Patent Description:
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 at any point before, during or after the culture process for various purposes (e.g., to take cell-associated measurements such as those relating to viability and density). One common way to take such an in-process sample from the fixed bed is to reach inside the bioreactor with a tool such as a tweezer during operation, and to manually extract a piece or portion of the bed. This operation is difficult as it requires careful dexterity. Also, it invariably causes undesirable perturbations that risk disrupting the cell culture environment, as well as the sample specimen. <CIT> describes a sampling device and method for sampling a bioreactor. The bioreactor of <CIT> is a packed bed bioreactor containing packed cell culture matrices. <CIT> describes a basket containing pre-embedded cell culture matrices to provide additional matrices for the cells to grow on besides the cell culture matrices inside the bioreactor. However, the sampling device of <CIT> cannot function adequately in combination with a structured fixed bed comprising at least two layers of material adjacent to each other, as the basket would severely disrupt the structured fixed bed due to the damage caused by the basket being inserted in the structured fixed bed.

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 where the freedom of movement and access of the operator for such a maneuver is limited. While a small scale bioreactor can be placed in such a containment unit, a large production-scale version cannot readily be placed in such cabinets to achieve this result. Additionally, sterility must be maintained during the whole operation, which means the operator collecting the sample has to follow precise aseptic operating procedures. This is challenging when having to introduce an extraction tool such as a tweezer.

Sterile (or aseptic) sensors, such as so-called "biomass" sensors have been proposed for assessing cell density. However, these 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 is far less accurate than direct examination of cells. Also, 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 a device that provides the ability to take a reliable sample from the structured fixed bed comprising at least two layers of material adjacent to each other and the cells associated therewith. The device would allow for the sample to be obtained in an easy and inexpensive manner, while maintaining aseptic conditions so as to protect against contamination (both internal to the bioreactor and external to it) and to avoid creating deleterious disruptions of the structured fixed bed cell culture environment.

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, while maintaining aseptic or sterile conditions, and also while avoiding creating deleterious disruptions of the structured fixed bed cell culture environment.

According to one aspect of the disclosure, a sampler is provided for a structured fixed bed comprising at least two layers of material adjacent to each other in which cells are grown, such as a cell culturing device or bioreactor. The sampler is proposed in several formats: (<NUM>) one which allows the operator to take a sample in a non-sterile manner (such as when the bioreactor is present in a laminar flow cabinet or a biosafety cabinet); (<NUM>) one which allows the operator to take a sample in a sterile manner; and (<NUM>) another which allows a larger volume or portion of the fixed bed to be extracted, an operation desirable at an or the end of a cell culture process. For the latter versions, the sterility of the sampling process itself ensures that the bioreactor is not constrained to being operated in a sterile environment, while ensuring protection both for the process and the environment. The sampler thereby improves the operability of the bioreactor by facilitating: (a) the operation required to take a sample from the structured fixed bed cell culture (manually, or possibly via partial or full automation) without deleterious disruption or effects, and (b) the containment and sterility constraints associated with fixed bed bioreactors, in such a way that it can be operated outside of a strictly sterile zone without risking contamination of the bioreactor itself or the sample by the environment, and the environment by the content of the bioreactor or the sample. However, a proposal is also made for a sampler that can be used in a situation where the cell culturing device (bioreactor) is operated in a sterile environment as well (e.g., a small size (e.g., <<NUM> m2 surface area) bioreactor that can be used in connection with an isolator, cabinet, or the like).

According to one aspect of the disclosure, an apparatus for use with a cell culture system, such as for example a bioreactor, fermenter, or the like, is disclosed. The apparatus comprises a structured fixed bed comprising at least two layers of material adjacent to each other including a removable sample portion for recovering a sample of cells from the cell culture system, the removable sample portion being located at least partially between the at least two layers.

The structured fixed bed comprises at least two layers of material adjacent to each other, the removable sample portion being located at least partially between the at least two layers. In some embodiments, the at least two layers comprise: (<NUM>) cell immobilization layers, and the removable sample portion has a first side in contact with a first cell immobilization layer and a second side in contact with a second cell immobilization layer; or (<NUM>) one cell immobilization layer and one spacer layer. In some embodiments, the removable sample portion comprises one or more fibers, and in some embodiments, removable sample portion comprise a non-woven material. In any embodiment, the removable sample portion may comprise a sheet of material. In some embodiments, the structured fixed bed comprises a cell immobilization layer, and the sheet of material forming the removable sample portion is in direct contact with the cell immobilization layer.

In some embodiments, the apparatus comprises a positioner mechanically connected to the removable sample portion by a connector. In some embodiments, the structured fixed bed comprises a plurality of removable sample portions. In some embodiments, the structured fixed bed comprises a roller or spiral bed, and in some embodiments the removable sample portion is adjacent to the structured fixed bed, and in some embodiments the removable sample portion is a perforated or pre-cut portion of the structured fixed bed.

The apparatus of any disclosed embodiment may be applied to a bioreactor. In one exemplary form, the bioreactor comprises an outer chamber for receiving the structured fixed bed with an upward flow of fluid. An inner chamber is provided for returning fluid flow to a lower portion of the bioreactor including an agitator.

According to another aspect of the disclosure, an apparatus for use with a bioreactor for growing a cell culture is provided. The apparatus comprises a structured fixed bed comprising at least two layers of material adjacent to each other and includes a removable sample portion comprising one or more fibers for recovering a sample of cells from the cell culture, the removable sample portion being located at least partially between the at least two layers.

The fixed bed comprises a structured fixed bed comprising at least two layers of material adjacent to each other. In some embodiments, the removable sample portion comprises a non-woven material. In some embodiments, the removable sample portion is between two layers of the fixed bed, and in some embodiments, the removable sample portion is adjacent to the fixed bed (or both approaches are combined). In these or other embodiments, the removable sample portion is a perforated or pre-cut portion of the structured fixed bed.

