Patent Publication Number: US-2020296905-A1

Title: Methods and Systems for Culturing Microbial and Cellular See Cultures

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. application Ser. No. 14/507,548, filed Oct. 6, 2014, which is incorporated herein by specific reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. The Field of the Invention 
     The present invention relates to methods and systems for culturing microbial and cellular seed cultures using disposable container assemblies. 
     2. The Relevant Technology 
     The growth of biological cells within a bioreactor requires critical control over a number of different process parameters. For example, as cells grow, they absorb oxygen from the surrounding media and release CO 2 . The concentration of oxygen and CO 2  within the media must be carefully monitored and regulated to ensure viability and optimal growth of the cells. Another factor that needs to be carefully monitored and controlled is the density of the cells within the culture. To make sure that all of the processing parameters are properly controlled, cells are typically grown in sequential stages of increasingly larger reactors. For example, a cellular seed culture may initially start by being grown in a small glass flask which is positioned on a shaker table. Once the cell density approaches a critical value, the culture is transferred to a larger bench top reactor where the culture is combined with additional media. 
     Although glass flasks are useful in growing a seed culture, they have some shortcomings. For example, between each use it is necessary to clean and sterilize the flask. This process is time consuming, labor intensive and requires the use of chemicals. Furthermore, because of potential failures in the cleaning process, this process has has an increased risk that the cell culture may be contaminated. 
     Glass flasks also take a large area to transport and store, are prone to breaking, are cumbersome to make sterile gas and fluid connections therewith, can be difficult to remove samples from in a sterile manner, and are not easily operated with multiple sensors. 
     Accordingly, what is needed in the art are systems and methods for growing cellular seed cultures that address all or some of the above shortcomings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Various embodiments of the present invention will now be discussed with reference to the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. 
         FIG. 1  is a perspective view of a seed culturing system incorporating features of the present invention; 
         FIG. 2  is a perspective view of a retainer of the system shown in  FIG. 1 ; 
         FIG. 3  is a perspective view of an alternative embodiment of the retainer shown in  FIG. 2 ; 
         FIG. 4  is a perspective view of another alternative embodiment of a retainer that is formed without lateral sidewalls; 
         FIG. 5  is a perspective view of yet another alternative embodiment of a retainer wherein the front and rear sidewalls are disconnected from each other; 
         FIG. 6  is a perspective view of the container assembly of the system shown in  FIG. 1 ; 
         FIG. 7  is a perspective view of a container assembly disposed within the retainer of  FIG. 2  wherein opposing tabs of the container assembly are secured together by fasteners; 
         FIG. 8  is a perspective view of an alternative embodiment of the container assembly shown in  FIG. 6 ; 
         FIG. 9  is a perspective view of a container assembly shown in  FIG. 6  having tubular baffles therein; 
         FIG. 10  is a perspective view of the container assembly shown in  FIG. 6  having wall baffles therein; 
         FIG. 11  is a perspective of a baffle which can be attached is a flexible bag by a fitting; 
         FIG. 12  is a perspective view of the assembled baffle and fitting shown in  FIG. 11 ; 
         FIG. 13  is a perspective view of an alternative container assembly having film baffles; 
         FIG. 14  is a perspective view of a retainer having baffles mounted directly onto the floor of the retainer; and 
         FIG. 15  is a perspective view of the mixer table shown in  FIG. 1  having a plurality of retainers and container assemblies mounted thereon and an incubator coupled thereto. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention is directed to methods and systems for culturing microbial or cellular seed cultures. For example, the inventive methods and systems can be used in culturing bacteria, fungi, algae, plant cells, mammalian cells, animal cells, insect cells, plant cells, protozoan, nematodes, and the like from a starter culture. 
     Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particularly exemplified apparatus, systems, methods, or process parameters that may, of course, vary. It is also to be understood that the terminology used herein is only for the purpose of describing particular embodiments of the present invention, and is not intended to limit the scope of the invention in any manner. 
     All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. 
     The term “comprising” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. 
     It will be noted that, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a “container assembly” can include one, two, or more container assemblies. 
     As used in the specification and appended claims, directional terms, such as “top,” “bottom,” “left,” “right,” “up,” “down,” “upper,” “lower,” “proximal,” “distal” and the like are used herein solely to indicate relative directions and are not otherwise intended to limit the scope of the invention or claims. 
     Where possible, like numbering of elements have been used in various figures. Furthermore, multiple instances of an element and or sub-elements of a parent element may each include separate letters appended to the element number. For example two instances of a particular element “91” may be labeled as “91A” and “91B”. In that case, the element label may be used without an appended letter (e.g., “91”) to generally refer to instances of the element or any one of the elements. Element labels including an appended letter (e.g., “91A”) can be used to refer to a specific instance of the element or to distinguish or draw attention to multiple uses of the element. Furthermore, an element label with an appended letter can be used to designate an alternative design, structure, function, implementation, and/or embodiment of an element or feature without an appended letter. Likewise, an element label with an appended letter can be used to indicate a sub-element of a parent element. For instance, an element “12” can comprise sub-elements “12A” and “12B.” 
     Various aspects of the present devices and systems may be illustrated by describing components that are coupled, attached, and/or joined together. As used herein, the terms “coupled”, “attached”, and/or “joined” are used to indicate either a direct connection between two components or, where appropriate, an indirect connection to one another through intervening or intermediate components. In contrast, when a component is referred to as being “directly coupled”, “directly attached”, and/or “directly joined” to another component, there are no intervening elements present. Furthermore, as used herein, the terms “connection,” “connected,” and the like do not necessarily imply direct contact between the two or more elements. 
     Various aspects of the present devices, systems, and methods may be illustrated with reference to one or more exemplary embodiments. As used herein, the term “exemplary” means “serving as an example, instance, or illustration,” and should not necessarily be construed as preferred or advantageous over other embodiments disclosed herein. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, the preferred materials and methods are described herein. 
     In certain embodiments, the inventive systems (or portion(s) thereof) are designed so that at least some components that contact the material being processed can be disposed of after each use. As a result, some embodiments of the present invention substantially eliminate the burden of cleaning and sterilization required by conventional glass or plastic systems. This feature also ensures that sterility can be consistently maintained during repeated processing of multiple batches. In view of the foregoing, and the fact that certain embodiments can be easily scalable, relatively low cost, and easily operated, some embodiments of the present invention can be used in a variety of industrial and research facilities that previously outsourced such processing. 
