Patent Publication Number: US-2023146372-A1

Title: Sampling apparatus for cell culture bed and related methods

Description:
This patent application claims the benefit of U.S. Provisional Patent Application. Ser. No. 63/276,621, filed Nov. 7, 2021, the disclosure of which is incorporated herein by reference. This application is related to International Patent Application WO 2019/175442, U.S. Provisional Patent Application Ser. Nos. 62/728,405, 62/644,014, Belgian Patent Application BE2018/5179, U.S. Patent Application Publication No. 2018/0282678, International Patent Application PCT/EP2018/076354, U.S. Provisional Patent Application 62/711,070, and U.S. Provisional Patent Application 62/725,545, the disclosures of which are incorporated herein by reference. 
    
    
     TECHNICAL FIELD 
     This document relates generally to the cell culturing arts and, more particularly, to a sampling device for a cell culture bed, such as for example, one in a bioreactor including a fixed bed. 
     BACKGROUND 
     Certain cell culturing devices, such as bioreactors, use a “fixed bed” for the growth of cells that are entrapped thereon or for the growth of adherent cells which attach and grow thereon. These high cell density bioreactors suffer from the inherent inability of its users to easily take samples during the culture process for various purposes (e.g., to take cell-associated measurements such as those relating to viability and density). Past approaches for in-process sampling from a fixed bed involve reaching inside the bioreactor with an extraction tool, such as tweezers, to manually extract a piece or portion of the bed or a sample thereof. This operation is difficult as it requires careful dexterity, and invariably causes undesirable perturbations that risk disrupting the cell culture environment, as well as the sample specimen. Sterility must also be maintained during the entire bioprocessing operation, which requires the operator collecting the sample to follow precise aseptic operating procedures, and may be particularly challenging when simultaneously introducing an extraction tool. 
     Furthermore, to maintain the necessary sterile conditions, the bioreactor is typically located inside a containment unit, such as a laminar flow cabinet or a biosafety cabinet. This limits an operator&#39;s access, as well as their freedom of movement. While a small scale bioreactor may be fully placed in such a containment unit, a large, production-scale version cannot readily be placed in such a cabinet to achieve this result. 
     Sterile (or aseptic) sensors, such as so-called “biomass” sensors have been proposed for assessing cell density. However, many known sensors lack sufficiently robust technology, and do not allow for the actual direct examination of the cells as a sample. Indirect measurement of cell characteristics by the biomass sensor may also be far less accurate than direct examination of cells. 
     Moreover, as opposed to the case of fluidized beds or classical agitated bioreactors where sampling of the liquid in which the cells reside is possible, a sterile liquid sensor in a fixed bed bioreactor yields only information from which an educated guess or estimate can be made regarding cell conditions based on detected byproducts (metabolites) of the growth process left in the fluid. Thus, current samplers and methods do not provide an accurate and timely tool for developing a reliable cell culture process for a fixed bed bioreactor. 
     Accordingly, a need is identified for an apparatus that provides the ability to take a reliable sample from the cell culture bed and the cells associated therewith. The apparatus would allow for the sample to be obtained in an easy and inexpensive manner, would protect against contamination (both internal to the bioreactor and external to it), and/or also avoid creating deleterious disruptions of the cell culture environment. 
     SUMMARY 
     An object of the invention is to provide a device that enables the taking of one or more samples from the cell culture in an easy, inexpensive, and reliable manner, without risking contamination, and also while avoiding creating deleterious disruptions of the cell culture environment. 
     According to a first aspect of the disclosure, a bioreactor with integrated cell sampling capability is provided. The bioreactor includes a housing including a sampling port. A fixed bed is positioned within the housing. A cell sampler extending through the sampling port and including a releasably attached sample portion positioned within or adjacent to the fixed bed. 
     In one embodiment, the releasably attached sample portion includes a substrate adapted for growing cells thereon during cell culture. The sampler includes a positioner adapted for positioning the releasably attached sample portion within or adjacent to the fixed bed. The releasably attached sample portion is connected to the positioner by a frangible connection. The positioner may be associated with the sampling port. 
