Abstract:
Embodiments described herein generally relate to systems and methods for promoting the expansion of high density non-adherent cells through the use of a cell growth chamber, a mass transfer device, and a fluid circulation loop. Improved cell growth is achieved in the cell growth chamber by using a chamber having a particular orientation and shape, e.g., conical, to create a media-rich reservoir for growing cells. By placing the chamber in a vertical position, the force of media flow along the chamber walls is substantially equal and opposite to the gravitational force on the cells. The interaction of these forces maintains the non-adherent cells in suspension. The use of the cell growth chamber in conjunction with the mass transfer device and fluid circulation loop(s) creates efficiencies by relying on the cumulative and combined features of the devices.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.S. Provisional Application Ser. No. 61/487,086, filed on May 17, 2011, and entitled, “A System for Expanding High Density Non-Adherent Cells.” The disclosure of the above-identified application is hereby incorporated by reference in its entirety as if set forth herein in full for all that it teaches and for all purposes. 
    
    
     BACKGROUND 
     Cell expansion systems can be used to grow stem cells, as well as other types of cells, both adherent and non-adherent. Adherent cells require a surface for the cells to attach to before they will grow and divide. Non-adherent cells will grow and divide while floating in suspension. 
     Cell expansion systems provide nutrients to the growing cells and remove metabolites, as well as furnish a physiochemical environment conducive to cell growth. Cell expansion systems are known in the art. 
     As a component of a cell expansion system, a bioreactor, or cell growth chamber, plays an important role in providing an optimized environment for the expanding cells. There are many types of bioreactors known in the art. Bioreactor devices include culture flasks, roller bottles, shaker flasks, stirred-tank reactors, air-lift reactors, and hollow fiber bioreactors. 
     SUMMARY 
     Embodiments of the present disclosure generally relate to providing an environment conducive to high density non-adherent cell growth. Numerous factors may influence cell growth, including, for example, temperature, the geometries of the cells, etc. In particular, non-adherent cells expand based at least in part on the amount, or volume, of cell growth media available to them, in which increasing volumes of media promote increased cell growth. Cell density may affect not only the ability of cells to grow, but also the cell characteristics themselves. Therefore, if large amounts, or numbers, of non-adherent cells are desired, a large amount, or volume, of fluid should generally be available. 
     Aspects of particular embodiments provide for the expansion of high density non-adherent cells through the combined use of a cell growth chamber, a mass transfer device, or bioreactor, and a fluid circulation loop(s). Cell growth in the cell growth chamber is particularly promoted by using a cell growth chamber having a specialized shape and/or particular orientation that relies on the use of gravity to create a media-rich reservoir for cells to grow in. By vertically positioning the cell growth chamber, gravitational forces cause cells to accumulate in the lower portion of the cell growth chamber, in which such lower portion includes a media-rich reservoir for cell expansion. 
     According to embodiments of the present disclosure, the use of a cell growth chamber in conjunction with a mass transfer device and a fluid circulation loop creates efficiencies in cell expansion by relying on the cumulative and combined features of the devices. For example, in an embodiment, a majority of cells in the cell growth chamber are caused to settle to the bottom, or lower portion, of the cell growth chamber by gravitational forces. While some cells may exit the top portion of the cell growth chamber with the circulating media, most cells will settle into the media-rich reservoir of the lower portion of the cell growth chamber where cells can thrive and grow. Meanwhile, oxygen- and nutrient-depleted media, with some cells according to embodiments, exits the cell growth chamber. This circulating media enters the intracapillary space of the mass transfer device while fresh media, oxygenated by an oxygenator, enters the extracapillary space of the mass transfer device. The circulating media is replenished by nutrients and oxygen diffusing through the extracapillary space into the intracapillary space. Waste in the circulating media may also be diffused from the media into the extracapillary space. The replenished and cleaned media then flows through the outlet port of the mass transfer device to travel to the cell growth chamber to replenish its reservoir of media. Cells in the cell growth chamber are thus able to receive the nutrients they need for increasing expansion while remaining in the media-rich lower portion of the cell growth chamber. 