In some embodiments, the sample portion may be in contact with the fixed bed in a bioreactor and need not be inserted or positioned within it. In some embodiments the sample portion may be positioned adjacent to and in contact with a surface of the fixed bed, such as a side surface or an upper surface thereof, and connected to the positioner. In some embodiments the sample portion comprises a folded portion. In some embodiments, the sample portion comprises a folded portion and a welded portion.

According to yet another aspect of the disclosure, an apparatus for sampling a cell culture is provided. The apparatus comprises a bioreactor comprising a structured fixed bed comprising at least two layers of material adjacent to each other including a removable sample portion. A sampler is associated with the bioreactor for recovering the removable sample portion from the structured fixed bed.

In some embodiments, the removable sample portion includes a positioner adapted for positioning the sample portion within the structured fixed bed, the positioner being accessible via a port in the bioreactor. In some embodiments, a support is associated with the port by way of a releasable connection, such as a bayonet fitting including a slot on the support and a post on the port, a threaded connection, or a releasable latch, the support being connected to the positioner. In some embodiments, the support is fixed to the positioner, or releasably connected to the positioner, such as by a flexible portion for releasably engaging a portion of the positioner for movement together in an axial direction, but allowing for the support to rotate without imparting rotation to the positioner. In some embodiments, the support comprises a frangible connection for separating the support into multiple portions for removal of the removable sample portion.

In some embodiments, the sampler comprises a cutter for forming the removable sample portion of the structured fixed bed. In some embodiments, the sampler comprises a groove for engaging a locking pin associated with the port for guiding the sampler into position.

In some embodiments, a container is provided for maintaining a sterile condition of the removable sample portion when removed from the bioreactor. In some embodiments, the container connects to the bioreactor via an aseptic connection so as to maintain a sterile condition within a compartment defined by the container and including the sampler, and also a sterile condition of the bioreactor. In some embodiments, the container comprises a septum for receiving a connector connected to the removable sample portion. In some embodiments, the container comprises a flexible sleeve, which in some embodiments is connected to a rigid elbow.

In some embodiments, the removable sample portion includes a positioner connected to the removable sample portion, the positioner being associated with an actuator for withdrawing the removable sample portion from the structured fixed bed. In some embodiments, the actuator comprises a stator and a rotor. In some embodiments, the actuator is connected to a sleeve for receiving a positioner connected to the sample portion. In some embodiments, the stator comprises a flexible bag surrounding the rotor. In some embodiments, the stator comprises a cradle for cradling the rotor.

In some embodiments, a controller is provided for controlling the actuator. In some embodiments, the controller is adapted for receiving an output signal from at least one sensor associated with the bioreactor.

In some embodiments, the removable sample portion is connected to a positioner forming a releasable connection with a cap for a port of the bioreactor. In some embodiments, the releasable connection comprises a magnetic coupling. In some embodiments, the port includes a receiver for receiving a projection of the positioner in a manner that prevents relative rotate.

In any disclosed embodiment, the structured fixed bed may comprises a roller or spiral bed. In some embodiments, the removable sample portion is adjacent to the structured fixed bed. In some embodiments, the removable sample portion is a perforated or pre-cut portion of the structured fixed bed.

Still further, the disclosure pertains to an apparatus for sampling a cell culture. The apparatus comprises a bioreactor comprising a structured fixed bed comprising at least two layers of material adjacent to each other including a removable sample portion and a sampler associated with the bioreactor for recovering the removable sample portion from the bed. The sampler comprises a positioner releasably connected to the removable sample portion.

In some embodiments, the positioner is releasably connected to the removable sample portion by a clamp. In some embodiments, the removable sample portion is within or adjacent to the structured fixed bed. In some embodiments, the removable sample portion is a perforated or pre-cut portion of the structured fixed bed.

Yet another aspect of the disclosure pertains to an apparatus for sampling a cell culture associated with a structured fixed bed comprising at least two layers of material adjacent to each other in a bioreactor having a port. The apparatus comprises a sampler associated with the bioreactor for recovering a sample portion of the bed via the port, the sampler including an actuator for withdrawing the sample portion from the bed.

In some embodiments, the actuator is connected to a container for receiving a positioner connected to the sample portion. In some embodiments, the actuator comprises a stator and a rotor. In some embodiments, the stator comprises a flexible bag surrounding the rotor.

In some embodiments, the stator comprises a cradle for cradling the rotor. In some embodiments, a controller is provided for controlling the actuator. In some embodiments, the controller is adapted for receiving an output signal from at least one sensor associated with the bioreactor.

The bed comprises a structured fixed bed including at least two layers, and the sample portion comprises a sheet of material located between at least two layers. In some embodiments, the removable sample portion is within or adjacent to the structured fixed bed. In some embodiments, the removable sample portion is a perforated or pre-cut portion of the structured fixed bed.

A further aspect of this disclosure pertains to a method of sampling a cell culture in cell culture system, such as a bioreactor having a structured fixed bed comprising at least two layers of material adjacent to each other. The method includes the step of recovering a removable sample portion from the structured fixed bed of the bioreactor. The structured fixed bed comprises at least two layers, and the method comprises positioning the removable sample portion at least partially between the at least two layers prior to the recovering step.

In some embodiments, the recovering step comprises withdrawing a positioner connected to the removable sample portion from the bioreactor until the removable sample portion is received in a container connected to the bioreactor by an aseptic connector. In some embodiments, the withdrawing step comprises using an actuator controlled by a controller based on a sensed condition of the bioreactor. In some embodiments, any or all of the following steps are performed: (<NUM>) counting the cells removed on the removable sample portion; (<NUM>) coloration of the cells or (<NUM>) extracting intra-cell viruses from the cells removed on the removable sample portion.

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:.