     In these or other embodiments, the bioreactor includes a cap for sealing the sampling port. The sampler may be releasable from a grip located external to the bioreactor. The sampler may be adapted for selectively forming a locking engagement with the bioreactor housing. The bioreactor housing includes a lid and the lid comprises the sampling port. 
     The fixed bed may comprise a plurality of layers. The releasably attached sample portion of the sampler may be positioned between the plurality of layers. 
     According to a further aspect of the disclosure, an apparatus for sampling a cell culture associated with a fixed bed in a bioreactor having a port is provided. The apparatus comprises a sampler including a releasably attached sample portion adapted for positioning in the bioreactor adjacent the fixed bed. The sample portion includes a substrate adapted for growing cells. 
     In one embodiment, the releasably attached sample portion includes a frame for receiving the substrate. The sampler includes a grip connected to a positioner for positioning the releasably attached sample portion in the bioreactor. The sampler may be adapted for selectively forming a locking engagement with the bioreactor. The sample portion may be releasably attached to the sampler by a frangible connection. 
     Still a further aspect of the disclosure relates to a kit for sampling a fixed bed of a bioreactor. The kit comprises a sampler for sampling the fixed bed, the sampler including a releasably attached sample portion. The kit further comprises a container adapted to detach the releasably attached sample portion from the sampler. 
     In one embodiment, the container includes an insert for receiving the releasably attached sample portion, the insert adapted to facilitate detaching the releasable sample portion from the sampler. The releasably attached sample portion may include a substrate for growing cells. 
     Yet another aspect of the disclosure relates to a method of obtaining a sample of a cell culture in a fixed bed bioreactor including a sampler at least partially positioned in or adjacent to the fixed bed to promote the growth of cells on a releasably attached portion of the sampler. The method comprises initiating the cell culture process in the bioreactor, and allowing the process to run for a period of time to permit cell growth in the fixed bed and the releasably attached portion. The method further comprises withdrawing the sampler from the bioreactor, and detaching the releasably attached portion from the sampler. In one embodiment, the detaching step comprises inserting the releasably attached portion into an opening of an insert in a container, and using the insert to break the portion from the sampler. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWING FIGURES 
       The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: 
         FIG.  1    is a schematic view of a first embodiment of a sampler for a cell culture system, such as a bioreactor, according to the disclosure. 
         FIG.  1 A  illustrates one example of a structured fixed bed in the form of a spiral bed. 
         FIG.  1 B  is a schematic view illustrating a sampler in use in an unstructured bed, where a positioner includes at least one fiber. 
         FIG.  1 C  illustrates alternate forms of a structured fixed bed. 
         FIGS.  2 ,  3 , and  4    illustrate a sampler according to one aspect of the disclosure. 
         FIGS.  5 ,  5 A,  5 B and  6    illustrate a positioner forming part of the sampler of  FIGS.  2 ,  3 , and  4   . 
         FIGS.  7  and  8    illustrate an embodiment of a container including an insert for receiving a sample portion of the sampler. 
         FIGS.  9 ,  9 A and  10    illustrate the manner in which the sample portion may be detached from the sampler when associated with the container and insert. 
         FIG.  11    is one example of a fixed bed bioreactor for possible application of the sampler. 
         FIGS.  12 ,  12 A, and  12 B  are partially cutaway, cross-sectional views of the bioreactor of  FIG.  11   . 
         FIGS.  13  and  14    illustrate a locked and unlocked condition of the sampler when associated with a bioreactor. 
         FIGS.  15  and  16    show alternate versions of bioreactors. 
     
    
    
     DETAILED DESCRIPTION 
     Reference is now made to  FIG.  1   , which illustrates a sampler  10  for a cell culturing apparatus, such as for example a bioreactor  12 , fermenter or the like, according to one aspect of the disclosure. For purposes of this example, the bioreactor  12  may be presumed to operate under external sterile conditions, such as in a containment unit (e.g., a laminar flow cabinet, external isolator or the like). However, as outlined further in the description that follows, the sampler  10  may also be used in other environments. 