     Depicted in  FIG. 1  is one embodiment of a culturing system  10  incorporating features of the present invention. In general, culturing system  10  comprises a mixer table  12 , one or more retainers  14  disposed on mixer table  12 , and a container assembly  16  at least partially disposed within each retainer  14 . Each of the elements will now be discussed below in greater detail. 
     Mixer table  12  comprises a base  18  having a platform  20  moveably mounted thereon. Platform  20  has a top surface  22  that is typically flat and disposed in a horizontal plane. Other contours, orientations and configurations can also be used. Although not required, in one embodiment a plurality of threaded holes  24  are uniformly formed over the entire area or a predefined area of top surface  22 . Holes  24  typically have a diameter in a range between about 0.2 cm to about 1 cm with about 0.2 cm to about 0.5 cm being more common. Other dimensions can also be used. As will be discussed below in greater detail, in one embodiment, holes  24  are used to secure each retainer  14  to platform  20  at a desired location. The number of holes  24  can depend on the size of platform  20  but a common platform  20  will often include more than 20 holes  24  and typically more than 100, 200 or 400 holes  24 . Other numbers can also be used. 
     Mixer table  12  has a drive motor or other mechanism that facilitates rapid displacement of platform  20  relative to base  18 . The displacement can be in a variety of different patterns but commonly results in platform  20  being moved within a single plane. For example, platform  20  can reciprocally move horizontally side-to-side, front-to-back, or combinations of the foregoing. Furthermore, platform  20  can move in a two-dimensional path such as circular path, elliptical path, or other random or repeating path. In other embodiments, platform  20  can be configured to rock or tilt back and forth or can be configured to move in a three dimensional path. For example, platform  20  can rock, tilt or be repeatedly raised and lowered while it is concurrently being moved horizontally such as discussed above. In view of the forgoing, mixer table  12  can comprise a conventional shaker table or rocker table. 
     Depicted in  FIG. 1  is one embodiment of a retainer  14 A used for securing container assembly  16  to moveable platform  20 . As depicted in  FIG. 2 , retainer  14 A comprises a floor  26  having a perimeter edge  28 . Floor  26  has a square or rectangular configuration. However, other configurations can also be used including circular, oval, and other polygonal or irregular configurations. Upstanding from floor  26 , typically at perimeter edge  28 , are a plurality of sidewalls  30 . In the depicted embodiment the plurality of sidewalls  30  include a front sidewall  30 A, a rear sidewall  30 B, and opposing lateral sidewalls  30 C and  30 D. Sidewalls  30  are depicted as upstanding from floor  26  and are also inwardly inclined towards floor  26 . Specifically, with floor  26  disposed in a horizontal position, sidewalls  30  can commonly incline back towards floor  26  at an angle in a range between about 2° to about 15° with about 3° to about 10° being more common. Other angles can also be used. The sloping of sidewalls  30  is not critical but assists in helping to retain container assembly  16  within retainer  14  and to maintain container assembly  16  in its preferred orientation. In alternative embodiments, sidewalls  30  can extend vertically or could slope outward away from floor  26 . 
     Each sidewall  30  has a top edge  32  that extends between opposing side edges  34 A and B. A slot  36  is formed between each adjacent pair of side edges  34  of adjacent sidewalls  30 . In the depicted embodiment, slots  36  extend the entire length of side edges  34  from top edge  32  to floor  26 . However, slots  36  need not extend to floor  26  and need not extend to top edge  32 . For example, sidewalls  30  could bridge slot  36  at top edge  32 , adjacent to floor  26  or at one or more locations therebetween so that slots  36  form one or more bounded openings. The function of slots  36  will be discussed below. 
     Retainer  14 A has an interior surface  38  that partially bounds a chamber  40 . That is, chamber  40  is at least partially bound by the interior surface of floor  26  and the interior surface of each of sidewalls  30 . Chamber  40  is sized to receive container assembly  16 . Extending through each sidewall  30  is a window  42  which permits visual inspection of container assembly  16  and which enables a port extending from container assembly  16  to pass therethrough. Windows are not necessary and can be eliminated. Extending through floor  26  are a plurality of spaced apart holes  44 . Holes  44  have a size and spacing comparable to holes  24  on platform  20  ( FIG. 1 ). Accordingly, to secure retainer  14  to platform  20 , floor  26  of retainer  14  is place on top surface  22  of platform  20  so that at least some of holes  44  are aligned with holes  24 . One or more fasteners  46  are then passed through select holes  44  and threaded into holes  24 , thereby securing retainer  14  to platform  20 . This mechanism for attachment makes it easy to move retainer  14 A to any desired location upon platform  20  and makes it easy to attach any number of retainers  14 A to platform  20  at any desired position and/or orientation. In alternative embodiments, however, it is appreciated that holes  24  and/or  44  could be replaced with clamps, brackets, straps, and other types of fasteners that permit retainers  14  to be removably secured to platform  20 . In other embodiments, one or more retainers  14  can be permanently secured to platform  20 , such as by welding or an adhesive. Retainers  14  disclosed herein are common made out of a metal, such as stainless steel or aluminum, but could also be made from plastic, composite, or other materials that will withstand the applied forces. 
     Depicted in  FIG. 3  is an alternative embodiment of a retainer  14 B. Like elements between retainers  14 A and  14 B are identified by like reference characters. Retainer  14 B is substantially identical to retainer  14 A except that slots  36  have been removed so that sidewalls  30 A-D combined to form a continuous sidewall  30  that encircles chamber  40 . Again in this depicted embodiment, floor  26  has four sides and four sidewalls  30 A-D upstanding therefrom. In alternative embodiments, retainers  14  can be formed with floor  26  having three, five, six, seven, eight, or more sides with a corresponding number of sidewalls upstanding therefrom. The sidewalls can be connected together or have slot(s) formed therebetween. In other embodiments, floor  26  could be circular, oval, or irregular and could thus have a complimentary circular, oval, or irregular sidewall, i.e., a single continuous sidewall, upstanding therefrom. Again, the one or more sidewalls can be vertical or slope inward. For example, a circular sidewall  30  of a retainer  14  could form a frustum of a cone, a cylinder, or other three dimensional configurations that bound chamber  40 . 
     Depicted in  FIG. 4  is another alternative embodiment of a retainer  14 C. Again, like elements between retainers  14 A and  14 C are identified by like reference characters. Retainer  14 C incudes floor  26  having front wall  30 A and opposing rear wall  30 B. However, lateral sidewalls  30 C and  30 D have been eliminated so that chamber  40  is only bounded between sidewalls  30 A and  30 B. Recessed on top edge  32  of front wall  30 A are a pair of grooves  48 A and B in which tubes from container assembly  16  can be received and secured by friction fit for desired alignment. Any desired number of grooves  48  can be formed. Upstanding from top edge  32  of rear sidewall  30 B is a stand  50  having groove  52  received thereon for engaging and supporting a filter or tube from a container assembly  16 . 