Reference is now made to <FIG>, which illustrates a sampler <NUM> for a cell culture system, such as for example a bioreactor <NUM>, fermenter or the like, according to one aspect of the disclosure. For purposes of this example, the bioreactor <NUM> is presumed to be in operation under external sterile conditions in a containment unit (such as a laminar flow cabinet, external isolator or the like). However, as outlined further in the description that follows, the sampler <NUM> may also be used in other environments.

The sampler <NUM> may comprise a support portion for associating with an opening in the bioreactor <NUM> providing access to an interior compartment thereof. In the illustrated embodiment, the support portion comprises a cover or cap <NUM> for sealing the opening in the bioreactor <NUM>, and thus maintains the sterility of the interior compartment, even when not in a containment unit. The support portion may support a removable sample portion <NUM> for positioning within a fixed bed <NUM> in the interior compartment of the bioreactor <NUM>.

The sample portion <NUM> 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 <NUM> may alternatively comprise a portion or part of the fixed bed <NUM> with perforations, tear away seams or other means for separating from the rest of the fixed bed. Alternatively, the sample portion <NUM> may be a separate structure for positioning within the fixed bed <NUM> during the process of assembling the bioreactor <NUM> (as outlined further in the following discussion). In this case the sample portion <NUM> may be adapted for detachment and removal from the rest of the fixed bed (such as by providing perforations or tear-away seams in the fixed bed, or pre-cutting the fixed bed or other means for separating samples to create one or more removable (detachable) portions).

The sample portion <NUM> may be removably attached to a positioner <NUM> to facilitate holding the sample portion <NUM> in an appropriate position in the fixed bed <NUM> and also removing it from the fixed bed <NUM>. Prepositioning of the sample portion <NUM> within the fixed bed <NUM> may be achieved by hand via the positioner <NUM>, or by using a tool, such as tweezers (not shown) to pass the sample portion <NUM> into the fixed bed <NUM> through a port <NUM> in the lid <NUM> of the bioreactor <NUM> (or by removing the lid entirely). This may be done prior to the commencement of the bioprocessing operation, even during assembly of the bioreactor or manufacturing and assembly of the fixed bed.

The fixed bed <NUM> may comprise any substrate for achieving cell growth or cell immobilization, and consists of 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) comprising at least two layers of material adjacent to each other. The material of the fixed bed <NUM> 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>, the structured fixed bed comprises a non-woven material 18a arranged in at least two layers on and within which cells grow. These layers are thus considered cell immobilization layers. In the particular form shown, the non-woven material 18a is arranged in a rolled or spiral fashion, with each of the two cell immobilization layers separated by spacer layers 18b, which promote culture and media flow between the various layers. The sample portion <NUM> may be a pre-defined portion of a cell immobilization layer. It may be a separate substrate positioned (as shown) between two of the layers of the bed <NUM> in rolled or spiral form, such as cell immobilization (or non-woven material) layers 18a. Alternatively, the sample portion may be located between a spacer layer and an adjacent cell immobilization layer. The fixed bed <NUM> 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 <NUM> may be associated with a fixed bed <NUM> of any known form (e.g., positioning within a fiber matrix, or an arrangement of layers that is not spirally wound as shown). <FIG> illustrates a non-structured fixed bed not being part of the invention, which may comprise a plurality of randomly oriented fibers 18c. In this embodiment, the sample portion <NUM> may comprise one of the fibers 18c. This fiber 18c may be connected to the positioner <NUM>, as indicated (or as a substrate of non-woven fibers). Alternatively, more than one fiber can be connected to the positioner <NUM> and act as the sample portion (such as a non-woven material, as noted previously).

While <FIG> illustrates a rolled, generally cylindrical arrangement for a structured fixed bed <NUM> with at least two layers (including a cell immobilization and spacer layers 18a, 18b), it should be appreciated that other forms may be used. Thus, with reference to <FIG>, the fixed bed <NUM> may comprise a plurality of concentric layers 18a that are continuous, and may be in any shape (e.g., circular, square). Alternatively, the layers 18a may be linear. In any of these cases, spacer layers 18b may or may not be provided, and the sampler portion <NUM> may be located at any location (or locations) within the bed <NUM>, without limitation, depending on the desired approach to sampling.

Returning to <FIG>, an anchorage point, such as a sidewall of the port <NUM> associated with the lid <NUM> of the bioreactor <NUM>, may be adapted to form a seal with the cover or cap <NUM> to ensure that sterility is maintained. The cap <NUM> may be releasably connected to the port <NUM>. This may be achieved by threading or other means of secure, but releasable connection (e. g, a bayonet fitting, as outlined further 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 <NUM> in the illustrated format, the positioner <NUM> may extend a distance sufficient to ensure that the sample portion <NUM> is at least partially positioned within the fixed bed <NUM>. As can be appreciated, this distance may vary depending on the size and shape of the bioreactor <NUM>, as well as the desired location for the sampling to occur, the type of bed, or the type of culture. The positioner <NUM> may be connected to the cover or cap <NUM> serving as the support, but may also be separate therefrom (such that the cap <NUM> may be removed while the positioner <NUM> remains in position, and can then be withdrawn and the cap replaced). A structured fixed bed is suited for the latter version, since the sample portion <NUM> may be pre-positioned within the structured fixed bed layers and held therein, and the positioner <NUM> later associated with the cap <NUM> (if desired).

In any disclosed embodiment, the spacer layers and/or the cell immobilization layers which make up respectively the spacer section and the immobilization section in the matrix assembly are preferably made of a biocompatible polymer selected from polyester, polyethylene, polypropylene, polyamide, plasma treated polyethylene, plasma treated polyester, plasma treated polypropylene or plasma treated polyamide. Said layers can be hydrophilic or hydrophobic. The cell immobilization layers are preferably hydrophilic.