     The sampler  10  may comprise a support portion for associating with an opening or port in the bioreactor  12  providing access to an interior compartment thereof. In the illustrated embodiment, the support portion takes the form of a cover or cap  14  for sealing the port in the bioreactor  12 , thus maintaining the sterility of the interior compartment, even when the bioreactor  12  is not in a containment unit. The support portion, or cover/cap  14  in this version, may support a sample portion  16  for positioning within a fixed bed  18  in the interior compartment of the bioreactor  12 . The sample portion  16  may be removable from both the fixed bed  18  and the support portion, as outlined further in the following description. 
     The sample portion  16  may comprise a substrate (such as, for example, a sheet of material, a sheet of flexible material, or the like, which sheet may be in the form of an elongated strip) which, like the fixed bed itself, is adapted to promote cell growth or cell immobilization/entrapment. The substrate may alternatively comprise one or more fibers, including those arranged as a piece of nonwoven material, as outlined further in the following description. The sample portion  16  may be a separate structure for positioning within the fixed bed  18  during the process of assembling the bioreactor  12  (as outlined further in the following discussion). In such case, the sample portion  16  may be adapted for detachment and removal from the remainder of the fixed bed  18 . 
     The sample portion  16  may be removably attached to a positioner  20 . The positioner  20  facilitates holding the sample portion  16  in an appropriate position in or adjacent to the fixed bed  18 , and also facilitates removing it from the fixed bed  18 . Prepositioning of the sample portion  16  within the fixed bed  18  may be achieved by hand via the positioner  20 , or by using a tool, such as tweezers (not shown), to pass the sample portion  16  into or adjacent the fixed bed  18  through a port  22  in the lid  24  of the bioreactor  12  (but could be achieved by removing the lid entirely). This placement may be done prior to the commencement of the bioprocessing operation, including possibly during assembly of the bioreactor (including after the sample portion  16  if separate is placed in the fixed bed) or as a part of the process of manufacturing and assembling the fixed bed  18 . 
     The fixed bed  18  may comprise any substrate for achieving cell growth or cell immobilization, and may comprise, for example, a structured fixed bed (which means that it is formed of an easily replicated, generally homogeneous, substantially fixed structure, and thus is not randomly oriented or unstructured, yet, as can be appreciated, could take a variety of sizes or shapes while meeting this qualification). The material of the fixed bed  18  may be woven, non-woven, a fiber matrix, or other forms, and may be formed of various polymer materials, including but not limited to polyethylene and polyethylene terephthalate. In one embodiment, as shown in a top view in  FIG.  1 A , the fixed bed  18  comprises a non-woven material  18   a  arranged in at least two layers on and within which cells grow, thereby forming a structured fixed bed. These layers are thus considered cell immobilization layers. 
     In the particular form shown, the non-woven material  18   a  is arranged in a rolled or spiral fashion, with each of the two cell immobilization layers separated by spacer layers  18   b , which promote culture and media flow between the various layers. The sample portion  16  may be a separate substrate positioned (as shown) between two of the layers of the bed  18  in rolled or spiral form, such as cell immobilization (or non-woven material) layers  18   a . Alternatively, the sample portion  16  may be located between a spacer layer and an adjacent cell immobilization layer. The fixed bed  18  may have only a single cell immobilization layer alternatively in roll or spiral with a single spacer layer. 
     However, the above is merely one example, and is not intended to limit the manner in which the sample portion  16  may be associated with a fixed bed  18  of any known form (e.g., positioning within a fiber matrix, or an arrangement of layers that are not spirally wound). For instance,  FIG.  1 B  illustrates a non-structured fixed bed, which may comprise a plurality of randomly oriented fibers  18   c . In this embodiment, the sample portion  16  may comprise one of the fibers  18   c . This fiber  18   c  may be connected to the positioner  20 , as indicated (or as a substrate of non-woven fibers). Alternatively, more than one fiber can be connected to the positioner  20  and act as the sample portion (such as a woven or non-woven material, as noted previously). 
