means is a 0.2-micron filter that is closed with a detachable filter cap 162. The first air filter 118 provides passage for displaced air from within the input tubing means 104 when the harvested cell solution is introduced into the at least one sterile, functionally closed container 102 through the input tubing means 104. The at least one middle tubing 120 and the at least one harvest tubing 122 are connected to the input tubing means 104. The input tubing means 104, in turn, is connected to the sterile weldable tubing 124 which is connected to the at least one cell suspension bag 106 and the at least one sterile medium bag 108. The fluid and air movement through the input tubing means 104 is based on both gravity and pressure. In the preferred embodiment, the at least one cell suspension bag 106 and the at least one sterile diluent bag 108 are hanging above the at least one sterile, functionally closed container 102. The gravity-based movement of the fluid can be supplemented or reduced by the addition of positive or negative air pressure to the pump connection tubing 156 from at least one pump (not shown) through the at least one pump connection 134. The at least one pump (not shown) may be an air compressor/vacuum pump that can be attached to the at least one pump connection 134 for applying pressure/vacuum to the fluid in the sterile, functionally closed container 102. Thus, the positive and negative air pressure along with the gravitational force provide the movement of the fluid through the input tubing means 104 to the at least one sterile, functionally closed container 102. The second air filter 158 is a 0.2-micron filter that is closed with the detachable filter cap 164 and configured to provide pressure/vacuum to the fluid contained in the at least one sterile, functionally closed container 102. The output tubing means 112 is connected between the at least one sterile, functionally closed container 102 and the at least one waste container 110 to form a closed system that is capable of fluid communication between components, while remaining isolated from the outside environment. Specifically, the output tubing means 112 is configured to transfer the at least one functionally isolated compartment to the at least one waste container 110.

[00055] FIG. 6 illustrates the at least one sterile, functionally closed container

102 in operation wherein the harvested cell suspension is being loaded into the main functionally isolated compartment and the wash medium is being loaded in the second functionally isolated harvest compartment in accordance with the preferred embodiment of the present invention. As a first step in the method, the at least one sterile, functionally closed container 102 is placed on the at least one upper platform 128, as shown in FIGS. 1 and 6, such that the at least one sterile, functionally closed container 102 is locked on the at least one upper platform 128 utilizing the at least one locking means 150. Preferably, an operator squeezes the at least one locking means 150 on the upper platform 128 so as to place the at least one sterile, functionally closed container 102 onto the at least one upper platform 128. After placing the at least one sterile, functionally closed container 102 onto the at least one upper platform 128, the operator releases the at least one locking means 150 and ensures the at least one sterile, functionally closed container 102 is locked in place. Then, the input tubing means 104 is connected between the at least one cell suspension bag 106, the at least one sterile wash medium bag 108 and the at least one sterile, functionally closed container 102. In detail, the input tubing means is connected to the at least one middle tubing 120 and the at least one harvest tubing 122 of the at least one sterile, functionally closed container 102. The input tubing means 104, in turn, is connected to the sterile weldable tubing 124 from the at least one cell suspension bag 106 and the at least one sterile medium bag 108. After that, the pump connection tubing 156 is connected to the second air filter 158 on the at least one sterile, functionally closed container 102. Then, the harvested cell suspension is loaded into the main rigid compartment of the at least one sterile, functionally closed container 102. The medium is loaded into the second rigid harvest compartment of the at least one sterile, functionally closed container 102 when the luer connection filter 116 attached to the sterile weldable tubing 124 is opened. In order to accelerate the fluid through the input tubing means 104 and into the at least one sterile, functionally closed container 102, the pressure/vacuum is applied to the at least one sterile, functionally closed container 102 to assist the flow. The at least one pump connection 134 is configured to provide the positive and negative air pressure via the pump connection tubing 156 to the fluid from the at least one pump (not shown). In an exemplary embodiment, this is done through a 5 -minute automated process wherein the first 10 mL of medium are transferred into the second rigid harvest compartment in both of the sterile, functionally closed containers 102 to await the concentrated and harvested cells, which may be of sufficient concentration to constitute a pellet. A second equal volume of the harvested cell suspension is transferred into the main rigid compartments of the sterile, functionally closed containers 102 in which the cell concentration takes place. In one embodiment, the harvested cell suspension is mesenchymal cells suspended in trypsin.

[00056] In another embodiment, a Y connection tubing is provided for the transfer of fluid from the at least one cell suspension bag 106 and the at least one sterile diluent bag 108 to the at least one sterile, functionally closed container 102. Here, the Y connection tubing, coupled with sensors, such as load cells, flow regulators and other suitable sensors, allows substantially 50% of the harvested cell suspension or wash medium to transfer to one functionally closed container 102a while the other 50% of the solution goes to the other functionally closed container 102b, thereby keeping the apparatus 100 balanced so that taring will not be required prior to the centrifugation.