     Depicted in  FIG. 5  is still another alternative embodiment of a retainer  14 D incorporating features of the present invention. Again, like elements between retainer  14 A and  14 D are identified by like reference characters. Retainer  14 D incudes front sidewall  30 A and rear sidewall  30 B. However, in contrast to the sidewalls being connected together by a single continuous floor  26  extending therebetween, each sidewall  30 A and  30 B has a separate floor portion  56 A and B, respectively, from which sidewalls  30 A and  30 B upstand. Holes  44  extend through each floor portion  56  to enable floor portions  56  to be selectively attached to platform  20 , as previously discussed, at a preferred spacing and orientation so that chamber  40  is formed between sidewalls  30 A and  30 B. It is appreciated that any number of separate sidewalls  30  having a separate floor portion  56  connected thereto can be separately mounted to platform  20  for forming the desired chamber  40 . 
     With regard to each of the above retainers  14 A- 14 C, it is appreciated that examples, materials, and alternative embodiments discussed for one are also applicable to the others and that features between the different retainers can be mixed and matched to form other retainers. Furthermore, other retainers not disclosed herein but that can support a container assembly  16  on platform  20  can also be used. 
     As depicted in  FIG. 1 , container assembly  16 A is positioned within chamber  40  of retainer  14 A so that retainer  14 A holds container assembly  16 A on platform  20  as platform  20  moves. In general, container assembly  12  is configured to hold a culture which includes a starter culture and growth media. Mixer table  12  rapidly moves container assembly  12  so that the culture is continuously uniformly mixed. Gas delivered into container assembly  12  interacts with the culture to oxygenate the culture. Depending on the application, the gas can also be used to strip CO 2 , regulate the pH and have other purposes. The culture typically continues to grow within container assembly  12  until the culture achieves a desired cell/microorganism density. The culture is then transferred to a larger container for subsequent growth and processing. 
     Turing to  FIG. 6 , container assembly  16 A comprises a flexible bag  68  having an upper end  70  to an opposing lower end  72 . Upper end  70  can terminate at an upper end wall  74  while lower end  72  can terminate at a lower end wall  76 . An encircling sidewall  75  can extend between end walls  74  and  76 . Flexible bag  68  also has an interior surface  78  that bounds a compartment  80 . Compartment  80  is configured to hold a fluid such as a biological culture. In the depicted embodiment, a culture  82  is shown housed within compartment  80  having a top surface  83 . A head space  84  occupies the remainder of compartment  80  above top surface  83  of culture  82 . 
     Culture  82  comprises a starter culture and a growth media. The starter culture comprises cells or microorganisms that are used to inoculate the growth media. The starter culture can comprise a colony of cells or microorganisms from a new line of cells or microorganisms or can be taken from an existing culture of cells or microorganisms. The starter culture can be in a frozen or at least partially frozen state, in a free flowing liquid state, or in a semi-sold state such as a colony growing on an agar plate. The starter culture typically has a volume in a range between about 0.25% to about 20% of the total culture volume with between about 1% to about 10%, about 1% to about 5% or about 5% to about 10% of the total culture volume being more common. Other percentages can also be used. As is known in the art, the growth media comprises a media broth which can be mixed with nutrients, vitamins, and/or other desired components to optimize the desired growth of the starter culture. Culture  82  comprising the combination of the starter culture and the growth media and typically has a volume in a range between 0.3 liters about to about 10 liters with between about 1 liter to about 7 liters or about 1 liter to about 5 liters being more common. The volume of culture  82  is often less than 10 liters, 7 liters, 5 liters or 3 liters. Other volumes can also be used. 
     Continuing with  FIG. 6 , flexible bag  68  is comprised of a flexible, water impermeable material such as a low-density polyethylene or other polymeric sheets or film having a thickness in a range between about 0.05 mm to about 5 mm with about 0.1 mm to about 1 mm being more common. Other thicknesses can also be used. The material can be comprised of a single ply material or can comprise two or more layers which are either sealed together or separated to form a double wall container. Where the layers are sealed together, the material can comprise a laminated or extruded material. The laminated material comprises two or more separately formed layers that are subsequently secured together by an adhesive. Examples of extruded material that can be used in the present invention include the HyQ CX3-9 and HyQ CX5-14 films available from Life Technologies Corporation out of Logan, Utah. The material can be approved for direct contact with living cells and be capable of maintaining a solution sterile. In such an embodiment, the material can also be sterilizable such as by ionizing radiation. Prior to use, container assembly  16 A is typically sealed closed and sterilized so that compartment  80  is sterile prior to the introduction of a seed culture. 
     In one embodiment, flexible bag  68  can comprise a two-dimensional pillow style bag. In another embodiment, flexible bag  68  can be formed from a continuous tubular extrusion of polymeric material that is cut to length. The ends can be seamed closed or panels can be sealed over the open ends to form a three-dimensional bag. Three-dimensional bags not only have an annular sidewall but also a two dimensional top end wall and a two dimensional bottom end wall. Three dimensional containers can comprise a plurality of discrete panels, typically three or more, and more commonly four or six. Each panel is substantially identical and comprises a portion of the sidewall, top end wall, and bottom end wall of the container. Corresponding perimeter edges of each panel are seamed together. The seams are typically formed using methods known in the art such as heat energies, RF energies, sonics, or other sealing energies. 
     In alternative embodiments, the panels can be formed in a variety of different patterns. Further disclosure with regard to one method of manufacturing three-dimensional bags is disclosed in United States Patent Publication No. US 2002-0131654 A1, published Sep. 19, 2002, which is incorporated herein by specific reference in its entirety. 
     It is appreciated that flexible bag  68  can be manufactured to have virtually any desired size, shape, and configuration. Flexible bag  68  is typically formed having a compartment  80  having a volume in a range between 0.5 liters and 15 liters with between 1 liter and 10 liters or between 1 liter and 7 liters being more common. Often the volume of compartment  80  is in a range between 1.1 to 10 times the volume of culture  82  initially formed within compartment  80  with between 1.5 to 6 or 2 to 4 times the volume of culture  82  being more common. Other volumes can also be used. As discussed below, having the volume of compartment  80  be greater than the volume of culture  82  disposed therein is more important when growing cells/microorganisms aerobically than when growing anaerobically. When growing aerobically, the extra volume, i.e., head space  84 , is used for holding the gas which oxygenates the cells/microorganisms during mixing of the culture. When growing the cells/microorganisms anaerobically, the gas is not used and thus a large head space  84  is not required. In this embodiment, the tube and port leading to the gas filter can be eliminated or the tube leading the gas filter can be clamped closed. 