The thickness of both layers will advantageously be between <NUM> and <NUM>, more preferably between <NUM> and <NUM> or between <NUM> and <NUM>. Suitable material for the cell immobilization layer may be a woven or nonwoven material. By preference, a nonwoven material is used. 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 used in the current invention may be chosen from a polyethyleteterepthalate nonwoven. The nonwoven material may be plasma treated to enhance cell adherence and flow.

The spacer layers may consist of 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 can take many forms depending on the application and type of bioreactor. In an embodiment of the current invention, 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. The alternately positioned sections may alternate in vertical position as shown in the figures (see further) or in a horizontal position according to the use of the matrix and/or to the bioreactor in which the matrix will be 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 required in order creating a stack of several immobilization and spacer sections. Ideally, the end configuration may comprise between <NUM> and 500alternations 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 number of layers used in both 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 <NUM> and <NUM>, more preferably between <NUM> and <NUM>, even more preferably between <NUM> and <NUM>.

As mentioned, the presence of the spacer sections creates space inside the matrix through which the culture medium flows. This provides improved circulation of the culture medium through the matrix thereby reaching all cultured cells. This effect is even more enhanced in the embodiment wherein the spacer section comprises one spacer layer and the immobilization section comprises two immobilization layers. The culture medium flowing inside the matrix via the spacer sections is tangentially oriented with respect to the cell immobilization sections.

Examples of releasable connections between the positioner <NUM> and the support for it (cap <NUM>) are shown in <FIG>, <FIG>, and <FIG>. In <FIG>, the cap <NUM> includes flexible projections 14a forming a receiver 14b for receiving and retaining a portion <NUM> of the positioner <NUM> (which portion <NUM> may also form part of a separate structure, such as a sleeve). As can be appreciated, the portion <NUM> within the receiver 14b is retained against movement in an axial direction of the positioner <NUM> unless sufficient force is generated to separate the projections, yet allows for free rotation. Hence, the cap <NUM> may be rotated for removal (if a bayonet or threaded connection) without causing the positioner <NUM>, and hence the sample portion <NUM>, to rotate relative to the fixed bed <NUM>, which might cause an undesirable disruption or result. The cap <NUM> can still be withdrawn in the vertical direction, though, including with the positioner <NUM> remaining attached, or separated therefrom (in which case it can be accessed and removed independently of the cap). Likewise, the positioner <NUM> with the sample portion <NUM> may be positioned in the bioreactor <NUM>, and the cap <NUM> then connected to the positioner <NUM>. Of course, the arrangement could also be reversed, such that the projections are on the sleeve/positioner, and the portion for engagement is on the positioner.

<FIG> illustrates a version in which a removable part 14c includes the projections 14a forming the receiver 14b. Fasteners, such as screws 14d, clips, or snaps, may be used to secure the part 14c to the cap <NUM>. Using this arrangement, the part 14c may be released before the cap <NUM> is removed, and the cap <NUM> may be associated with part 14c once in place. Seals, such as O-rings 14e, may also be provided for sealing the interface created with the exterior environment through the openings for the fasteners/screws 14d.

<FIG> illustrates another version in which the part 14c is adapted for passing through an opening O in the cap <NUM>. An external fastener, such as a threaded nut 14f may then be used to secure the detachable part 14c to the cap <NUM>, such as by threading along a shank <NUM> thereof. In this manner, the part 14c may be selectively associated with the cap <NUM> during placement or removal, and without disturbing the position of the positioner <NUM> and sample portion <NUM>.

As indicated, cells may be cultured in the fixed bed <NUM>, as well as on the sample portion <NUM> as a result of its positioning. When external examination of the cell growth is desired, the sample portion <NUM> may be removed from its location in the fixed bed <NUM>. In one example, this may be achieved by removing the cap <NUM> from the port <NUM>. This removal is shown in <FIG> as involving rotation of the cap <NUM> in one direction (note arrow R) to release a connection (as discussed below), and then raising it in a vertical direction V. Port <NUM> could also be associated with a sidewall of the bioreactor <NUM> or any other part, in which case the movement would be primarily horizontal. Likewise, the cap or cover <NUM> could also form a friction fit with an associated part of the bioreactor <NUM>, such as the port <NUM> or another part of the lid <NUM> for providing access to the fixed bed <NUM>, in which case only vertical movement may be used to remove the cover <NUM>.

The positioner <NUM> may be removed from bioreactor <NUM> as well to recover the sample portion <NUM> from the fixed bed <NUM>, without requiring an enhanced level of coordination or dexterity, and without the need for using a tool that can disrupt the surrounding material of the fixed bed <NUM>. Examination of the sample portion <NUM> may then be done external to the bioreactor <NUM> to evaluate the cells thereon, which would be directly representative of the corresponding cell growth in the fixed bed <NUM>. Optionally, a second cover or cap <NUM> may then be used to seal port <NUM>, and a new sample portion <NUM> may also be positioned within the fixed bed <NUM> via an associated positioner in case a desire exists to repeat the sampling operation.

Turning to <FIG>, a second embodiment of the sampler <NUM> is shown, for possible use in environments where the bioreactor <NUM> is internally sterile, but the external environment is not sterile (e.g., where the bioreactor <NUM> is of a large scale or size, making use of a conventional isolator or cabinet difficult, costly or impossible). In this case, the sample portion <NUM> (as well as the bioreactor <NUM>) must be protected against contamination during removal and prior to inspection of the sample portion.

In this embodiment for maintaining the sterile conditions, the basic parts are essentially the same as above, but an additional partition or barrier, optionally in the form of a flexible sleeve <NUM>, is provided. This sleeve <NUM> forms a sterile barrier between the anchorage point, such as port <NUM>, and the cover or cap <NUM>. This sleeve <NUM> may be formed of an elongated piece of flexible material, such as a thin, flexible polymer material, and may be arranged so as to not interfere with the connection between the cap <NUM> and the port <NUM>.