     Additional efficiencies are created in embodiments which rely on the cumulative and combined features of the devices. For example, the different characteristics and features of the cell growth chamber, mass transfer device, and fluid circulation loop allow for different cell types and/or sizes to flourish in environments conducive to handling their particular cell properties. In an embodiment, cells of a large diameter or weight, for example, tend to settle into the lower portion of the cell growth chamber at greater rates and at greater volumes than cells of a smaller diameter or weight. 
     The disclosure relates to a closed cell expansion system including a cell growth chamber, in which the cell growth chamber comprises two frustoconical shaped sections. The system also comprises a mass transfer device and a first fluid circulation loop fluidly associated with the cell growth chamber and the mass transfer device, in which non-adherent cells expand in at least two of the cell growth chamber, the mass transfer device, and the first fluid circulation loop. The non-adherent cells expand in a media that travels through the cell growth chamber, the mass transfer device, and the first fluid circulation loop. 
     In at least one embodiment, the two frustoconical shaped sections are joined at a maximum cross-sectional area. In at least one embodiment, the two frustoconical shaped sections taper in opposite directions toward an inlet and an outlet, in which the inlet and the outlet are disposed on opposite ends of the cell growth chamber. 
     In at least one embodiment, the inlet is positioned at a bottom portion of the cell growth chamber, and the cell growth chamber is oriented such that a direction of gravitational force is substantially from the outlet to the inlet. In at least one embodiment, a force of media flow from the inlet into the cell growth chamber is substantially equal to the gravitational force, in which the interaction of the force of the media flow and the gravitational force maintains the non-adherent cells in suspension in the cell growth chamber. In at least one embodiment, the cell growth chamber is formed from a unitary form. In at least one embodiment, the cell growth chamber is formed from a biocompatible polymeric material. In at least one embodiment, a semi-permeable material positioned substantially at the outlet of the cell growth chamber at least partially blocks the non-adherent cells from exiting the outlet. 
     In at least one embodiment, the mass transfer device comprises a housing having an intracapillary portion and an extracapillary portion. In at least one embodiment, the intracapillary portion is fluidly associated with the first fluid circulation loop. In at least one embodiment, the mass transfer device comprises an intracapillary inlet fluidly associated with an in-flow of the first circulation loop. 
     In at least one embodiment, the mass transfer device comprises a first end cap disposed at a first end of the housing and a second end cap disposed at a second end of the housing. In at least one embodiment, the mass transfer device comprises a plurality of hollow fibers potted to the first end cap and the second end cap. In at least one embodiment, the media flows through the plurality of the hollow fibers. In at least one embodiment, the plurality of the hollow fibers forms a membrane. In at least one embodiment, the plurality of the hollow fibers comprises a plurality of pores that allow small molecules to diffuse between the intracapillary portion and the extracapillary portion, in which the non-adherent cells are not small molecules. In at least one embodiment, the plurality of the hollow fibers is made from a biocompatible polymeric material. In at least one embodiment, the membrane allows for removal of metabolites from the media and replacement of nutrients in the media, in which the nutrients promote cell growth. 
     In at least one embodiment, the cell expansion system further comprises a second fluid circulation loop fluidly associated with the extracapillary portion of the mass transfer device, in which the second fluid circulation loop includes a second media that travels through the second fluid circulation loop. In at least one embodiment, the second fluid circulation loop comprises an oxygenator that adds at least a first gas to the second media. In at least one embodiment, a second gas purged from the system vents to the atmosphere via an exit port of the oxygenator. In at least one embodiment, a nutrient is introduced to the extracapillary portion of the mass transfer device through the second fluid circulation loop. In at least one embodiment, a first flow direction of the first fluid circulation loop and a second flow direction of the second fluid circulation loop are co-current. 