     Although flexible bag  68  can be any shape, in one embodiment flexible bag  68  is configured to rest against the one or more of sidewalls  30  of retainer  14  when received within chamber  40 , thereby helping to support container assembly  16  and maintain it in its desired orientation during use. 
     As also shown in  FIG. 6 , container assembly  16 A further comprises a plurality of ports  86  secured to flexible bag  68  and communicating with compartment  80 . Ports  86  can comprise a conventional barbed port, tube port, or any other type of tubular fitting for coupling a tube to flexible bag  68 . In the depicted embodiment, three port  86 A-C are formed on flexible bag  68  at upper end  70  with tubes  88 A-C coupled therewith, respectively. Tubes  88  and other tubes discussed herein can comprise any form of tubular member but typically comprise flexible tubing or hose. Tubes  88  can have a variety of different uses. By way of example and not by limitation, tube  88 A can be used for delivering the starter culture and growth media into compartment  80  for forming culture  82 . In other embodiments, the starter culture and growth media can be combined to form culture  82  outside of flexible bag  68  and then delivered into compartment  80  through tube  88 A. Tube  88 A can also be used for removing culture  82  from compartment  80 . Tube  88 B can be used for delivering a gas into compartment  80 . The gas typically comprises air or oxygen and is often mixed with other gases such as nitrogen. Tube  88 C can be used for removing gas from compartment  80  or can enable gas to transfer between the surrounding environment and head space  84  by passing through a gas filter. In the depicted embodiment, each of tubes  88 A-C communicates directly with head space  84  through ports  86 A-C, respectively. In alternative embodiments, as discussed below, one or more of tubes  88  could communicate directly with culture  82  by one or more of ports  86 A-C being positioned at lower end  72 , lower end wall  76 , or sidewall  75 . 
     Any number of tubes  88  can be coupled with flexible bag  68  and each tube can have one or multiple different uses. For example, because flexible bag  68  is relatively small and holds a relatively small amount of fluid, flexible bag  68  can be manually picked up and moved even when filled with a desired volume of culture. Thus, when it is desired to test culture  82  or to remove culture  82  from flexible bag  68 , i.e., when culture  82  has achieved a desired cell or microorganism density, flexible bag  68  can be manually lifted and then inverted so that culture  82  passes to one of tubes  88  disposed at upper end  70  or upper end wall  74  such as tube  88 A. A syringe can couple with tube  88 A and be used to draw out a sample of culture  82  through tube  88 A for testing. Alternatively, tube  88 A can be coupled with a container and then used to dispense culture  82  into the container under gravity flow. The container could be smaller than flexible bag  68  if only colleting a sample or larger than flexible bag  68  if dispensing for subsequent processing. This coupling with a syringe or other container typically occurs within a sterile hood and container assembly  16  or at least flexible bag  68  and/or tube  88 A can be manually moved into the sterile hood during such coupling and dispensing. However, in an alternative embodiment, a drain tube  96  can be coupled with flexible bag  68  at lower end  72  or lower end wall  76  through a port  86 D. Drain tube  96  can be used for draining or drawing culture  82  out of compartment  80  without the need for inverting or compressing flexible bag  86 . Drain tube  96  could be used while container assembly  16 A remains supported on platform  20  or could be used after container assembly  16 A is manually removed from platform  20 . Likewise, a tube  88 AA can be coupled to sidewall  75  of flexible bag  68  through a port  86 AA and be used for drawing samples of culture  82  out of flexible bag  68 . Tube  88 AA can pass through window  40  of retainer  14 A ( FIG. 2 ). In other embodiments, a dip tube could be disposed within compartment  80  and coupled with tube  88 A. The dip tube could be used for drawing or pushing culture out of compartment  80 , such as by compressing flexible bag  68 , without the need for inverting container assembly  16 . 
     Because of the relative small volume of culture  82  within flexible bag  68 , it is typically not necessary to use a sparger disposed at lower end  72  of flexible bag  68  for delivering gas into compartment  80 . That is, the gas is typically used for oxygenating the culture, stripping CO 2  from the culture and regulating the pH of the culture. In large scale bioreactors and fermenters, this is typically accomplished by continuously delivering a gas at the bottom of the container so that the gas passes up through the culture in small and/or large bubbles accomplishing a mass transfer between the culture and the gas before the gas enters the head space in the container. An impeller or other mixing element is typically used to concurrently mix the culture so that the mass transfer occurs throughout the culture. As used herein, the term “sparger” is intended to broadly cover fritted, porous, perforated and other gas permeable structures through which gas can be passed to disperse small bubbles of gas into a liquid and also includes pipes, tubes, ports and other structures that can be used to deliver larger bubbles into a liquid. 
     In one embodiment of the present invention, gas can be fed into compartment  80  through tube  88 B, or other tube  88 ,so that gas enters directly into headspace  84  in compartment  80  without passing through culture  82 . Furthermore, in contrast to continuously delivering gas into compartment  80  under a forced gas flow or positive gas pressure during the growth of phase of culture  82 , because culture  82  is a relatively small volume and is only grown within container assembly  16  for a relatively short time period, the gas can be delivered to fill or sufficiently fill head space  84  at the start of the growth phase but not be subsequently delivered under a forced gas flow or positive gas pressure during the subsequent growth phase. Mixing culture  82  within compartment  80  through the use of mixer table  12  is sufficient to cause homogeneous mixing of culture  82  and to achieve sufficient mass transfer between culture  82  and the gas within head space  84  without the need for an applied forced gas flow or positive gas pressure. However, a filter, such as filter  120  depicted in  FIG. 8  and discussed below, can be mounted on the end of tube  88 C. During the growth phase, air can freely transfer between the surrounding environment and compartment  80  by passing through filter  120  to assist in maintaining the desired oxygenation of culture  82 . 
     Accordingly, in one embodiment of the present invention where flexible bag  68  is filled with gas prior to delivery of the starter culture, culture  82  can be grown in flexible bag  68  without supplying a forced flow of gas into flexible bag  68  during growth of culture  82  therein. Alternatively, where the starter culture is delivered into flexible bag  68  prior to filling flexible bag  68  with a gas, culture  82  can be grown in flexible bag  68  by supplying a forced flow of gas into the compartment of the flexible bag  68 , during growth of culture  82 , for a period of time that is less than 15% and more commonly less than 10%, 5% or 1% of the total time period that culture  82  is grown within flexible bag  68 . 