When it is desired to take a sample of the fixed bed <NUM> during the culturing process, the cap <NUM> is released and removed. Consequently, the sample portion <NUM> is withdrawn from the fixed bed <NUM> via positioner <NUM>, but remains under sterile conditions as a result of the surrounding sleeve <NUM> and the seals established with cap and port. A portion of the sleeve <NUM> may then be sealed, either mechanically or by welding (e.g., using heat), as indicated by arrow S in <FIG>, which also seals a remaining portion of the sleeve for covering the opening to the bioreactor <NUM> (e.g. port <NUM>). Other examples of sealing means in the form of an aseptic connection include the Quickseal or Clipster technologies distributed by Sartorius (see, e.g., <CIT> and <CIT>, the disclosures of which is incorporated herein by reference), or a mechanical seal between inner portions of the sleeve <NUM> could be provided, similar to a "Zip-Lock" style of resealable, flexible bag. In any case, the sample portion <NUM> remains under sterile conditions in a compartment thus formed as a result of the severing/sealing of the sleeve, yet can be transported for further examination or processing, and the contents of the bioreactor <NUM> also remain under sterile conditions with the cap or cover replaced. In situations where the external environment is sterile, then the sleeve <NUM> may be omitted, yet the same parts used to perform sampling in the desired manner.

It is sometimes desirable to sample a portion of the fixed bed itself, such as by cutting it out and extracting it - at the end of a cell culture process when more cells are needed for sampling. One could attempt to add material or the number of fibers to the sample portion to increase the number of cells available for analysis in a sample. However, achieving the required scale may be difficult using this methodology. In this regard, a third embodiment of the sampler <NUM>, which is shown in <FIG>, may receive and recover or extract a relatively larger sample portion 216a of the fixed bed <NUM> in order to more effectively harvest a larger number of cells. In the illustrated embodiment, the sample portion <NUM> is connected to the cover or cap <NUM> associated with a port <NUM>.

In one embodiment, the sampler <NUM> includes a cutter <NUM> at one end portion for engaging with a portion of the fixed bed <NUM> to sever or detach it and form the sample portion 216a. The distal end of the cutter <NUM> may be adapted for cutting the material of the fixed bed <NUM>, and may have a pointed end, blade, or both, essentially like a needle, and the interior of the sampler <NUM> may be at least partially tubular or hollow for capturing the portion of the fixed bed <NUM> and removing it once detached. Initially, the cutter <NUM> may be held in a position at least partially within the bioreactor <NUM>, but spaced from the fixed bed <NUM>, such as by retainer in the form of a locking pin <NUM>. The locking pin <NUM> may associate with a rail or channel <NUM> of the cutter <NUM> extending generally in a direction corresponding to the direction of relative movement with the bioreactor <NUM>.

The sample portion 216a may be used in a situation where the bioreactor <NUM> is in a sterile environment, but may otherwise include a sleeve <NUM> for maintaining such an environment in the space surrounding the cutter <NUM>. When it is desired to remove a sample of the fixed bed <NUM>, the cutter <NUM> may be manipulated such that the locking pin <NUM> (shown at the home position) travels within the channel <NUM> until the cutter <NUM> at least partially plunged into the material of the fixed bed <NUM> (note down arrow D and rotational arrow R), thus capturing a "core" sample of the fixed bed in an interior hollow compartment of the cutter <NUM>. As can be appreciated, the travel distance of the cutter <NUM> may be regulated by simply altering the dimensions and geometry of the channel or groove <NUM>. The cutter <NUM> may then be withdrawn from the port <NUM> (up arrow U and opposite rotation arrow L, but again the movement could be up only in the event of a different connection, such as a friction fit), and the sleeve <NUM> severed/sealed in the manner described above to maintain the sterile condition (both with respect to the cutter <NUM> and the bioreactor <NUM>).

In another possible version, as shown in <FIG>, the sampler <NUM> may be pre-positioned within the fixed bed <NUM> (structured or otherwise). Within its interior, the sampler <NUM> may include a pre-cut or pre-assembled substrate or material (e.g., a nonwoven material) serving as a sample portion <NUM> for entrapping or growing cells, and thus the body <NUM> of the sampler may function as a positioner (similar to positioner <NUM> of sampler <NUM>). The material forming the body <NUM> of the sampler <NUM> may include perforations P to allow for cell culture fluid to flow into the interior of the body and thus to any cells entrapped or grown on the material of the sample portion <NUM>. Removal of the sample portion <NUM> from the fixed bed <NUM> may be done in the manner shown in <FIG>, if maintaining sterility is required, or in any other manner shown herein.

Still another option is for the <FIG> version to be used without providing a cutter <NUM> on the sampler <NUM>, as shown in the side and top views <FIG>. Rather, a portion of the fixed bed <NUM> may be pre-positioned and adapted for removal as the sample portion <NUM> when received in the interior of the sampler <NUM>. The interior of the body of the sampler <NUM> may include grippers, barbs or other engaging structures to engage and remove the sample portion <NUM> Again, sterile conditions may be maintained by using the sleeve <NUM> shown in <FIG>.

<FIG> illustrate one exemplary embodiment of the cover or cap <NUM> (or <NUM>, <NUM>) for use in connection with the port <NUM> (or <NUM>, <NUM>). The cap <NUM> may include a labyrinth (e.g., L-shaped, X-shaped, S-shaped, or like serpentine) slot <NUM> for mating with a corresponding post 22a extending radially from an upstanding portion of the port <NUM>, and thus form a releasable bayonet-style fitting. The cap <NUM> may also include a tab <NUM> to facilitate manual grasping for removal, and may be reusable/resealable. As can be appreciated, a plurality of these caps <NUM> may be provided on a single lid <NUM> to create a variety of sampling options at different locations within the fixed bed, as shown in <FIG>.