     The disclosure further relates to a closed cell expansion system, in which the system comprises a cell growth chamber having a first volume, wherein a first number of cells is grown in the first volume, and the cell growth chamber comprises two frustoconical shaped sections. The system also includes a mass transfer device comprising an intracapillary portion and an extracapillary portion, in which the intracapillary portion has a second volume, and wherein a second number of cells is grown in the second volume. The system also comprises a first fluid circulation loop fluidly associated with the cell growth chamber and the mass transfer device, in which cells expand in the cell growth chamber, the mass transfer device, and the first fluid circulation loop, and wherein the cells expand in a media that travels through the cell growth chamber, the mass transfer device, and the first fluid circulation loop, in which the first number of cells is greater than the second number of cells. 
     In at least one embodiment, the first and second volumes are different. In at least one embodiment, the cell growth chamber provides a reservoir of the media to promote high density cell growth. In at least one embodiment, the two frustoconical shaped sections are joined at a maximum cross-sectional area. In at least one embodiment, the two frustoconical shaped sections taper in opposite directions toward an inlet and an outlet, in which the inlet and the outlet are disposed on opposite ends of the cell growth chamber. In at least one embodiment, the inlet is positioned at a bottom portion of the cell growth chamber, in which the cell growth chamber is oriented such that a direction of gravitational force is substantially from the outlet to the inlet. In at least one embodiment, a force of media flow from the inlet into the cell growth chamber is substantially equal to the gravitational force, in which interaction of the force of the media flow and the gravitational force maintains the non-adherent cells in suspension in the cell growth chamber. 
     In at least one embodiment, the cell expansion system further comprises a second fluid circulation loop fluidly associated with the extracapillary portion of the mass transfer device, in which the second fluid circulation loop includes a second media that travels through the second fluid circulation loop. In at least one embodiment, the second fluid circulation loop comprises an oxygenator that adds at least one gas to the second media. 
     The disclosure further relates to a method of growing cells in a closed cell expansion system. The method includes the steps of providing a first volume of media in a cell growth chamber; growing a first number of cells in the first volume; fluidly associating the cell growth chamber with a mass transfer device and with a first fluid circulation loop; providing a second volume of media in an intracapillary portion of the mass transfer device; growing a second number of cells in the second volume; providing a third volume of media in the first fluid circulation loop; and growing a third number of cells in the third volume, in which the first, second, and third number of cells are different. 
     In at least one embodiment, the media flows through the cell growth chamber, the mass transfer device, and the first fluid circulation loop. In at least one embodiment, the method further comprises orienting the cell growth chamber such that a flow of the media is equal to and opposite in direction to a gravitational force on the cells in the cell growth chamber. In at least one embodiment, the media comprises one or more from the group consisting of: a fluid, a gas, a nutrient, a metabolite, an ion, and a lactate. 
     The disclosure further relates to a closed cell expansion system including a cell growth chamber, in which the cell growth chamber comprises an inlet and an outlet disposed on opposite ends of the cell growth chamber, the inlet being positioned at a bottom portion of the cell growth chamber, and the cell growth chamber being oriented such that a direction of gravitational force is substantially from the outlet to the inlet. The system further comprises a mass transfer device and a first fluid circulation loop fluidly associated with the cell growth chamber and the mass transfer device, in which non-adherent cells expand in at least two of the cell growth chamber, the mass transfer device, and the first fluid circulation loop, and wherein the non-adherent cells expand in a media that travels through the cell growth chamber, the mass transfer device, and the first fluid circulation loop. 
     In at least one embodiment, a force of media flow from the inlet into the cell growth chamber is substantially equal to the gravitational force, in which interaction of the force of the media flow and the gravitational force maintains the non-adherent cells in suspension in the cell growth chamber. In at least one embodiment, the cell growth chamber is formed from a biocompatible polymeric material. In at least one embodiment, a semi-permeable material positioned substantially at the outlet of the cell growth chamber partially blocks the non-adherent cells from exiting the outlet. In at least one embodiment, the mass transfer device comprises a housing having an intracapillary portion and an extracapillary portion. In at least one embodiment, the intracapillary portion is fluidly associated with the first circulation loop. 