     Again, sufficient mass transfer between the gas and culture  82  can be achieved in the manner discussed above because of the relatively small volume of culture  82  that is being treated and the relatively short growth period. As such, in one embodiment of the present invention, container assembly  16  is void of any sparger located at lower end  72  of flexible bag  68  which could dispense gas bubbles directly into the culture  82  within compartment  80 . Likewise, container assembly  16  can also be void of any mixing element within compartment  80  that is configured to directly mix culture  82  within compartment  80  by movement of the mixing element within compartment  80 . Such mixing elements could include an impeller, paddle, stir bar, or other elements that can achieve mixing by having a driven movement, e.g., driven by a rotating drive shaft, reciprocating shaft, magnetic mixer or other mechanism that engages with the mixing element. 
     Avoiding the use of a sparger and mixing element simplifies the manufacture, use and operation of container assembly  16  and decreases manufacturing and operation costs. For example, by having all tubes  88  connect at upper end  70  of flexible bag  68 , container assembly  16 A can be easily positioned within chamber  40  of retainer  14  without tubes  88  interfering with retainer  14 , without the need to modify retainer  14  or mixer table  12  to receive one or more tubes  88  and without the risk of one or more tubes  88  being kinked or pinched. 
     In an alternative embodiment and use, however, as depicted in  FIG. 6 , a sparger  92  coupled with a gas line  94  can be coupled with flexible bag  68  at lower end  72  or lower end wall  76  so that gas passing out of sparger directly enters into and is required to pass through a portion of culture  82  before reaching head space  84 . The gas can be delivered continuously through sparger  92  or can be delivered continuously directly into head space  84  through a tube  88  during the growth phase of culture  82 . Furthermore, independent of or in conjunction with sparger  92 , a mixing element having a driven movement could be disposed within compartment  80  for mixing culture  82  therein. 
     In large volume bioreactors and fermenters where a culture is grown for an extended period of time, it is critical that the chemical and physical properties of the culture be continuously monitored and regulated to maintain the viability of the culture. To that end, sensor such as dissolved oxygen sensors, pH sensors, temperature sensor, carbon dioxide sensors, cell mass sensors, nutrient sensors and the like and combinations of the foregoing are disposed within and/or coupled to the container of conventional bioreactors and fermenters to sense the chemical and physical properties of the culture therein. 
     In contrast, because the present invention is directed to the initial growth of a starter culture which is relatively small and is grown over a relatively short period of time before being transferred to a larger container, it is less critical to continuously monitor the physical and chemical properties of the culture. That is, those skilled in the art can safely maintain desired physical and chemical properties of an initial start-up culture without having to continuously monitor the properties through sensors. Where some monitoring is required, periodic samples of culture  82  can be taken and the desired properties measured. 
     As such, in one embodiment of the present invention, container assembly  16 A can be void of any sensors that directly measure the physical or chemical properties of culture  82  within compartment  80 . Thus container assembly  16 A can be void of any dissolved oxygen sensors, pH sensors, temperature sensors, mass sensors, and/or nutrient sensors that are disposed within, coupled to or interact with container assembly  16 A or flexible bag  68  to sense corresponding properties of the culture within compartment  80 . This configuration of system  10  simplifies the operation of the system and minimizes costs. In other embodiments, however, one or more of the above sensors can be coupled with or otherwise interact with flexible bag  68  to detect the corresponding properties of the culture within compartment  80 . The sensors can be connected to flexible bag  68  through one or more ports mounted on flexible bag  68 . Examples of how sensors can be mounted to flexible bag  68  through ports is disclosed in U.S. Pat. No. 7,487,688 issued Feb. 10, 2009 and U.S. Pat. No. 7,901,934 issued Mar. 8, 2011, which are incorporated herein by specific reference. 
     One of the unique features of one embodiment of the present invention is that because container assembly  16  is formed from polymeric sheets and flexible tubing, container assembly  16  is easy and inexpensive to manufacture. As such, container assembly  16  can be disposed of after a single use. This disposable avoids the need for cleaning and re-sterilization and thereby minimizes any risk of contamination. This is in contrast to conventional glass flasks which must be cleaned and sterilized between each use. Furthermore, in contrast to conventional glass and plastic flasks which occupy a large volume during transport and storage and are prone to breaking, container assembly  16  can be collapsed for transport and storage so as to occupy minimal space and there is minimal risk of breaking. 
     In one embodiment of the present invention, means are provided for removably securing container assembly  16  to retainer  14 . By way of example and not by limitation, as also depicted in  FIG. 6 , container assembly  16 A further comprises a plurality of tabs  98  outwardly projecting from flexible bag  68 . Tabs  98  can be made from the same material as flexible bag  68 , i.e., polymeric sheet or film, and can be attached to flexible bag  68  by having one end welded or otherwise secured between edges of adjoining sheets forming flexible bag  68 . Any desired numbers of tabs  98  can be attached to flexible bag  68 . For example, container assembly  68  can have at least two, four, six, eight, ten or more tabs  98  projecting therefrom. 
     In one embodiment of the present invention, means are provided for securing tabs  98  to retainer  14  which means comprises part of the means for removably securing container assembly  16  to retainer  14 . By way of example and not by limitation, each tab  98  is shown having an opening  100  extending therethrough. Returning to  FIG. 2 , outwardly projecting from exterior surface of front sidewall  30 A are a plurality of fasteners  102 . Specifically, fasteners  102 A and B are disposed adjacent to side edge  34 A while fasteners  102 C and D and disposed along opposing side edge  34 B. Corresponding fasteners also project from the exterior surface of rear sidewall  30 B. 
     During use, as depicted in  FIG. 1 , container assembly  60 A is received within chamber  40 . Tabs  98 A and B are passed through a slot  36 A and folded over fasteners  102 A and  102 B, respectively, so that fasteners  102 A and  102 B pass through corresponding openings  100  in tabs  98 A and B. Likewise, tabs  98 C and D are passed through a slot  38 B and folded over fasteners  102 C and  102 D, respectively, so that fasteners  102 C and  102 D are received within the openings  100  thereof. Four tabs located on the opposing side of flexible bag  68  are likewise passed through corresponding slots on retainer  14 A and engage fasteners  102  projecting from rear sidewall  30 B. As a result of the engagement between tabs  98  and fasteners  102 , container assembly  16 A/flexible bag  68  is secured to retainer  14 . This securing between retainer  14  and container assembly  16 A helps to prevent container assembly  16 A from separating from retainer  14 A during movement of mixer table  12  and helps to prevent container assembly  16 A from rotating out of proper alignment within chamber  40  of retainer  14 A. Furthermore, securing container assembly  16 A to retainer  14 A helps to optimize mixing of culture  82  within container assembly  16 A during movement of mixer table  12 . 