Referring to <FIG>, and using the <FIG> embodiment as an example, an arrangement for a temporary support <NUM> associated with a port <NUM> in the bioreactor lid <NUM> is illustrated. The support <NUM> is arranged to receive the sampler <NUM>, which includes an oversized head 200a for engaging the support <NUM>. The sleeve <NUM> is connected between the bioreactor lid <NUM> and the sampler <NUM>, such as along the underside surface of the oversized head 216a. The pin <NUM> and groove <NUM> from <FIG> can also be seen, which correspond to the sampler <NUM> in the home or raised configuration.

When it is desired to sample the fixed bed <NUM>, the support <NUM> may be removed to allow for the sampler <NUM> to advance. This may be achieved by cutting through the sidewall, but in one particular embodiment, a frangible connection is established by a pull <NUM> embedded in the sidewall that, when manipulated, breaks the corresponding connection between portions of the support <NUM> along a vertical line. This allows for the support <NUM> to be removed from the supporting position, and the sampler <NUM> may then be used as per the indication in <FIG>, yet reliable support is provided for the sample portion prior to use and the sterile conditions are fully maintained. Instead of a pull <NUM>, 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.

<FIG> shows an embodiment of the sample portion <NUM> of the <FIG> embodiment, and the positioner <NUM>, which is shown as being an elongated rod-like structure. The sample portion <NUM> may comprise the same or a similar material as the material of the fixed bed <NUM>, such as a nonwoven fabric of one or more fibers. In a simple form, as shown in <FIG>, the sample portion <NUM> may be temporarily affixed to the positioner <NUM> using a fastener, such as a crimped piece of metal <NUM> (see, e.g. <FIG>) to form a secure connection. While shown having a generally rectangular shape, the sample portion <NUM> in this or any embodiment may comprise any shape, thickness or dimension. This would permit cell association therewith in relation to the cell density of the entire fixed bed, without limitation. As shown in <FIG>, the positioner <NUM> may also be associated with the sleeve <NUM>, which as described above may be connected to the port of the bioreactor (not shown).

As indicated above, the sample portion <NUM> may be removably attached to the positioner <NUM> in a variety of ways, but which may be characterized as forming a clamp for clamping the two structures together. As shown in <FIG>, a distal end of the positioner <NUM> may comprise a removable portion <NUM> that may be fastened in place by fasteners F passing through the parts to sandwich a portion of the sample portion <NUM> therebetween. <FIG> shows an embodiment in which a clip <NUM> is clipped over the sample portion (not shown) and an oversized distal end portion 20a of the positioner <NUM>, which may be T-shaped. <FIG> shows that the portion <NUM> may be secured to the positioner <NUM> by welding or adhering (e.g., gluing), rather than via removable fasteners. <FIG> shows that the sample portion <NUM> could be molded directly to a distal end of the positioner <NUM>, such as by overmolding. A spring clamp <NUM> may also be used to clip the sample portion <NUM> to the positioner <NUM> as shown in <FIG>, and the arrangement of <FIG> uses a toothed wedge <NUM> and a pivotable clamp <NUM> to hold the sample portion <NUM> in place.

In a further example, <FIG> shows that the positioner <NUM> may be provided with a clamp in the form of a malleable end 20b (e.g., formed of Aluminum or other soft material) and clamped onto the sample portion <NUM> (which may have an oversized head 16a to facilitate the connection). <FIG> illustrates the manner in which a cap <NUM> may be connected to the positioner <NUM> after the installation of the bioreactor lid <NUM>, with the positioner extending through a port <NUM> therein to engage with the cap <NUM>, and thus facilitate removal of the sample portion <NUM>. <FIG> shows an arrangement wherein foldable wings 20c at the end of the connector may be folded into position over the sample portion <NUM> to temporarily hold it in place. Still further, the sample portion <NUM> may be held in position on the positioner <NUM> by positioning it in a bore <NUM> at the distal end of positioner <NUM>, the bore including a flexible clamp <NUM>, which is forced to a clamped position by an actuator <NUM>, as shown in <FIG>. <FIG> shows a similar arrangement in which an outer part <NUM> is used to cause an inner part <NUM> (either of which may connect to positioner, not shown) to clamp down onto the sample portion <NUM> and thus retain it in position (and which arrangement can be reversed by flexing the inner part to release the connection). <FIG> illustrates that a fastener <NUM> associated with the positioner <NUM> may be used to urge a holder <NUM> into a clamped position.

A method of assembling a bioreactor is also described. Using the <FIG> example simply for purposes of illustration, the bioreactor <NUM> may be assembled by positioning the material of the structured fixed bed <NUM> in an interior compartment thereof. In the case of a structured bed, such as in <FIG>, the sample portion <NUM> may be positioned in the bed, and attached to the positioner <NUM> before or thereafter. The lid <NUM> may be applied to the bioreactor <NUM> such that the positioner passes into or through port, and the cap <NUM> put in place (including by connecting it to the positioner <NUM>, if desired).

Referring now to <FIG>, a further embodiment of a sampler <NUM> for a bioreactor <NUM> is illustrated. In this embodiment, the sample portion <NUM> is positioned in the fixed bed <NUM>, as per the other embodiments, and attached at a proximal end to a positioner <NUM> in the form of a flexible wire (which may be metal, plastic, or the like). A sleeve <NUM> may be sealed to a port <NUM> of the bioreactor <NUM> at one end, which guides the positioner <NUM> to connect with a sealed cap <NUM> for sealing with the proximal end of the sleeve <NUM>. A coupling <NUM> may be formed by male and female parts <NUM>, <NUM> for securing the cap <NUM> to the sleeve <NUM>, and thus forming a seal that maintains the sterile condition of the bioreactor <NUM>.