     This Summary is included to provide a selection of concepts in a simplified form, in which such concepts are further described below in the Detailed Description. This Summary is not intended to be used in any way to limit the claimed subject matter&#39;s scope. Features, including equivalents and variations thereof, may be included in addition to those provided herein. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Embodiments of the present disclosure may be described by referencing the accompanying figures. In the figures, like numerals refer to like items. Further, optional steps or components are illustrated in a dashed-line format. 
         FIG. 1  is a schematic illustration of the mass transfer device in accordance with embodiments of the present disclosure. 
         FIG. 2  is a schematic illustration of the cell growth chamber in accordance with embodiments of the present disclosure. 
         FIG. 3  is a schematic illustration of a high density cell expansion system in accordance with embodiments of the present disclosure. 
         FIG. 4  depicts a flow diagram showing the operational characteristics of a process for growing cells in a cell expansion system in accordance with embodiments of the present disclosure. 
     
    
    
     DETAILED DESCRIPTION 
     The following Detailed Description provides a discussion of illustrative embodiments with reference to the accompanying drawings. The inclusion of specific embodiments herein should not be construed as limiting or restricting the present disclosure. Further, while language specific to features, acts, and/or structures, for example, may be used in describing embodiments herein, the claims are not limited to the features, acts, and/or structures described. A person of skill in the art will understand other embodiments, including improvements, that are within the spirit and scope of the present disclosure. 
     Embodiments of the present disclosure are generally directed toward a closed system  200  (see  FIG. 3 ) for continuous high density cell expansion, in particular, a system for expanding non-adherent cells. A closed system means that the contents of the system are closed, or not directly exposed, to the atmosphere. The system contains at least a cell growth chamber  12  (also referred to herein as a “cell expansion chamber”) and a mass transfer device  100 . 
     With reference to  FIG. 1 , an example mass transfer device  100  which may be used with the present disclosure is shown in front side elevation view. Mass transfer device  100  has a longitudinal axis LA-LA and includes housing  104 . In at least one embodiment, mass transfer device housing  104  includes four openings or ports: intracapillary (IC) inlet port  108 , intracapillary (IC) outlet port  120 , extracapillary (EC) inlet port  128 , and extracapillary (EC) outlet port  132 . 
     A plurality of hollow fibers  116  are disposed within mass transfer device housing  104 . The material used to make the hollow fibers  116  may be any biocompatible polymeric material which is capable of being made into hollow fibers. The terms “hollow fiber,” “hollow fiber capillary,” and “capillary” are used interchangeably. A plurality of hollow fibers are collectively referred to as a “membrane.” 
     In embodiments, the ends of hollow fibers  116  are potted to the ends of the mass transfer device  100  by a connective material (also referred to herein as “potting” or “potting material”). The potting can be any suitable material for binding the hollow fibers  116 , provided that the flow or travel of culture media (and cells if desired) into the hollow fibers is not obstructed. Exemplary potting materials include, but are not limited to, polyurethane or other suitable binding or adhesive components. End caps  112  and  124  respectively, are disposed at each end of the mass transfer device. According to embodiments, the media includes one or more of the following, for example: a fluid, a gas, a nutrient, a metabolite, an ion, a lactate, and/or an oxygen atom, for example. 
     Small molecules (e.g., ions, water, oxygen, a metabolite, lactate, etc.) can diffuse through pores in the hollow fibers from the interior or IC space of the hollow fiber to the exterior or EC space, or from the EC space to the IC space, according to embodiments. 
     Use of a mass transfer device  100 , such as the one described, allows for the simultaneous and continuous removal of waste products from the cell growth media and the replacement of nutrients in the cell growth media throughout the cell expansion process. 