     While  FIG.  1 A  illustrates a rolled, generally cylindrical arrangement for a structured fixed bed  18  (including cell immobilization and spacer layers  18   a ,  18   b , which combination is optional), it should be appreciated that other forms may be used (including, for example, just one cell immobilization layer or multiple spacer layers, either of which may be folded like an accordion). Thus, with reference to  FIG.  1 C , the fixed bed  18  may comprise a plurality of concentric layers  18   a  formed of a continuous material, and may be in any shape (e.g., circular, square, etc.). Alternatively, instead of being non-linear, the layers  18   a  may instead be linear. In any of these cases, spacer layers  18   b  may or may not be provided, and the sample portion  16  may be located at any location (or locations) within the bed  18 , without limitation, depending on the particular application and/or the desired approach to sampling. 
     Returning to  FIG.  1   , an anchorage point may be adapted to form a seal with the cover or cap  14 . This anchorage point may comprise, for instance, a sidewall of the port  22  associated with the lid  24  of the bioreactor  12 . The cap  14  associated with the sampler  10  may be releasably connected to the port  22 . This may be achieved by threading or other means of secure, but releasable connection (e.g., a bayonet fitting, as further outlined in the following description, or a door with a releasable latch (push-pull type of arrangement, or even a friction or interference fit)). 
     To ensure that cell growth is achieved on the sample portion  16  in the illustrated format, the positioner  20  may extend a distance sufficient to ensure that the sample portion  16  is at least partially positioned within or adjacent to the fixed bed  18 , such as between two adjacent layers (e.g., between cell immobilization layers  18   a  arranged in direct contact, or separated by a spacer layer  18   b ). As can be appreciated, this distance may vary depending on the size and shape of the bioreactor  12 , as well as the desired location for the sampling to occur, the type of bed, or the type of culture. 
     The positioner  20  may be connected to the cover or cap  14  serving as the support, but may also be separate therefrom (such that the cap  14  may be removed while the positioner  20  remains in position, which positioner  20  can then be withdrawn and the cap  14  replaced). The former version may be applied more readily to a non-structured fixed bed, such as shown in  FIG.  1 B , which allows the positioner  20  to be passed through the port  22  for positioning in the non-structured material within the non-structured fixed bed. A structured fixed bed is better suited for the latter version, since the sample portion  16  may be pre-positioned within or adjacent to the structured fixed bed layers and held in place thereby, and the positioner  20  later associated with the cap  14  (if desired). 
     In any disclosed embodiment, the spacer layers and/or the cell immobilization layers which make up respectively the spacer section (when present) and the immobilization section in the matrix assembly may comprise a biocompatible polymer. As examples the biocompatible polymer may be selected from polyester, polyethylene, polypropylene, polyamide, plasma treated polyethylene, plasma treated polyester, plasma treated polypropylene or plasma treated polyamide. The layers can be hydrophilic or hydrophobic, with a preference for the former in terms of the cell immobilization layers. 
     The thickness of the one or more layers may vary depending on the application, and may be between 0.05 mm and 3 mm, between 0.1 and 2 mm, or between 0.1 and 1 mm. Suitable material for the cell immobilization layer may be a woven or nonwoven material. A nonwoven, contrary to a woven material, is a fabric which is not created by weaving or knitting and does not require converting the fibers to yarn. Nonwovens are broadly defined as sheet or web structures bonded together by entangling fiber or filaments (and by perforating films) mechanically, thermally or chemically. The nature of the nonwoven material used in the current application may be of any origin, either comprising of natural fibers or synthetic fibers. By preference, the nonwoven is made of a polymer, such as polyester or polypropylene. The cell immobilization layers may comprise a polyethylete terepthalate (PET) nonwoven. The nonwoven material may be plasma treated to enhance cell adherence and flow. 
     The one or more layers may comprise a substrate including a plurality of openings configured to allow flow of at least one of cell culture media, cells, or cell products through the thickness of the substrate. The substrate can be at least one of a molded polymer lattice sheet, a 3D-printed lattice sheet, and a woven mesh sheet. The substrate may have a regular, ordered structure and provide a surface for cell adhesion, growth, and eventual cell release. The substrate may comprise a mesh fabricated from monofilament or multifilament fibers of polymeric materials compatible in cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride, polyethylene oxide, polypyrroles, and polypropylene oxide. Mesh substrates may have a different patterns or weaves, including, for example knitted, warp-knitted, or woven (e.g., plain weave, twilled weave, dutch weave, five needle weave). A non-woven mesh may have a fiber diameter of about 20 μm, a thickness of about 0.18 mm, and a porosity of 91%. A woven mesh substrate may have a diameter of about 160 μm and an opening diameter of 250 μm. 