[00057] Once the contents of the at least one cell suspension bag 106 have been substantially equally divided into each of two sterile, functionally closed containers 102, the sterile weldable tubing 124, connecting the at least one cell suspension bag 106 and the input tubing means 104 which connects to the two sterile, functionally closed containers 102, is sealed and separated. After the wash medium is transferred to the two sterile, functionally closed containers 102, the sterile weldable tubing 124 of the at least one sterile diluent bag 108 is removed from the luer connection filter 116. Preferably, the remaining input tubing means 104 is left in place so the apparatus 100 remains sterile.

[00058] As shown in FIGS. 7 and 8, the at least one sterile, functionally closed container 102 along with the input tubing means 104 are transferred to the centrifuging means 166 for the first concentration of the cells. The contents in the at least one sterile, functionally closed container 102 are centrifuged, and sedimented cells are directed into the at least one functionally isolated compartment of the at least one sterile, functionally closed container 102 based on the output of the at least one detector. The at least one detector includes any detecting means, including but not limited to optical absorbance detectors, optical scattering detectors, optical reflectance detectors, optical imaging detectors, fluorescence detectors, luminescence detectors, ultrasound detectors, conductance detectors, resistance detectors, impedance detectors, acoustic detectors, density detectors, chemical detectors, gas detectors, radiation detectors, temperature detectors, density detectors, and/or viscosity detectors. The centrifugation occurs for an appropriate amount of time and the appropriate gravitational force (G force) level (greater than 1) to cause the sedimenting of cells within the two sterile, functionally closed containers 102. The full details of the operation of the containers 102 are described in U.S. Patent 8,747,289 B2, as attached to this document in Appendix A in its published format. The at least one detector is configured to optically track the migration of the cells for each individual blood sample and to then deplete certain cell types by diverting them into the first and second compartments of the at least one sterile, functionally closed container 102 during centrifugation. As described in that document and based on the outputs of the one or more detectors within each sterile, functionally closed container 102a and 102b, the sedimenting cells are directed by the automated means into one or more of the functionally isolated compartments during centrifugation, the results of which are shown in FIG. 9, wherein approximately 2xl0 ⁸ concentrated mesenchymal stem cells (-0.4 mL) harvested are shown in the second harvest compartment of the at least one sterile, functionally closed container 102.

[00059] In one embodiment, before placing the at least one sterile, functionally closed container 102 in a centrifuge cup, the at least one sterile, functionally closed container 102 is connected with a control module. In operation, the at least one sterile, functionally closed container 102 and the control module are releasably locked together. The control module may include optical and gravitational sensing means for controlling the activity in the at least one sterile, functionally closed container 102.

[00060] FIG. 10 illustrates a front perspective view of the apparatus 100 wherein the apparatus 100 is configured to prepare a second concentration of cells. In this step, the sedimented cells are unloaded, substantially free of supernatant, from their respective functionally isolated compartments. This is accomplished by employing one or more functionally closed, sterile devices to remove the cells of interest from the functionally isolated compartments of the at least one sterile, functionally closed container 102. The functionally closed, sterile devices may be integral to or separate from the at least one sterile, functionally closed container 102 and preferably utilize positive pressure along with gravity to move the liquid. In one embodiment, the at least one pump (not shown) is utilized for providing the positive pressure. The pressure and gravitational force may also be utilized to remove the at least one cell dissociation reagent or at least one waste medium in which the growth factors have been consumed and the exudate from the expanded cells, from the main rigid compartments of each of the sterile, functionally closed containers 102a, 102b to the at least one waste container 110. In one embodiment the sterile, functionally closed container 102 is rotated to one side at least 30 degrees and at most approximately 90 degrees in order to assist the removal of the at least one cell dissociation reagent or at least one waste medium in which the growth factors have been consumed and the exudate from the expanded cells. This is accomplished by the programmable rotary motion of the at least one upper platform 128. In another embodiment, a waste tube instead of the at least one waste container 110 provides a liquid connection for the at least one cell dissociation reagent to be utilized elsewhere or stored.

[00061] The cells of interest are then re-suspended in a fresh medium, which may contain growth factors, at an appropriate cell density as shown in FIG. 10. In this step, the harvested cells initially remain in 10 mL of medium while the at least one cell dissociation reagent or at least one waste medium in which the growth factors have been consumed and the exudate from the expanded cells (now substantially void of cells) is transferred from the main rigid compartments of each of the sterile, functionally closed containers 102a, 102b to the at least one waste container 110. Then, the new wash medium, which may contain growth factors, (approximately 240 mL or less) is introduced into the main rigid compartment of each sterile, functionally closed container 102a and 102b. Next, the 1 mL of concentrated cells (suspended in lOmL of new medium) is transferred from the second harvest compartment to the main compartment of one container 102b to another container 102a. Preferably, this is accomplished via the positive or negative pressure from the at least one pump or other pressure source (not shown). Then, 10 mL of new wash medium is transferred to the harvest compartments of each sterile, functionally closed container 102a and 102b where it awaits the introduction of the 1 mL of concentrated cells. As before, the harvested cell suspension and wash medium are transferred between the functional isolated compartments of each sterile, functionally closed container 102a and 102b by the programmable administration of pressure/vacuum and the opening and closing of the plurality of pinch valves.