     In embodiments, the system  200  (see  FIG. 3 ) also includes a cell growth or cell expansion chamber  12 . As shown in  FIG. 2 , a schematic of a possible embodiment of a cell growth chamber which may be used with the present disclosure is depicted. In an embodiment, cell growth chamber  12  includes two frustoconical shaped sections  25 ,  27  joined together at a maximum cross-sectional area  23  of the cell growth chamber  12 . The interior of the cell growth chamber  12  tapers (decreases in cross-section) from the maximum cross-sectional area  23  in opposite directions toward inlet  30  and outlet  32 . According to an embodiment, inlet  30  is positioned at the bottom, or a bottom portion, of the cell growth chamber. 
     The cell growth chamber  12  may be constructed from a unitary piece of plastic or from separate pieces joined together using a fixative or other sealing methods. It may be made of any biocompatible material capable of being assembled into the frustoconical shape, according to an embodiment. 
     The conical shape of the cell growth chamber  12  helps to keep the cells suspended within the chamber  12 . The flow of media, or force of media flow, along the walls of the cell growth chamber  12  from the inlet  30  of the chamber  12  through the interior to the outlet  32  is substantially equal and opposite to the gravitational pull, or gravitational force, on the cells. In an embodiment, the flow of media along the walls of the cell growth chamber  12  from the inlet  30  of the chamber  12  through the interior to the outlet  32  is constant. The interaction of the force of the media flow and the gravitational force helps to keep the cells suspended, i.e., maintains the non-adherent cells in suspension, within the cell growth chamber  12 . 
     The cells may be retained within the cell growth chamber  12  by blocking at least the outlet port  32  of the cell growth chamber  12  with some type of semi-permeable material which allows fluid to flow there through, yet retains cells within the chamber  12 . 
     For non-adherent cells, the rate limiting step for cell expansion is the amount, or volume, of cell growth media available to the cells, according to embodiments. Therefore, the cell expansion chamber  12  acts not only as a place for the cells to grow, but also as a media reservoir to encourage high density cell growth by providing as much media to the cells as possible. In an embodiment, for example, a greater number of cells grows in a larger volume of media. To achieve maximal growth, the chamber should be made as large as possible, including with respect to corresponding volume, for example, within the constraints of system  200 . 
     In embodiments, cells may additionally be grown inside the lumen or IC space of the hollow fibers of the mass transfer device  100  and also within the associated tubing of the first fluid circulation loop  202  (see  FIG. 3 ). In such embodiments, cells are not only grown within the cell growth chamber  12 , but are also circulated throughout the first fluid circulation loop  202  in a volume of media, from the cell growth chamber  12  through the IC space of the mass transfer device  100 , and back to the cell growth chamber  12 . In embodiments, the volumes of media in the mass transfer device and in the first fluid circulation loop are different from the volume of media in the cell growth chamber. For example, the cell growth chamber may comprise a first volume of media, the mass transfer device may comprise a second volume of media, and the first fluid circulation loop may comprise a third volume of media. In an embodiment, the first, second and third volumes of media are different. In another embodiment, the first, second, and third volumes of media are the same. 
     A schematic of one possible embodiment of a cell expansion system  200  containing both the mass transfer device  100  and the cell growth chamber  12  as described above is shown in  FIG. 3 . 
     First fluid flow path  206  is fluidly associated with mass transfer device  100  and cell growth chamber  12  to form first fluid circulation path  202  (also referred to herein as the “intracapillary loop” or “IC loop” or “first fluid circulation loop”). In an embodiment, a single mass transfer device  100  and cell growth chamber  12  are used. In another embodiment, multiple mass transfer devices and multiple cell growth chambers are used. Fluid flows or travels into mass transfer device  100  through inlet port  108 , and exits via outlet port  120 . The fluid path between the inlet port  108  and the outlet port  120  defines the intracapillary portion  126  of the mass transfer device (see  FIG. 1 ). The intracapillary inlet  108  is fluidly associated with an in-flow of the first fluid circulation loop  202 . Fluid flows into cell growth chamber  12  through inlet port  30  and exits via outlet port  32 . It should be noted that the cell growth chamber may be located anywhere within the IC loop. Pressure gauge  210  measures the pressure of media leaving mass transfer device  100  and entering cell growth chamber  12 . IC circulation pump  212  controls the rate of media flow through first fluid circulation loop  202 . Media entering the IC loop may enter through valve  214 . As those skilled in the art will appreciate, additional valves and/or other devices can be placed at various locations to isolate and/or measure characteristics of the media along portions of the fluid paths. Accordingly, it is to be understood that the schematic shown represents one possible configuration for various elements of the cell expansion system, and modifications to the schematic shown are within the scope of embodiments of the present disclosure. 