     It is noted that tabs  98 A and C are located at upper end  70  of flexible bag  68  while tabs  98 B and D are located at lower end  72  of flexible bag  68 . Positioning and using tabs  98  at upper end  70  as opposed to just lower end  72  optimizes the retaining of container assembly  16 . Alternatively, for smaller flexible bag  68 , i.e., typically 6 liters or less, one tab  98  located midway between depicted tabs  98 A and B and one tab located midway between tabs  98 C and  98 D could be sufficient to secure those vertical corners of flexible bag  68 . A single tab  98  could likewise be used on the other corners. 
     In the depicted embodiment, fasteners  102  comprise a linear stem  103  having an enlarged rounded head  104  disposed at the free end thereof. In alternative embodiments, fasteners  102  could be in the shape of hooks, elbows, curved arms or other projections that are configured to be received within openings  100 . In still other embodiments, fasteners  102  could be in the form of screws, bolts, pegs, pins, or the like that pass through openings  100  and thread or otherwise engage with sidewalls  30 . In yet other embodiments, openings  100  can be eliminated and the fasteners could comprise clamps, wedges, clips, or other types of fasteners that directly engage tabs  100 . In other embodiments, other conventional fasteners such as buckles, Velcro (i.e., hook and loop), snaps, buttons, springs, ties, line and the like could be used to connect tabs  100  to retainer  14  or used to container assembly  16  to retainer  14  without the use of tabs  100 . By forming slots  26  through which tabs  98  can pass, container assembly  16 A can be secured to retainer  14  at both upper end  70  and lower end  72 . However, in the alternative embodiments where slots  26  are not provided, such as in retainer  14 B depicted in  FIG. 3 , fasteners  102  can be disposed adjacent to top edge  32  of sidewall  30 . For example, fasteners  102 A and  102 B are disposed on the exterior surface of front wall  30 A adjacent to top edge  32 . Tabs  98  at upper end  70  of flexible bag  68  can then pass over top edge  32  and then fold over fasteners  102 A and  102 B so that the fasteners are received within openings  100  of each tab  98 . Again, other fasteners as previously discussed can be used to connect tabs  98  to retainer  14 B. 
     Returning to retainer  14 C depicted in  FIG. 4 , in contrast to fasteners  102  being in the form of cylindrical pegs, retainer  14  has fasteners  102  in the form of rounded projections that are formed by cutting U-shape slots  104  through front sidewall  30 A and rear sidewall  30 B. Specifically, fasteners  102 A and E are formed adjacent to side edge  34 A while fasteners  102 G and H are formed adjacent to side edge  34 B. With container assembly  16 A disposed within compartment  40 , tabs  98 A and B ( FIG. 6 ) can fold around side edge  34 A and manipulate so that fastener  102 E and F side into openings  100  on tabs  98 A and B. Likewise, tabs  98 C and D ( FIG. 6 ) can fold around side edge  34 B so that fasteners  102 G and H slide into openings  100  of tabs  98 C and D. Tabs  98  on the back side of container assembly  16 A are similarly attached to retainers  102  on rear sidewall  30 B. In this configuration, tabs  98  secure container assembly  16 A to retainer  14 C so that the lateral sidewalls extending between front sidewall  30 A and rear sidewall  30 B are not required. Again it is appreciated that virtually any type of projection that can be received within opening  100  of a tab  98  can be used as a fastener  102 . Likewise, other types of connection can be used to secure container assembly  16 A to retainer  14 C. 
     In another embodiment as depicted in  FIG. 7 , with tabs  98 A and B passed through slot  36 A and tabs  98 C and D passed through slot  36 B, a fastener  102 I extends between tabs  98 A and C on the outside of front sidewall  30 A while a fastener  102 J extends between tabs  98 B and D on the outside of front sidewall  30 A. Although fasteners  102 I and J are not secured to retainer  14 A, connecting tabs  98  together secures container assembly  16 A to retainer  14 A. In the embodiment depicted, fastener  102 I comprises a first strap  190  that is secured to tab  98 A and a second strap  191  that is secured to tab  98 C. Straps  190  and  191  are removably secured together by a fastener  192  such as hook and loop (Velcro), snaps, hook, buckle, clamp or the like. In another embodiment, straps  190  and  191  can form one continuous strap that is looped through tabs  98 A and C with the opposing ends connected together by fastener  192 . To illustrate a further contrasting example, fastener  102 J comprises a spring  194  having opposing ends connected to tabs  98 B and D. Those skilled in the art will appreciate that a variety of different types of fasteners such as straps, cinches, lines and the like could be used to replace fastener  102 H and/or I that secure tabs  98  together. 
     Fasteners  102 I and J can also be used to secure together opposing tabs  98  on the outside of rear sidewall  30 B. Fasteners  102 I and J are particularly well suited for connecting container assembly  16  to retainers  14 C and  14 D depicted in  FIGS. 4 and 5 , respectively, where the retainers do not include lateral sidewalls  30 C and D. 
     Depicted in  FIG. 8  is an alternative embodiment of a container assembly  16 B incorporating features of the present invention. Like elements between container assembly  16 A and  16 B are identified by like reference characters. Container assembly  16 B comprises flexible bag  68  depicted in a collapsed position. Flexible bag  68  comprises a top sheet  122 , an opposing bottom sheet  124  having a complementary configuration and two folded over side sheets  125  and  126  in the form of gussets that are disposed between sheets  122  and  124  along opposing sides thereof. The sheets are welded together along the perimeter edges so that the flexible bag  68  can expand. A tube  88 A is coupled with flexible bag  68  by a port  86 E. Tube  88 A has a Y-connector  108  formed on the end thereof which couples tubes  110 A and B to tube  88 A. Tube  110 A has a clamp  112 A which can be opened and closed to selectively open and close the fluid pathway extending through tube  110 A. Mounted on the end of tube  110 A is a fitting  114  which can be used to selectively couple with a separate container for either delivering fluid into flexible bag  68  or dispensing fluid out of flexible bag  68 . A removable cover  116 A is disposed over fitting  114  to maintain fitting  114  sterile after container assembly  16 B has been sterilized as a fully assembled system. During use, cover  116 A is removed within a sterile hood for coupling to the desired container or fluid source. 