     The spacer layers may comprise a (biocompatible) polymer with mesh size as described above. In one embodiment, the spacer layer is a synthetic woven fabric or structure. In another embodiment, the spacer layer is a bearing structure. Such structure may be produced from a biopolymer (e.g. alginate). Other suitable material for this purpose is silica, polystyrene, agarose, styrene divinylbenzene, polyacrylonitrile or latex. The spacer layer may be gamma irradiated in order to reduce bioburden. 
     The design of the matrix assembly may take many forms depending on the application and type of bioreactor. In one embodiment, the immobilization section and spacer section are alternately positioned. “Alternately positioned” means that each spacer section is followed by a cell immobilization section which is itself followed by a spacer section. These alternately positioned sections may alternate in vertical position or in a horizontal position according to the use of the matrix and/or to the bioreactor in which the matrix is introduced. In this embodiment, one or more layers of cell immobilization layers are superimposed on one or more spacer layers (or vice versa). This configuration may be repeated several times if deemed desirable in order creating a stack of several immobilization and spacer sections. Ideally, the end configuration may comprise between 1 and 500 alternations of the above described layering. The stacked layers may be positioned in a frame or cassette or sealed/connected at their circumference. In another embodiment, the achieved stack can be rolled around an axis or core to achieve a spiral configuration. 
     The substrate used for either the immobilization section and spacer section can be chosen based on the application, characteristics of the layers (dimensions, size, etc.) and desired result. Hence, the number of layers within either immobilization section or spacer section may be between 1 and 20, between 1 and 10, or between 1 and 5. 
     The optional spacer section(s), if present, create space inside the matrix assembly through which the culture medium flows. This provides improved circulation of the culture medium through the matrix assembly, thereby ensuring it reaches all cultured cells. This effect is even more enhanced in the embodiment wherein the spacer section comprises one spacer layer and the cell immobilization section comprises two immobilization layers. The culture medium flowing inside the matrix assembly via the spacer sections is tangentially oriented with respect to the cell immobilization sections. 
     According to one aspect of the disclosure, and with reference to  FIG.  2   , a sampler  100  for sampling a cell culture may be provided. The sampler  100  may include a positioner  120  having a removable portion  102  depending therefrom, with a sample portion  116  at a distal end portion thereof. This removable portion  102  may comprise an elongated pair of spaced apart members  102   a ,  102   b  connected at one end to a body  104 . This arrangement allows the members  102   a ,  102   b  to move or flex inwardly toward each other as shown in  FIG.  3   , but could also be a one-piece structure and adapted for coupling to and decoupling from the sample portion  116 . 
     At the other, proximal end, the members  102   a ,  102   b  may be adapted to connect with a part by which the sampler  100  may be grasped, such as a grip  106 . Such grip can include any handle or similar mode of grasping by an operator. Specifically, each member  102   a ,  102   b  may include head portions  102   c ,  102   d  forming barbs or catches. When inserted into a passage  108  in the grip  106 , the head portions  102   c ,  102   d  engage a ledge  106   a  in a normal or home position (see also  FIG.  12   ). Simultaneously depressing or squeezing the members  102   a ,  102   b  toward one another, as indicated by opposed action arrows A in  FIG.  3   , allows the head portions  102   c ,  102   d  to move radially inwardly for passing outwardly in an axial direction through the passage  108 , thereby releasing the removable portion  102  from the grip  106 . 
     Initial or reinsertion of the removable portion  102  to connect with the grip  106  proceeds in a similar manner, with initial depression of the members  102   a ,  102   b , but with insertion inwardly in the axial direction and then release to form the connection. As can be appreciated, this allows for reuse of the sampler  100  by adding a replacement removable portion  102  once used. This also potentially allows for retrofitting in connection with the grip  106  (which as outlined herein may also form part of a cap for sealing a port on the bioreactor). 