     Samples of media can be obtained from a sample port  216  or a sample coil  218  during operation. Pressure/temperature gauge  220  allows measurement of media pressure and temperature during operation. 
     Cells grown/expanded in cell growth chamber  12  or in the entire IC loop  202  including mass transfer device  100  can be flushed out of the IC loop  202  into harvest bag  299  through valve  298 . 
     Fluid in second fluid circulation path  204  (also referred to herein as the “extracapillary loop” or “EC loop” or “second fluid circulation loop”) enters mass transfer device  100  via EC inlet port  128 , and leaves mass transfer device  100  via EC outlet port  132 . The fluid path between the EC inlet port  128  and the EC outlet port  132  defines the EC portion  136  of the mass transfer device  100  (see  FIG. 1 ). 
     Pressure/temperature gauge  224  measures the pressure and temperature of the media before the media enters the EC space of the mass transfer device  100 . Pressure gauge  226  measures the pressure of media after it leaves the mass transfer device  100 . Samples of media can be obtained from sample port  230  or a sample coil (not shown) during operation, according to embodiments. 
     After leaving EC outlet port  132  of mass transfer device  100 , fluid in second fluid circulation path  204  passes through EC circulation pump  228  to oxygenator  232 . Media flows into oxygenator  232  via inlet port  234 , and exits oxygenator  232  via outlet port  236 . Oxygenator  232  adds oxygen and other gases, as desired, to the media. The oxygenator  232  can be any appropriately sized oxygenator known in the art. Gas flows into oxygenator  232  via inlet port  238  and out of oxygenator  232  through outlet port  240 . Filters (not shown) may be associated with ports  238  and  240  respectively to reduce or prevent contamination of oxygenator  232  and associated media. Air or gas purged from the system  200  can vent to the atmosphere via exit port  240  of oxygenator  232 . 
     In the configuration depicted in  FIG. 3 , fluid media in first fluid circulation path  202  and second fluid circulation path  204  flows through mass transfer device  100  in the same direction (a co-current configuration). However, cell expansion system  200  can also be configured to flow fluid in an opposite or counter-current direction. 
     In an embodiment, cells (from bag  262 ) to be expanded and IC media from bag  246  are introduced to first fluid circulation path  202  via a valve(s). In an embodiment, valve  264  and/or valve  250  may be used, respectively, for example. Fluid containers  244  (reagent) and  246  (IC media) may be fluidly associated with either first fluid inlet path  242  via valves  248  and  250 , respectively, or second fluid inlet path  274  via valves  270  and  276 . First and second sterile sealable input priming paths  208  and  209  are provided. In embodiments, air removal chamber  256  is fluidly associated with first fluid circulation path  202 . 
     According to embodiments of the present disclosure, EC media (from bag  268 ) or wash solution ((if used) from bag  266 ) may be added to either the first or second fluid flow path. Fluid container  266  may be fluidly associated with valve  270  that is fluidly associated with first fluid circulation path  202  via distribution valve  272  and first fluid inlet path  242 . Alternatively, fluid container  266  can be fluidly associated with second fluid circulation path  204  via second fluid inlet path  274  and second fluid flow path  284  by opening valve  270  and closing distribution valve  272 . Likewise, fluid container  268  is fluidly associated with valve  276  that may be fluidly associated with first fluid circulation path  202  via first fluid inlet path  242  and distribution valve  272 . Alternatively, fluid container  268  may be fluidly associated with second fluid inlet path  274  by opening valve  276  and closing distribution valve  272 . 