     Tube  110 B also has a clamp  112 B mounted thereon for selectively opening and closing the pathway extending through tube  110 B. A fitting  118  is disposed on the end of tube  110 B and is used for coupling with a gas source for delivering a gas into flexible bag  68 . As with fitting  114 , a cover  116 B is removably disposed over fitting  118 . A clamp  112 C is disposed on tube  88 F and is likewise used for selectively opening and closing the passageway through tube  88 F. A sterilizing gas filter  120  is disposed on the end of tube  88 F. As previously discussed, in one embodiment of use the gas source is merely used to fill head space  84  ( FIG. 6 ) with gas and is then turned off during the growth phase of culture  82 . In this case, gas filter  120  prevents the over pressurization of flexible bag  68  during initial filling by allowing gas to escape and also permits the free flow of gas between the environment and head space  84  by allowing gas to pass through filter  120 . Filter  120 , however, prevents any contaminants in the environment from passing into container  68  through tube  88 F. As such, filter  120  can comprise a gas sterilization filter such as a filter having a maximum pore size that is 2 microns or less. 
     In an alternative method of use, the gas source can be used to deliver a continuous or substantially continuous gas stream to head space  84  during the growth phase of culture  82 . For example, the gas can be delivered into the compartment of flexible bag  68 , during growth of culture  82 , for a period of time that is greater than 70% and more commonly greater than 80% or 90% of the total time period that culture  82  is grown within flexible bag  68 . As gas pressure builds up within flexile bag  68 , gas can pass through tube  88 F and out filter  120  into the surround environment. Filter  120  thus enables gas to safely escape from flexible bag  68  without permitting contaminants in the environment from passing into container  68  through tube  88 F. 
     A removable cover  116 C is also disposed over filter  120  which is removed prior to use. In view of the foregoing, it is appreciated that a variety of different tube configurations and fittings can be used on each container assembly  16 B. In this embodiment, it is noted that no tubes or spargers are disposed at the lower end of flexible container  68 . 
     In one embodiment of the present invention, baffles can be used in association with the container assembly  16  to both improve mixing of the culture within compartment  80  and to improve mass transfer between culture  82  and the gas within container assembly  16 . For example, depicted in  FIG. 9  is container assembly  16 A having baffles  130 A disposed therein. In general, baffles  130  discussed herein are secured to flexible bag  68  at lower end  72  and more commonly on lower end wall  76 . However, baffles  130  can also be mounted on sidewall  75 . Baffles  130  can be attached by adhesive, welding, mechanical connection or using other conventional techniques. In the embodiment depicted, baffle  130 A comprises a cylindrical tube having an interior surface  132  that bounds a passage  134  extending between opposing end faces  136  and  138 . Although container assembly  16 A is shown as having four baffles disposed within compartment  80 , it is appreciated that one, two, three, five, six, seven, or more or at least each of the foregoing numbers of baffles can be used. In part, baffles  130  help to improve the mixing of culture  82  ( FIG. 6 ) within flexible bag  68  as culture  82  is being moved by the movement of mixer table  12 . Baffles  130  help to ensure that culture  82  is uniformly mixed and that the desired mass transfer between the gas and culture  82  is achieved. 
     Baffles  130  can be disposed randomly within compartment  80  or placed at predetermined locations. For example, in the embodiment depicted in  FIG. 6 , baffles  130  are positioned so that a central longitudinal axis of baffles  130  extends perpendicular to the adjacent side wall of flexible bag  86 . This positioning helps to increase turbulent flow as the fluid flows over and around baffles  130 . In other embodiments, the central longitudinal axis of baffles  130  can be disposed to form an inside angle with the adjacent sidewall of flexible bag  86  that is in a range between 45° and 90° and more commonly in a range between 60° and 90° or 75° and 90°. In another embodiment, baffles can be disposed so that a central longitudinal axis of each baffle  130  is disposed so as to be tangential to a common radius on lower end wall  76  of the flexible bag. The radius can be based on a point that is centrally located on lower end wall  76 . For example, in the embodiment depicted in  FIG. 10  and discussed below, the longitudinal axis of baffles  130 B are disposed tangential to a radius R. This orientation helps to redirect fluid that is flowing out towards the sides of flexible bag  86 . In other embodiments, baffles  130  need not be linear but can be curved, bent, or have an irregular pattern. 
     As previously mentioned, it is appreciated that baffles  130  can be any desired configuration that will improve mixing of the culture. For example, depicted in  FIG. 10  are baffles  130 B disposed at lower end  72  or lower end wall  76  of a flexible bag  68 . In contrast to baffles  130 A, baffles  130 B comprise a base  144  secured to flexible bag  68  in one or more of the manners previously discussed and a wall  146  upstanding therefrom. In the depicted embodiment, wall  146  has a rectangular configuration. In other embodiments, however, wall  146  could be square, semi-circular, polygonal, irregular, or any other desired configuration. Alternative embodiments as to numbers, positioning, use, and the like for baffle  130 B are the same as previously discussed with regard to baffle  130 A. 
     As previously mentioned, baffles  130  can be attached to flexible bag  68  using a variety of different techniques. In one embodiment, as depicted in  FIG. 11 , a fitting  152  is initially attached to flexible bag  68 . Fitting  152  comprises an elongated stem  154 , a flange  156  that encircles and radially outwardly projects from a first end of stem  154  and a tapered barb  158  that encircles and radially outwardly projects from the opposing end of stem  154 . As with other conventional ports, fitting  152  can be attached to lower end wall  72  of flexible bag  68  by passing stem  152  through an opening  160  on lower end wall  72  and then welding or otherwise securing flange  56  to the bottom surface of the lower end wall  72 . As a result, stem  154  upwardly projects into compartment  80  of flexible bag  68 . 
     In this embodiment, a baffle  130 C comprises a base  162  having a wall  146  upstanding therefrom. Base  162  comprises a collar having a bottom surface  164  with an opening  166  formed thereon. Opening  166  is configured to receive stem  154  and barb  158  in locking engagement. That is, barb  158  can form a friction fit within opening  166  or can engage with ribs or other features within opening  166  so that baffle  130 C locks to fitting  152  as depicted in  FIG. 12 . Barb  158  is one example of a locking feature that can be formed on stem  152  for engaging base  162 . In other embodiments, barb  158  can be replaced with other locking features such as ribs, knobs, catches, and other projections that extend from stem  154  and engage base  162 . 