     As perhaps best understood from  FIGS.  5 ,  5 A, and  5 B , the sample portion  116  includes a substrate  116   a  for growing cells, which may be as previously described. The substrate  116   a  may be positioned within an opening O or window in a frame  116   b  of the sample portion  116 , and may be recessed within it (see  FIGS.  5 A,  5 B , showing recessed substrate mounting portions  116   c  associated with the frame  116   b , and  FIG.  6   , noting thin profile of frame  116   b  when including the recessed). This may promote fluid flow around or along the substrate  116   a  when positioned within a cell culture bed, such as between layers of a fixed bed. A tip  116   d  of the sample portion  116  may be chamfered, pointed or sharpened to facilitate insertion between adjacent layers of the fixed bed with minimal or no disruption. The frame  116   b  may also be sufficiently rigid to facilitate insertion into the cell culture bed, such as between layers of it, without bending or folding, thereby ensuring that the substrate  116   a  remains present in the optimum position for cell attachment and growth once positioned in the bed. 
     In order to undergo further processing, the sample portion  116  may be releasably attached to the sampler  100 . In one example, this releasability is achieved by providing a frangible connection  124  between one end of the sample portion  116  and the body  104  of the removable portion  102 . This frangible connection  124  may be achieved by providing two connecting portions of material that can be easily broken away to separate the sample portion  116  from the body  104 . This allows for the simple and rapid detachment, and thus, release, of the sample portion  116  once removed from the cell culture, such as in a manner outlined further in the following description. As noted above, replacement of the removable portion  102  with a new sample portion  116  then allows for the sampling to be repeated anew, if desired. 
     In order to recover cells from the sample portion  116  to understand the cell density or other aspects of the culture, a container  200 , such as an EPPENDORF tube, may be provided for receiving the sample portion  116 , possibly along with a solution for promoting detachment of cells from the substrate  116   a . A removable insert  202  may be adapted for positioning within the container  200 . This insert  202  may comprise a passage, such as a slot  203 , for receiving the sample portion  116 . The insert  202  may be sized and shaped to occupy a substantial volume of the interior compartment of the container  200  (together with the sample portion  116  when present; see  FIG.  10   ), thereby reducing the amount of cell detachment solution required to be present without hampering cell detachment. 
     The insert  202  may also be adapted to assist in releasing the sample portion  116  from the sampler  100 . For example, as shown in  FIGS.  7  and  8   , upper end portions  202   b  of the insert  202  may be chamfered, such that a ledge  202   c  is created. Thus, when the sample portion  116  still connected to the sampler  100  is inserted into the insert  202 , as shown in  FIG.  9   , the removable portion  102  of the sample portion  116  may be moved in a direction toward and/or away from the ledges  202   a  (note arrow B in  FIG.  9 A ), possibly in a repeated fashion. The resulting bending of the material assists in breaking the two apart, such as along the frangible connection  124 , and thereby releases the sample portion ( 116 ′ in  FIG.  10   ) into the container  200 . The container  200  including the insert  202 , sample portion  116 , and any detachment solution may then undergo further processing, such as collecting the removed cells using centrifugation. Instead of a frangible connection  124 , any form of releasable attachment may serve to provide the releasability of the sample portion  116  from the sampler  100 . 
       FIG.  11    shows a bioreactor  12  with a fixed bed  18  including a sample portion  16  connected to a positioner  20 . The positioner  20  may be connected to or accessible via a cap  14  covering a port on the bioreactor lid  24 . This figure illustrates in particular one exemplary flow pattern through the bioreactor, which may be caused by an internal agitator, such as an impeller I. The flow arrangement may be such that fluid passes downwardly (arrow D) through an inner chamber, and then in a forward run (arrow F) to pass upwardly (arrow U) through the fixed bed  18  located outboard of the inner chamber. Flow is then returned (arrow R) via an internal column, where it may be exposed to a gas (air). As can be appreciated, with the same structural arrangement, the flow could be reversed. 