     An optional heat exchanger  252  may be provided to warm media, reagent or wash solution. 
     In the IC loop  202 , fluid is initially advanced by the IC inlet pump  254 . In the EC loop  204 , fluid is initially advanced by the EC inlet pump  278 . An air detector  280 , such as an ultrasonic sensor, may also be associated with the EC inlet path  284 , according to embodiments. 
     First and second fluid circulation paths  202  and  204  are connected to waste line  288 . When valve  290  is opened, IC media can flow through waste line  288  to waste bag  286 . Likewise, when valve  292  is opened, EC media can flow through waste line  288  to waste bag  286 . 
     In accordance with embodiments of the present disclosure, expanded cells are harvested via cell harvest path  296 . Here, cells from cell expansion chamber  12  and, optionally, mass transfer device  100  and associated tubing can be harvested from the IC loop by pumping the IC media containing the cells through cell harvest path  296  and valve  298  to cell harvest bag  299 . 
     In embodiments, various components of the cell expansion system  200  are contained or housed within an incubator  300 , wherein the incubator maintains cells and media at a desirable temperature. The size of cell growth chamber  12 , and the volume of media it may contain, for example, is dependent upon the size of the incubator, according to embodiments. However, in other embodiments, the cell expansion system  200  may be placed in a larger temperature controlled space such as a warm room, in which case the size of the cell growth chamber  12  is not necessarily limited and may have a range of possible dimensions. 
     As consistent with  FIGS. 1 ,  2 , and  3  described above,  FIG. 4  provides example operational steps  302  for growing cells in a cell expansion system, in accordance with embodiments of the present disclosure. START operation  304  is initiated, and process  302  proceeds to provide  306  a first volume of media in a cell growth chamber. In an embodiment, such first volume is controlled by the size of the cell growth chamber. In another embodiment, one or more pumps and/or one or more valves, as described above, may control the amount of the first volume of media. A first number of cells is then grown  308  in the first volume. Next, process  302  proceeds to fluidly associate  310 ,  312  the cell growth chamber with a mass transfer device and a first fluid circulation loop, as described above according to embodiments. 
     Proceeding to operation  314 , a second volume of media is provided in the mass transfer device, according to embodiments described above. In an embodiment, such second volume is controlled by the size of the mass transfer device. In another embodiment, one or more pumps and/or one or more valves, as described above, may control the amount of the second volume of media. In an embodiment, such second volume of media is in an intracapillary portion of the mass transfer device, as shown by optional step  316 . Next, process  302  proceeds to operation  318 , in which a second number of cells is grown in the second volume. 
     In an embodiment, a third volume of media is provided  320  in the first fluid circulation loop, as described above in accordance with embodiments of the present disclosure. In an embodiment, such third volume is controlled by the size of the first circulation loop. In another embodiment, one or more pumps and/or one or more valves, as described above, may control the amount of the third volume of media. Next, a third number of cells is grown in the third volume of media  322 . In an embodiment, process  302  then terminates at END operation  324 . 
     With respect to the process illustrated in  FIG. 4 , the operational steps depicted are offered for purposes of illustration and may be rearranged, combined into other steps, used in parallel with other steps, etc., according to embodiments of the present disclosure. Further, fewer or additional steps may be used in embodiments without departing from the spirit and scope of the present disclosure. 
     It will be apparent to those skilled in the art that various modifications may be made to the apparatus, systems, and methods described herein. Thus, it should be understood that the embodiments are not limited to the subject matter discussed in the Specification. Rather, the present disclosure is intended to cover modifications, variations, and/or equivalents. The acts, features, structures, and/or media are disclosed as illustrative embodiments for implementation of the claims. The invention is defined by the appended claims.