In order to sample the fixed bed <NUM>, the sample portion <NUM> may be withdrawn by disconnecting the cap <NUM>, which may or may not be connected to the positioner <NUM>. If connected, the cap <NUM> may be pulled to withdraw the sample portion <NUM> from the fixed bed <NUM>, through the internal passage of sleeve <NUM>, and eventually out the proximal end thereof for access by the operator (or one or both the ends of the sleeve <NUM> may be sealed with the sample portion <NUM> therein). If not connected, then the cap <NUM> may be removed, and the positioner <NUM> manually withdrawn from the sleeve <NUM> to withdraw the sample portion <NUM>. In the event of the wholesale withdrawal of the sample portion <NUM>, the cap <NUM> may then be replaced to maintain the sterile condition, or the cap <NUM> may be placed on the port <NUM> if the sleeve <NUM> is removed.

The overhead space may in some circumstances be limited (such as when the bioreactor <NUM> is placed in a laminar flow cabinet), and so the sleeve <NUM> may be flexible and thus able to assume a depending position, as shown. This allows the operator to pull the positioner <NUM> for recovering the sample portion <NUM>, without the need to access a space above the bioreactor <NUM>. Instead of a fully flexible sleeve, it can be appreciated from <FIG> that the sleeve may include one or more ends, such as flexible ends 328a, 328b, which may be connected to one or more rigid portions, such as intermediate portion 328c or "elbow.

Turning now to <FIG>, it is possible to use an actuator <NUM> for pulling the positioner <NUM> through the sleeve <NUM> to recover the sample portion <NUM>. The actuator <NUM> may comprise a handle <NUM> connected to a rotor <NUM>, to which a proximal end of the positioner <NUM> is connected (such as by winding the wire forming it in this embodiment). A stator <NUM> may receive the rotor <NUM>, and include a seal <NUM> for permitting relative rotation while maintaining the sterile condition. A connector, such as a hose barb <NUM>, may also be provided on the stator <NUM> for coupling with the sleeve <NUM>.

Withdrawal of the sample portion <NUM> from the bed <NUM> is achieved by using the handle <NUM> to rotate the rotor <NUM>, which may be manually done or automated. The flexible wire serving as positioner <NUM> is thus wrapped around the rotor <NUM>, and the sample portion <NUM> withdrawn into the sleeve <NUM> (which, as noted above, may be sealed off distal of the location of the sample portion <NUM> therein to preserve sterility). As can be appreciated, this arrangement allows for the sterile condition to be maintained while the sample portion <NUM> is recovered.

An alternate version is shown in <FIG>. The components are essentially the same, as indicated, but the "stator" <NUM> is connected (such as by welding) to a flexible bag <NUM>, which surrounds the rotor <NUM> and maintains a sterile condition. The stator <NUM> may take the form of a cradle for rotatably supporting the rotor <NUM>. Recovering the sample portion <NUM> may be accomplished by using handle <NUM> to rotate the rotor <NUM> through the flexible bag <NUM>, which as in the earlier-described embodiment pulls the positioner <NUM> (by winding the wire on rotor <NUM>).

A further embodiment of a sampler <NUM> for sampling a fixed bed <NUM> in a bioreactor <NUM> is shown with reference to <FIG>. In this embodiment, the sample portion <NUM> is connected to a positioner <NUM> (which could be any positioner shown herein). The positioner <NUM> is releasably connected to a cap <NUM> associated with a port <NUM> of the bioreactor <NUM> (such as by engaging trunnions 422a that form a bayonet-style fitting; see, e.g., <FIG>). The releasable connection may be established by a magnetic coupling created by corresponding magnet 414a in the cap <NUM> and a magnetic material 420a (including possible a ferromagnetic material) on the adjacent surface of the positioner <NUM>. In one embodiment, the magnetic coupling is formed once the positioner <NUM> is in place in the bioreactor <NUM> and the cap <NUM> already in place, with the magnet 414a then introduced to the cap so as to avoid disrupting the positioner <NUM>.

To allow for rotation of the cap <NUM> relative to the port <NUM>, without interfering with (i.e., inducting rotation in) the positioner <NUM> and the attached sample portion <NUM>, the positioner <NUM> and the port <NUM> may have matching surfaces. Specifically, the positioner <NUM> may include an outer projection 420b for aligning with a matching inner receiver 422b within the port <NUM>, at a height corresponding to the formation of the magnetic coupling. The projection 420b and receiver 422b may be square in cross-section, and thus create an anti-rotation feature (but of course other shapes could also be used to achieve the same result). Hence, the cap <NUM> may be rotated for removal without rotating the positioner <NUM>, which is retained by the engagement between the receiver 422b and projection 420b. Once the cap <NUM> is released, it may be removed from the port <NUM> to withdraw the positioner <NUM> and thus the sample portion <NUM> from the bioreactor <NUM>.

Referring now to <FIG>, one particular example of a bioreactor <NUM> including one or more samplers <NUM> is shown (in the instant case, two are shown, but as noted above, any number may be provided). The bioreactor <NUM> includes one or more fixed beds, such as two vertically stacked fixed beds 518a, 518b in the illustrated example, which are arranged in an outer chamber 512a of the bioreactor <NUM> and may be the spiral beds shown in <FIG> and otherwise described herein. An inner chamber 512b is also provided for circulating fluid to or from the fixed bed(s), which fluid may be caused to flow by an associated agitator, such as an impeller <NUM> located in a lower compartment 512c of the bioreactor <NUM>. The flow may be in a vertical direction within the fixed bed(s), such as from top to bottom or bottom to top. The fixed bed(s) could also be provided in the inner chamber 512b, with the outer chamber 512a serving to deliver fluid to and from the fixed bed(s) in the inner chamber.

In view of the substantially uniform conditions provided by a fixed bed in the form described herein, sampling any portion of the bed will typically provide a good indication of the cell growth conditions present at any portion therein. In any case, as shown in <FIG>, it is possible to selectively position sample portions 516a, 516b at different locations (e.g., heights or depths in the vertical orientation shown) in the fixed bed(s), including in different stacked beds 518a, 518b, as shown, with positioner 520b and associated sample portion 516b simply being withdrawn through the bed(s) (such as along a pre-defined passage or tunnel) for recovery or inspection. The positioners <NUM> may be any of the embodiments described herein (including with sleeves, not shown), and the use before, during, and after bioprocessing may also be as described herein.