     As can be appreciated from the cross-sectional view of  FIGS.  12 ,  12 A, and  12 B , the sampler  100  may be integrally formed with one or more of the caps  14  for closing the ports of the bioreactor  12 . For example, the cap  14  may comprise the grip  106  for receiving the removable positioner  120  connected to the sample portion  116  positioning within the cell culture, such as between layers of a fixed bed  118  (illustrated as a spiral configuration). The cap  14  may further include a seal  140  for sealing the associated port when positioned therein to assist in maintaining aseptic conditions. 
     With further reference to  FIGS.  13  and  14   , the sampler  100  including the grip  106  may have a locked position in which it is secured within a port  122  ( FIG.  13   ), and an unlocked position where it may be withdrawn from the port ( FIG.  14   ), which may be associated with a lid or cover of the bioreactor. As can be seen in  FIGS.  12 B and  14   , the portion of the sampler  100  within the port  122  may include a connector, such as a radially outwardly directed projection  148  adapted to engage a mating connector, such as a radially inwardly directed projection  150  within the port  122  when the sampler  100  is rotated to a certain position, thereby preventing withdrawal. Rotation of the sampler  100  within the port  122  then releases the engagement between the projections  148 ,  150  from engagement and allows for the withdrawal of the sampler  100 . Since the removable portion  102  is not directly attached to the grip  106 , interference with the position of the sample portion  116  in the bed is not impacted by this rotation. 
     Likewise, during installation, the sampler  100  may be inserted into the port  122  with the projections  148 ,  150  out of alignment. The sampler  100  may then be rotated to form the locking engagement, again without disturbing the bed. The locked and unlocked positions may be indicated by indicia, such as on the grip  106 , to facilitate the correct positioning for achieving the desired positioning. 
     While the bioreactor  12  is shown in a vertical orientation, it could be used in any orientation including for any layers present. For example, in  FIG.  15   , the bulk flow direction of the bioreactor  302  is in a direction from the inlet  310  to the outlet  312 , and, in this example, the first and second major sides of the layers  308  are perpendicular to the bulk flow direction. The sampler  100  may in such case may be passed through a lid or cover  24  through openings in the layers  308 . 
     In contrast, the example shown in  FIG.  16    is of an embodiment in which the bioreactor  322  and stack of layers  328  within the culture space  324  have first and second sides that are parallel to a bulk flow direction, which corresponds to a direction shown by the flow lines into the inlets  330  and out of the outlets  332 . The sampler  100  may pass through a port  22  in a sidewall of the bioreactor  322  to position the sample portion  116  between adjacent layers, but could also pass through the lid as noted. 
     A method of obtaining a sample of a cell culture in a fixed bed bioreactor may include using a sampler at least partially positioned in or adjacent to the fixed bed to promote the growth of cells on a releasably attached portion of the sampler. The method may involve initiating the cell culture process in the bioreactor, and allowing the process to run for a period of time to permit cell growth in the fixed bed and the releasably attached portion. The sampler may then be withdrawn from the bioreactor, and the releasably attached portion detached from the sampler (which may involve inserting the releasably attached portion into an opening of an insert in a container, and using the insert to break the portion from the sampler). 
     As used herein, the following terms have the following meanings: 
     “A”, “an”, and “the” as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a compartment” refers to one or more than one compartment. 
     “About,” “substantially,” or “approximately,” as used herein referring to a measurable value, such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/−20% or less, preferably +/−10% or less, more preferably +/−5% or less, even more preferably +/−1% or less, and still more preferably +/−0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier “about” refers is itself also specifically disclosed. 
     “Comprise”, “comprising”, and “comprises” and “comprised of” as used herein are synonymous with “include”, “including”, “includes” or “contain”, “containing”, “contains” and are inclusive or open-ended terms that specifies the presence of what follows and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein. 
     While certain preferred embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. For example, other forms of frangible connections may also be used, such as for example a perforated tear line, zipper, latch and hinge (living or otherwise), or other similar arrangements. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the protection under the applicable law and that methods and structures within the scope of these claims and their equivalents be covered thereby.