[00062] The sterile, functionally closed containers 102a and 102b are then placed back in the centrifuging means 166 again for an appropriate amount of time and at an appropriate G force level (greater than 1) to allow the sterile, functionally closed containers 102a and 102b to function as described in U.S. Pat. 8,747,289 B2. Although in the preferred embodiment the process described herein is repeated only twice, additional passes are contemplated, and there is no upper limit on the number of times the process can be repeated.

[00063] After the final centrifugation and as shown in FIGS. 11 and 12, the at least one harvest tubing that is pressed into tubing holders positioned on the containers 102a, 102b (FIG. 11) is released (FIG. 12) for sterile connection to a syringe or to other equipment for further processing.

[00064] In one embodiment, the apparatus 100 comprises a computer that is embedded in or connected to the automated workstation 114 for carrying out the methods described herein and includes a processor which executes program instructions stored in a memory and a data input element for inputting program instructions for controlling the flow of the fluid to the at least one sterile, functionally closed container. The program instructions are stored on an electronic apparatus-readable medium for implementing the steps of the methods described herein.

[00065] FIG. 13 illustrates a plot of a time along the X-axis versus gravitational force along the Y-axis. This plot depicts a processing report 168 for one exemplary round of centrifugation. Here, after a brief ramping up period, centrifugation is shown at 1000 Gs for nearly 25 minutes, followed by a reduction in Gs and a first harvest, followed by another increase in Gs, a second reduction in Gs and a second harvest. Additional details regarding this process are described in U.S. Pat. 8,747,289. FIG. 14 illustrates a summary of results for washing and concentrating Mesenchymal Stem Cells (MSCs) derived from the TerumoBCT QuantumR Cell Expansion System, showing a graph 169 of MSC recovery following concentration. FIG. 15 illustrates the summary of results for washing and concentrating Mesenchymal Stem Cells (MSCs) derived from the TerumoBCT QuantumR Cell Expansion System, showing a graph 170 of MSC viability following concentration and washing. The result, after centrifugation and resupp lying the 1 mL of remaining cells with approximately 250 mL of new diluent, is the amount of the at least one cell dissociation reagent the cells are exposed to has been reduced by approximately 250: 1. After a second passage through the apparatus 100, according to the preferred method, the ratio is 62,500:1. [00066] In certain embodiments, the cells types used for processing may include without limitation human cells, mammalian cells, vertebrate cells, invertebrate cells, primate cells, animal cells, plant cells, eukaryotic cells, prokaryotic cells, protozoan cells, bacterial cells, viruses, prions, live cells, dead cells, dying cells, diseased cells, healthy cells, differentiated cells, undifferentiated cells, stem cells, progenitor cells, blood cells, immune system cells, cardiomyocytes, neurons, hepatocytes, pancreatic cells, photoreceptor cells, muscle cells, kidney cells, endothelial cells, epithelial cells, fibroblasts, cell lines, and/or bone marrow cells.

[00067] In certain embodiments, the at least one functionally closed, sterile devices may include without limitation one or more tubings, valves, bags, bottles, syringes, pressure pumps, vacuum pumps, siphons, propellers, impellers, and/or pistons.

[00068] In certain embodiments, the efficiency of collection of viable cells is greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, greater than 98%, or greater than 99%.

[00069] In certain embodiments, the total cell processing time employing the container of the method is less than one hour, less than 45 minutes, less than 30 minutes, less than 25 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes.

[00070] In certain embodiments, the volume capacity of the container is greater than 100 mL, greater than 200 mL, greater than 300 mL, greater than 400 mL, greater than 500 mL, greater than 600 mL, greater than 700 mL, greater than 800 mL, greater than 900 mL, or greater than 1 L.

[00071] In certain embodiments, a kit is provided comprising one or more containers, one or more devices, and/or one or more buffers.

[00072] In certain embodiments, the at least one sterile, functionally closed container is single-use. In certain other embodiments, the at least one sterile, functionally closed container is reusable.

[00073] In certain embodiments, the at least one sterile, functionally closed container is cGMP-compliant and suitable for preparing cells for clinical use. In certain other embodiments, the container is not cGMP-compliant and not suitable for preparing cells for clinical use.

[00074] In certain embodiments, the input tubing means is bifurcated tubing and provides to load two containers simultaneously with cell suspension, such that the two containers receive substantially equal volumes of cell suspension and therefore do not require taring prior to centrifugation.

[00075] Furthermore, and as described above, the apparatus and method provide a means of sterile passage of the cell suspension and wash medium from the beginning of the process to the completion in one or more sealable storage containers.

[00076] The foregoing description of the preferred embodiment of the present invention has been presented for the purpose of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teachings. For example, all the processes described in the method as performed by the operator or technician may be instead automated and/or otherwise performed automatically by the apparatus itself or a robotic assistant. It is intended that the scope of the present invention not be limited by this detailed description but by the claims and the equivalents to the claims appended hereto.