     Depicted in  FIG. 13  is another embodiment on container assembly  16 A having flexible baffles  130 D disposed therein. Each baffle  130 D comprise a sheet of polymeric film, such as the same film used to make flexible bag  68  or film consisting of just one material such as HDPE (high-density polyethylene). Each baffle  130 D has a lower end  200  secured to lower end wall  76 , an upper end  202  secured to upper end wall  74 , an outside edge  204  secured to sidewall  75 , and an inside edge  206  that is freely disposed within compartment  80  of flexible bag  68 . Baffles  130 D can be secured to flexible bag  68  by welding, adhesive, or the like. As platform  20  ( FIG. 1 ) is moved, baffles  130 D help to uniformly mix culture  82  within compartment  80 . 
     In contrast to positioning baffles  130  within compartment  80  of flexible bag  68 , baffles  130  can also be secured to floor  26  of retainer  14  or directly onto platform  20  of mixer table  12  so that once container assembly  16  is received within chamber  40  of retainer  14 , flexible bag  68  sits on top of baffles  130 . For example, as depicted in  FIG. 14 , baffles  130 B are disposed on floor  26  of retainer  14 C. Again, baffles  130  can be a variety of different configurations and can be used in a variety of different numbers and different orientations, each as discussed above. As a result of flexible bag  68  sitting on baffles  130 B, lower end wall  76  flexes up over top of baffles  130 B, thereby effectively functioning as baffles within flexible bag  68 . It is appreciated that baffles  130  can be located on any of the floors  26  previously discussed with regard to retainers  14  or can be mounted directly onto platform  20  between the retainer walls as depicted in  FIG. 5 . 
     As depicted in  FIG. 15 , because container assemblies  16  are relatively small, it is appreciated that a plurality of different retainers  14  and corresponding container assemblies  16  can be simultaneously mounted on platform  20  of mixer table  12 . Each container assembly  16  can then concurrently culture a separate culture therein. In the depicted embodiment, four retainers  14  and four corresponding container assemblies  16  are disposed on platform  20 . In other embodiments, 2, 3, 5, 6, 7 or more or at least each of the foregoing numbers of retainers  14  and container assemblies  16  can be disposed on platform  20  for concurrently culturing a separate culture. 
       FIG. 15  also shows that an incubator  172  can operate with mixer table  12  for controlling the temperature of culture  82  within container assemblies  16 . Incubator  172  comprises a hood  174  that bounds an enclosure  176  and a temperature regulating source  178  for heating and/or cool enclosure  176  or items within the enclosure  176 . Temperature regulating source  178  can comprise a radiant heat source, heated forced air, electrical heating element, a heated fluid system or any other type of heating system that can be used to warm culture  82  within container assembly  16 . Typically, heat source  178  will warm the air within enclosure  176  which will in turn warm culture  82 . Temperature regulating source  178  can also comprise an air conditioner, a chilled fluid system, heat sink or other systems that can be used to cool culture  82  within container assembly  16 . The cooling is typically accomplished by cooling the air within enclosure  176 . 
     Hood  174  can be hingedly mounted or otherwise movably mounted to mixer table  12  so that it can be moved from an open position wherein container assemblies  16  can be accessed and a closed position wherein hood  174  covers retainers  14  and container assemblies  16 . When in the closed position, a controller coupled with temperature regulating source  178  can be used to regulate the temperature within enclosure  176 . 
     During one typical method of operation, the desired number of retainers  14  are mounted on platform  20  of mixer table  12  following of which a container assembly  16  is received within chamber  40  of each retainer  14  and is secured to each retainer  14 . A growth media is dispensed into each container assembly  16  through a tube  88 . A gas is also delivered into each container assembly  16  through a tube  88  so as to fill head space  84 . Hood  174  is typically closed and incubator  172  is used to warm the growth media to the desired temperature. Where desired, the growth media and/or gas can be delivered into container assembly  16  prior to positioning container assembly  16  within chamber  40 . Once the desired temperature is reached, the starter culture is dispensed into the growth media of each container assembly  16 . Mixer table  12  is activated so that platform  20  moves, thereby mixing the culture within each container assembly  16  so that the culture is continuously uniformly mixed and so that the culture is sufficiently oxygenated through mass transfer with the gas in head space  84 . 
     The culture is allowed to grow under these conditions until a predetermined condition is met. For example, the culture may be allowed to grown for a predetermined period of time or the culture may be allowed to grown until a predetermined cell/microorganism density is achieved. The density can be determined by periodically taking a sample of the culture from each container assembly, such as by using one of methods previously discussed, and testing the sample. For example, the sample can be extracted out through a tube  88  at the upper end or side of container assembly  16  or through a tube  96  at the lower end of container assembly  16 . In one method, the sample is taken by first removing container assembly  16  from retainer  14 . Container assembly  16  can then be manually placed within a sterile hood and/or inverted following which the sample is dispend or drawn from of container assembly  16 . Container assembly  16  is then returned to retainer  14  for further processing. In other embodiments, the sample can be drawn from container assembly  16  while container assembly  16  remains resting on platform  20 . In this embodiment, mixer table  12  can be deactivated prior to taking the same and then reactivated after the sample has been taken. 
     The sample can also be tested to determine the other physical and/or chemical properties discussed herein. Based on the results of such testing, needed components can be added to the culture or the operating conditions, such as temperature, rate of mixing or gas content, can be altered. In contrast to or in addition to taking samples, sensors can be mounted on or otherwise associated with container assembly  16  for continuously monitoring the various or select chemical and/or physical properties of the culture. 
     Based on the volumes and conditions discussed herein, culture  82  comprising a microbial culture will typically be grown in container assembly  16  for a period of time ranging from about 1 hour to about 48 hours with about 4 hours to about 24 hours or about 4 hours to about 16 hours or about 4 hours to about 8 hours being more common before reaching the desired density. For mammalian or insect cell culture, culture  82  will typically be grown in container assembly  16  for a period of time ranging from about 1 day to about 14 days with about 2 days to about 3 days or about 3 days to about 5 days being more common before reaching the desired cell culture density. Longer culture times may be achieved with a nutrient feed. Culture  82  may be both fully grown and removed from container assembly  16 , such as by being transferred to another container, with in the above time periods. Other time periods can also be used. 
     Once the predetermined condition is satisfied, mixer table  12  is turned off. Each container assembly  16  can then be manually removed from the corresponding retainer  14  and then coupled to a secondary container through one of tubes  88 . Where further processing is desired, the secondary container is typically larger than container assembly  16 /flexible bag  68  and forms a portion of a bioreactor or fermenter. Culture  82  from container assembly  16  can then be dispensed, such as through gravity flow or pumping, into the secondary container through a tube  88  or  96 . Where needed, container assembly  16  can be inverted prior to dispensing. Container assembly  16  can then be discarded and the process repeated. Other alternative embodiments and steps for the present invention can also be performed as previously discussed herein. 
     The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.