As can be appreciated, the disclosed sampler provides a unique opportunity to facilitate any of the steps herein by automation. For example, with reference to <FIG>, this may be achieved as noted above by providing a system including a bioreactor <NUM> and an actuator <NUM>, such as a motor or robotic device, to move (raise or lower) the positioner <NUM> to withdraw the sample portion <NUM> from the fixed bed <NUM> for analysis. A controller <NUM>, such as a programmable logic controller, computer, or the like, may also be used to control the actuator <NUM> to activate, which could be done based on any number of parameters, such as time since the commencement of a cell culturing operation. The controller <NUM> may also receive outputs from one or more sensors <NUM> associated with the bioreactor <NUM> for sensing one or more parameters, such as pH, cell density, or the like, which are indicative of the state of the cell culturing operation. When one or more of the parameters reaches a pre-determined level, the controller <NUM> may actuate the associated actuator <NUM> to cause the positioner to withdraw the sample portion.

Turning now to <FIG>, an exemplary use of the sampler <NUM> is shown. In <FIG>, it can be seen that the lid <NUM> of the bioreactor <NUM> includes a port <NUM>, to which a partition or barrier is provided in the form of a container or vial <NUM>. With combined reference to <FIG> and <FIG>, it can be understood that the vial <NUM> includes a septum <NUM> through which the positioner in the form of a flexible wire <NUM> may pass for connecting to the sampler portion <NUM> located in the fixed bed <NUM> (such as between two cell immobilization layers thereof). The septum may <NUM> form part of a removable cap or cover <NUM>, which may be crimped on (<FIG>) or threaded onto (<FIG>) the vial <NUM>. An aseptic connection <NUM>, such as a Quickseal device, is provided between the vial <NUM> and the port <NUM>, and the wire <NUM> extends through it.

When it is desired to sample the cell culture associated with the bed <NUM>, the positioner or wire <NUM> may be drawn through a continuous passage formed by the vial <NUM> and the connection <NUM>, as indicated by action arrow A in <FIG>. While a manual operation is shown, it could instead be automated, as indicated previously. Also, whether manual or automated, the decision to retrieve the sampler <NUM> could be based on the output of one or more sensors associated with the bioreactor <NUM>.

The wire <NUM> may be advanced until the sampler portion <NUM> is drawn into the vial <NUM>, as shown in <FIG>. Once fully located therein, the aseptic connection <NUM> may be disconnected, which in the case of the Quickseal device involves the use of a cutter C to crimp and sever the connection, thus separating the vial <NUM>, as indicated in <FIG> (note connection portions 756a, 756b), while still maintaining sterile conditions. The remaining portion 756a of the now-separated connection <NUM> may then be removed (<FIG>), such as by twisting (arrow T) and the vial <NUM> sealed using a cap <NUM>, as indicated in <FIG>, possibly after insertion of a reagent or the like. The vial <NUM> may then be used to study the growth of cells on the sampler portion <NUM>, and thus give an accurate indication of the condition of the cell culture in the fixed bed <NUM> of the bioreactor <NUM>. As examples, the study may involve counting the cells, coloration of the cells to evaluate some parameters (confluence, viability. ), or extraction of intra-cell viruses.

The vial <NUM> should of course have a volume that is greater than the volume of the sampler portion <NUM> it receives. It may in some cases be desirable to maintain a specific relationship between the volume of the vial <NUM> and the size of sampler portion <NUM> in terms of the surface area on which cells are grown. For example, the volume of the vial <NUM> may range from <NUM>-<NUM> milliliters, and the sampler portion <NUM> may have a surface area of about <NUM> to about <NUM><NUM> and, more specifically, about <NUM><NUM> for optimal results based on the number of cells present/unit volume of the vial.

In any of the foregoing embodiments, the sample portion <NUM> may simply be in contact with the fixed bed <NUM> in a bioreactor (not shown), and need not be inserted or positioned within it. For example, as shown in <FIG>, the sample portion <NUM> may be positioned adjacent to and in contact with a surface of the fixed bed <NUM>, such as a side surface (<FIG>) or an upper surface (<FIG>) thereof, and connected to the positioner <NUM>, which again may be a flexible wire. As illustrated, a plurality of sample portions <NUM> may be provided, and may be on the surface(s) of the fixed bed <NUM>, between layers of it, or both. The attachment may be achieved by a connector, such as a frangible one (adhesive). <FIG> further illustrates that a retainer 16a may be provided for retaining the sample portion <NUM> in place adjacent to the fixed bed <NUM>. <FIG> illustrates the above-mentioned situation where the sample portion <NUM> forms part of the fixed bed <NUM> and is pre-cut or provided with perforations for ease of detachment. The pre-cut sample portion may be located anywhere in the fixed bed and in the illustrated embodiment the pre-cut sample is located close to the top of the bed. The bed <NUM> could be provided with one or more receivers, markers, indentations, notches, openings, voids, receiving portions, indicators, markings, guides or the like to indicate placement of a separate sample portion <NUM>. When sampling of the cell culture is desired (which again may be done from any location in the bed <NUM> depending on the location(s) of the sample portion(s) <NUM>), the positioner <NUM> is actuated (manually or automatically) to withdraw the sample portion from the bioreactor.

Claim 1:
An apparatus for use with a cell culture system, comprising:
a fixed bed including a removable sample portion for recovering a sample of cells from the cell culture system, characterized in that, the fixed bed is a structured fixed bed comprising at least two layers of material adjacent to each other, the removable sample portion being located at least partially between the at least two layers.